Breast cancer is becoming more prominent in women today. As of now, there are no effective treatments in treating metastatic breast cancer. We have tested the cytotoxic and anti-migration effects of BHAQ, a synthesized anthraquinone, on two breast cancer cell lines, MCF-7 and MDA-MB231. Anthraquinones are an interesting class of molecules that display a wide spectrum of biological applications, including anticancer properties. Cellular inhibition was tested through a MTT assay, double acridine orange/propidium iodide staining and FACS cell cycle analysis. Inhibition of migration was tested by the wound healing method, and migration through a Boyden chamber. BHAQ was cytotoxic towards both cell lines in a dose dependent and possibly cell-dependent manner. Additionally, BHAQ also inhibited the migration of the highly metastatic MDA-MB231 cell line. Cancer is the number one killer disease in the history of humankind. Among all types of cancers, breast cancer is becoming more prevalent in women . In fact50value, which is the concentration of the drug that can inhibit or kill 50% of the total cells. The results from this assay also imply that this drug affects the mitochondrial potential of these cancer cell lines [50 values obtained at 48 h post-treatment in MCF-7, MDA-MB231 and MCF-10A. BHAQ had a slightly lower IC50 value in MDA-MB231 than MCF-7. BHAQ also exhibited a marginally higher inhibition as compared to tamoxifen in both cell lines. Doxorubicin’s IC50, was extremely low as compared to both BHAQ and tamoxifen in both MCF-7 and MDA-MB231. Nevertheless, both doxorubicin and tamoxifen exhibited higher cytotoxicity in the non-cancerous breast cell line, MCF-10A than BHAQ. The selectivity index (SI) of each drug was calculated by obtaining the ratio of IC50 in the non-cancerous breast cell line/IC50 in the cancerous cell line [The MTT assay was performed as a preliminary study of the cytotoxic activity of BHAQ. After treatment with BHAQ, the yellow tetrazolium dye MTT will be reduced by viable cells to a purple formazan substance . This prll lines . Table 1ell line . The SI 25 .All chemicals used to synthesize BHAQ were obtained from Sigma Chemical Company . Dimethylsulfoxide, DMEM, DMEM/F12, insulin, hEGF, RPMI, acridine orange, propidium iodide, RNAse, Triton-X, methanol and crystal violet were all also purchased from Sigma Chemical Company. Tryple E on the other hand was purchased from Invitrogen . For measurement of absorbance, we used a microtiter plate reader from Bio-Tek Instruments . For all the FACS analysis, the experiment was done using a FACS Calibur flow cytometer . For imaging analysis, we used a fluorescent microscope by Nikon . The NMR analysis was performed using a 500 MHz Varian INOVA NMR Spectrometer . Mass spectra were acquired using a ThermoFinnigan model LCQ2O and acetonitrile (30:70), retention time (tR) 9.17 min. The percentage purity of 99.6% was automatically calculated by the peak purity method. Proton NMR data showed no extra peaks in the BHAQ spectra. Yellow crystals, mp 188–189 °C. 1H-NMR (acetone-6d): δ H 12.63 , 8.33 , 7.96 , 7.86 , 7.52 . δ C 188.4 (C9), 181.6 (C10), 163.2 (C1), 135.1 (C7), 134.8 (C6), 134.3 (C14), 133.3 (C12), 133.2 (C11), 131.8 (C13), 127.9 (C8), 127.3 (C5), 127.1 (C2), 123.1 (C4), 115.3 (C3); m/z (EI) 304 , 302 , 276 (7), 274 (6), 248 (11), 246 (14), 223 (18), 195 (15), 167 (21), 139 (100), 113 (14), 97 (33), 83 (29),69 (96); m/z (%): 301.9558 .3-Bromo-1-hydroxy-9-10-anthraquinone (BHAQ) was synthesized by Friedel Crafts acylation. A mixture of anhydrous aluminium chloride  and sodium chloride  were melted at 125–130 °C. Phthalic anhydride  and 3-bromophenol (40.5 mmole) were mixed well and added slowly into the molten mixture. The reaction temperature was raised to 165–175 °C and maintained for 45–60 min. After being cooled, the deep red solid product was decomposed by adding a mixture of ice water (250 mL) and conc. hydrochloric acid (250 mL). The crude mixture was dissolved in distilled water and organic layer was extracted with ethyl acetate, washed with brine and dried over anhydrous sodium sulfate. The crude products were purified by flash silica gel column chromatography with elution of the ethyl acetate/hexane as yellow needles. The BHAQ purity was determined by HPLC using a JASCO-HPLC equipped with ChromNAV-software . HPLC conditions were as follows: column XBridge RP-18  kept at ambient temperature, injection volume of 20 μL, flow rate at 1 mL/min. and detector wavelength set at 366 nm, mobile phase H2.The cell lines MCF-7, MDA-MB231 and MCF-10a were obtained from the ATCC collection . MCF-7 was maintained in RPMI supplemented with 10% fetal bovine serum, while MDA-MB231 was maintained in DMEM, also supplemented with 10% FBS. MCF-10A on the other hand, was maintained in DMEM-F12 media supplemented with hydrocortisone (0.5 μg/mL), insulin (10 μg/mL), hEGF (20 ng/mL) and 10% FBS. All the cells were kept in a 37 °C incubator with 5% CO5 cells/well. The range of treatment for BHAQ, tamoxifen and doxorubicin was at 30 µg/mL followed by 2-fold dilutions. The cell viability was measured at 48 h post-treatment. MTT solution  was added at a volume of 20 µL in each well and was incubated for three hours. Then, the solution was removed, and 100 µL of DMSO was added to solubilize the crystals. The plates were then read using a microtiter plate reader at the wavelength of 570 nm (Bio-tek Instruments). Triplicates were carried out for each cell line. The following formula was used to determine the percentage of viable cells:The cells were seeded in a 96-well plate at a concentration of 0.8 × 105 cells/well. Both MCF-7 and MDA-MB231 were treated with 3 different concentrations of BHAQ for 48 h. MCF-7 was treated with BHAQ at these concentrations; IC25 (12.3 µM), IC50 (29.4 µM) and IC75 (71.2 µM) and IC50 of doxorubicin (0.41 µM). MDA-MB231 on the other hand, was treated with BHAQ at the following concentrations; IC25 (3.3 µM), IC50 (24.4 µM) and IC75 (92.7 µM) and IC50 of doxorubicin (0.60 µM). The harvested cells were trypsinized and centrifuged at 2,000 rpm for 5 min. The resulting pellets were resuspended in 100 µL PBS and stained with 10 µg/mL of each dye. Acridine orange is a stain that is permeable to viable cells and can stain the cell’s DNA directly [Staining of cells with acridine orange and propidium iodide was used to determine the cell viability of MCF-7 and MDA-MB231. The cells were seeded in a 6-well plate at the concentration of 2.3 × 105 cells/well. Both MCF-7 and MDA-MB231 were treated with three different concentrations of BHAQ for 48 h. MCF-7 was treated with BHAQ at these concentrations; IC25 (12.3 µM), IC50 (29.4 µM) and IC75 (71.2 µM) and IC50 of doxorubicin (0.41 µM). MDA-MB231 on the other hand, was treated with BHAQ at the following concentrations; IC25 (3.3 µM), IC50 (24.4 µM) and IC75 (92.7 µM) and IC50 of doxorubicin (0.60 µM). The cells were trypsinized and centrifuged at 2,000 rpm for 5 min. The resulting pellets were fixed in 70% (v/v) ethanol and stored at −20 °C. After a week, the fixed cells were washed with PBS and treated with RNAse and Triton-x, and were then stained with PI. Afterwards, the cells were subjected to flow cytometric analysis using the flow cytometer.The cells were seeded in a 6-well plate at the concentration of 2.3 × 1025 (3.3 µM), IC50 (24.4 µM) and IC75 (92.7 µM) and IC50 of doxorubicin (0.60 µM). A wound was introduced using a yellow tip pipette in the center of the well. Pictures of the wounded area were captured using an inverted microscope (Nikon) every 3 h, up until 24 h post-treatment.Monolayer cells of MDA-MB231 were grown to confluency in a 6-well plate. MDA-MB231 was treated with BHAQ at the following concentrations; IC5 cells/well. At the bottom of the membrane, 3T3 conditioned medium was added as a chemoattractant together with different concentrations of BHAQ IC25 (3.3 µM), IC50 (24.4 µM) and IC75 (92.7 µM) and IC50 of doxorubicin (0.60 µM). The following day the membrane was fixed with methanol and stained with crystal violet. The dye was then extracted using 30% acetic acid and the concentration was measured at 590 nm. The percentage of migrated cells was measured using the same formula as MTT.MDA-MB 231 cells were serum-starved 24 h prior the assay. The cells were collected and inserted into an 8 µm insert membrane  at a concentration of 3 × 10p-value < 0.05 was considered statistically significant.Data was expressed as mean ± standard deviation (S.D.) of at least three independent experiments. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test. This was accomplished via the GraphPad Prism, V6.0 . * in vivo analysis should be done to assess the antitumor and antimetastatic effects of BHAQ. Overall, BHAQ may be a highly promising candidate for developing a novel anti-cancer drug.Through our results it can be concluded that BHAQ is an interesting molecule that could be a potential anti-cancer agent. It is cytotoxic towards both breast cancer cell lines MDA-MB231 and MCF-7, but is more selective towards the non-cancerous breast cell line MCF-10A compared to tamoxifen and doxorubicin. The morphological changes in treated cells indicate that BHAQ induces apoptosis instead of necrosis, which is a more desirable method of cell death when developing drugs. Furthermore, BHAQ induces a G1 arrest in MCF-7, and this property can be a new target approach in further studies. BHAQ also possesses potential anti-migration properties, which can be further explored to its advantage. Although doxorubicin is more potent than BHAQ, due to doxorubicin’s poor selectivity, BHAQ may be a better compound. Nevertheless, further


Komagataella phaffii for use as an effective whole cell biocatalyst. We introduced into the yeast a transgene encoding a Chromobacterium violaceum ω-transaminase for transcription in response to methanol induction. The strain was then assessed with respect to its growth performance and biotransformation of a fed ketoalcohol substrate to an amino-alcohol. In the resultant strain, BG-TAM, methanol induction did not compromise cell growth. Successful bioconversion of fed substrates to the by-product, acetophenone, indicated transaminase activity in shake flask-cultivated BG-TAM cells. We then used bioreactor cultivation to exploit the high levels of biomass achievable by Komagataella phaffii. In a 900 μL reaction the BG-TAM strain at OD600 = 1024 achieved up to 0.41 mol mol−1 (molproduct molsubstrate−1) yield on substrate (Yp/s) for production of 1-methyl-3-phenylpropylamine and a space time yield (STY) of 0.29 g L−1 h−1 for production of 2-amino-1,3,4-butanetriol. We have shown that transamination, an important step for bespoke synthesis of small molecule medicines, is biologically realisable using enzymes with a broad substrate range, such as ω-transaminases, within living yeast cells that are fed low-cost substrates for bioconversion.We have engineered strain BG-10 of the methylotrophic yeast  The ini phaffii  has been phaffii , support phaffii . K. phafChromobacterium violaceum (C. violaceum) has shown promising results in the production of chiral amino-alcohols, such as 2-amino-1,3,4-butanetriol (ABT), using bacterial whole cells , when fed low-cost substrates.In this work, we engineered the 22.1Komagataella phaffii strain BG-10 and plasmid pJ902-15 were purchased from ATUM. K. phaffii biomass concentration was measured as optical density at 600 nm (OD600) using a spectrophotometer. The C. violaceum CV2025 ω-transaminase gene (NCBI sequence reference WP_011135573.1) was obtained from GenScript. Zeocin was purchased from Thermo Fischer Scientific.2.2C. violaceum CV2025 gene was codon-optimised for expression in K. phaffii using the OptimumGene™ algorithm by GenScript. The optimised coding sequence is available as a public data set  and subsequently plated onto YPD agar plates supplemented with 1 M sorbitol and 200–1000 μg mL−1 of zeocin to select for stable integrants.The data set . The 1.4AOX1 locus in zeocin-resistant colonies using the primer, CCAAAGACGAAAGGTTGAATG, which was designed to anneal within the AOX1 promoter and the primer, GATAATTCGACAACAGCAGG, designed to anneal within the codon-optimised C. violaceum CV2025 gene at a position predicted to be 300 base pairs downstream of the AOX1 promoter only in transformants. Transformant colonies, which were both zeocin resistant and positive by colony PCR, were designated ‘BG-TAM’ and a master cell bank of clones was generated and cryopreserved at -80 °C.Colony PCR was used to confirm the presence of integrated pJ-CV2025 in the 2.3K. phaffii BG-10 and BG-TAM strains were grown for 18 h in 250 mL baffled flasks at 30 °C with 250 rpm to an OD600 ∼ 10 in 50 mL of BMGY medium , a base medium for preparation of minimal and synthetic defined yeast media. Cells were harvested by centrifugation at 5,000 rpm and 4 °C for 5 min, washed in phosphate buffer and resuspended in 50 mL BMMY medium (same constituents as BMGY but substituting 1% v/v glycerol for 2% v/v methanol) to an OD600 = 5. 2% v/v methanol was added every 24 h to maintain induction of the AOX1 promoter. Samples were harvested after 48 h of induction and used for bioconversion measurements.2.4600 in the range of 800–1000 and DCW of 150–200 g L−1 during K. phaffii cultivation by 600 = 60 was reached. This was used as inoculum for 600 mL of basal salt medium  solution. PTM1 had the following constituents: 6.0 g L−1 CuSO4·5H2O, 0.08 g L−1 Nal, 3.0 g L−1 MnSO4·H2O, 0.2 g L−1 Na2MoO4·2H2O, 0.02 g L−1 H3BO3, 0.5 g L−1 CoCl2, 20.0 g L−1 ZnCl2, 65.0 g L−1 FeSO4·7H2O, 0.2 g L−1 biotin and 5.0 mL L−1 of 96% v/v H2SO4. Cultivation parameters and methanol induction conditions followed Invitrogen guidelines for Pichia fermentation  for 6 h, followed by a methanol fed-batch phase using 100% methanol supplemented with 12 mL L−1 PTM1 trace salts at the following feed rates: 3.6 mL h−1 L−1 for 1 h (M1), 7.3 mL h−1 L−1 for 2 h (M2), and final feed rate of 10.9 mL h−1 L−1, which was maintained for 48 h. Samples for growth rate monitoring were harvested at t = 0 and t = end of each feeding phase. The measurement of dry cell weight was carried out by putting the cell pellet of 1 mL of culture in an oven (100 °C) and measuring weight in an analytical balance every 24 h until constant (∼96 h). Samples for bioconversions were harvested after the initial batch stage and every 24 h after methanol induction, the supernatant removed and the cell pellet stored at -20 °C.Cultivation in bioreactor was performed using a Multifors 1L device (INFORS HT). The Multifors 1L device has been reported previously to achieve ODentation  with cul2.5All bioconversion reactions were carried out in 900 μL final volume using whole cells at 30 °C and 500 rpm in a thermomixer (Eppendorf ThermoMixer® C). Pelleted shake flask and bioreactor samples were thawed and re-suspended to 600 μL in a pH 7.5 solution of 0.2 mM pyridoxal phosphate (PLP) in 200 mM, 4-(2-hydroxyethyl)-1-piperazineethanesulfonate (HEPES) buffer to allow the PLP cofactor to associate with transaminase within cells. These cell suspensions were then incubated in 1.5 mL glass vials for 20 m, followed by the addition of 300 μL of substrate solution, consisting of 200 mM HEPES buffer pH 7.5, 0.2 mM pyridoxal phosphate and substrate levels needed to provide the final concentrations indicated in the Results section. Aliquots of 100 μL were taken from the bioconversion reactions at different time points, centrifuged at 12,000 rpm for 10 m at 4 °C, before the supernatant was removed, retained and stored at -20 °C for further analysis.2.6An integrated Dionex Ultimate 3000 HPLC system fitted with an ACE 5 C18 reverse phase column  controlled by Chromeleon 7 software (Thermo Fisher Scientific) was used to quantify MBA, ACP, PB, MPPA and ABT. Samples were quenched and diluted with 0.1% v/v TFA prior to analysis. To analyse MBA and ACP, two mobile phases were used: 100% acetonitrile (A) and 0.1% (v/v) trifluoroacetic acid (B). A gradient was run from 15% A/85% B to 72% A/28% B over 9 min, followed by a re-equilibration step for 2 m . UV detection was carried out at 260 nm, with detection times of 3.6 m for MBA, and 7.8 m for ACP.For MPPA and PB analysis, two mobile phases were used: 0.1% (v/v) trifluoroacetic acid (C) and 0.095% trifluoroacetic acid/80% acetonitrile in MilliQ water (D). A gradient of 40% C/60% D to 100% D was run over 15 min, followed by 2.5 m wash with 100% D, and a subsequent re-equilibration step for 2.5 m . UV detection was carried out at 254 nm, with retention times of 2.7 m for MPPA, and 7.7 m for PB.−1) and stored in dry conditions for no longer than 2 weeks. Typically, a volume of 75 μL of AQC solution was added to 150 μL of bioconversion supernatant previously diluted in 0.02 M borate buffer pH 8.8, reaching a final volume of 225 uL. This mixture was centrifuged prior to analysis. To analyse ABT, two mobile phases were used: 140 mM sodium acetate, pH 5.05 (E) and 100% acetonitrile (F). A gradient of 85% E/15% F to 100% F over 10 m (flow rate 0.5 mL min−1), followed by a 2.5 m wash with 40% E/60% F and a 2.5 m wash with 100% E (flow rate 1 mL min−1). A re-equilibration step was run for 5 min. UV detection was carried out at 254 nm, with a retention time of 6.1 min. For quantification of ERY, an Aminex HPX-87H reverse phase column  was used. The system was run at 60 °C with 0.6 mL min−1 isocratic flow of 0.1% trifluoroacetic acid as mobile phase. UV Detection was carried out at 210 nm with a retention time of 11.5 min. The molar extinction coefficient (ε) at wavelength of 260 nm was determined to be 4878.05 M−1 cm−1 for MBA and 4405.29 M−1 cm−1 for ACP. At 254 nm ε was determined to be 171.32 M−1 cm−1 for PB, 168.86 M−1 cm−1 for MPPA and 2890.17 M−1 cm−1 for derivatised ABT.For ABT measurement, 6-aminoquinolyl-n-hydroxysuccinimidyl carbamate (AQC) was used as derivatization agent. AQC, provided by Dr Fabiana Subrizi from UCL Department of Chemistry, was solubilized in dry acetonitrile  when an ODA concentration of 0.5 M MBA and ERY abolished production of both ACP a and ABT4−1 (molproduct molsubstrate−1), which is comparable to the Yp/s of 0.52 mol mol−1 reported by K. phaffii strain engineered to overexpress transketolase, an enzyme type that typically exhibits high activity in bioconversions. As ω-transaminases have been shown to be responsive to mutational approaches to increase their performance .This work was supported by the UK The authors declare no conflict of interest.No additional information is available for this paper.


The hazard ratio (HR) and 95% CI for any cancer mortality among individuals with body mass index 20 to 24.9 who used aspirin 3 or more times per week should have read “HR, 0.86; 95% CI, 0.79-0.92.” This article has been corrected.1In the Original Investigation titled “Association of Aspirin Use With Mortality Risk Among Older Adult Participants in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial,”


Aspergillus nidulans: 8-methoxycichorine (4), 8-epi-methoxycichorine (5), and N-(4’-carboxybutyl) cichorine (6). The structure of the new compounds was clearly determined by a combination of nuclear magnetic resonance (NMR) analysis and high-resolution electrospray ionization (HRESIMS). The phytotoxic bioassay was studied on leaves from Zea mays and Medicago polymorpha L. at the concentration of 5 × 10−3 M by using a moist chamber technique. Novel phytotoxins 8-methoxycichorine (4), 8-epi-methoxycichorine (5), and N-(4’-carboxybutyl) cichorine (6) exhibited a better phytotoxic effect than cichorine.Fungi are a source of novel phytotoxic compounds to be explored in the search for effective and environmentally safe herbicides. The genetic inactivation of the biosynthetic pathway of the new phytotoxin cichorine has led to the isolation of three novel phytotoxins from the fungus  Herbicides have been used widely in the field of agriculture to kill weed pests and increase crop yield production. However, most of the available herbicides are synthetic chemical compounds, causing serious pollution of the ecological system due to their large-scale usage and difficult degradation ,2. MeanwStreptomyces bacteria. It is one of the most successful commercial herbicides because it is a broad-spectrum herbicide, and is widely used to control important weeds such as Ipomoea aquatica, Sesbania aculeata (Wild.) Pers., Polygonum pensylvanicum and Cyperus esculentus + . Compound 2 was identified by comparison of the 1H-, 13C-NMR chemical shifts and HRESIMS with the authentic sample.cichorine (3): pale yellow amorphous solid; UV (MeOH) λmax (log ε): 212 (4.00), 252 (3.32), 295 (3.01) nm; HRESIMS, m/z 194.0815 [M + H]+ . Compound 3 was identified by comparison of the 1H-, 13C-NMR chemical shifts and HRESIMS with the authentic sample.8-methoxycichorine (4): yellow amorphous solid; m.p. 287 °C; c 0.5, MeOH); UV (MeOH) λmax (log ε) 215 (4.07), 259 (3.37), 298 (3.10) nm; CD (MeOH) λ (∆ε) 284 (+3.05), 352 (+2.06) nm; IR (ZnSe) νmax 3394 (br), 1665, 1572 cm−1; 1H- and 13C-NMR data, see m/z 246.0751 [M + Na]+ .epi-methoxycichorine8- (5): yellow amorphous solid; m.p. 287 °C; c 0.5, MeOH); UV (MeOH) λmax (log ε) 215 (4.07), 259 (3.37), 298 (3.10) nm; CD (MeOH) λ (∆ε) 284 (−3.02), 352 (−2.08) nm; IR (ZnSe) νmax 3393 (br), 1666, 1579 cm−1; 1H- and 13C-NMR data, see m/z 246.0751 [M + Na]+ .N-(4’-carboxybutyl)cichorine (6): white amorphous solid; m.p. 470 °C; UV (MeOH) λmax (log ε) 213 (4.03), 254 (3.33), 295 (3.05) nm; IR (ZnSe) νmax 3374, 1778, 1662, 1572 cm−1; 1H- and 13C-NMR data, see m/z 294.1348 [M + H]+ .σ is the width of the band at 1/e height. tEi and Ri are the excitation energies and rotatory strengths for transition i, respectively. In the current work, the value of σ was fixed at 0.09 eV.The ground-state geometries were optimized with density functional theory (DFT) calculations using Turbomole 6.5 with the basis set def-SV(P) for all atoms at the DFT level, using the B3LYP functional. The ground states were further confirmed by a harmonic frequency calculation. The calculated ECD data corresponding to the optimized structures were obtained using time-dependent density-functional theory (TDDFT) with the basis set def2-TZVPP for all atoms at the DFT level, using the B3LYP functional. The ECD spectra were simulated by overlapping each traction, where Zea mays and Medicago polymorpha L., respectively. All the used leaves were cut from three or four-week-old plants with the size 1.5 cm × 1.5 cm. All the tested compounds were prepared with the concentration at 5 × 10−3 M. The pricks were made using the tip of a 10 µL Hamilton syringe, and a droplet (10 µL) of test solution was applied in the center of the leaves from the upper side. The diameters of the lesions were measured after 48 h of incubation in a moist, sterile chamber at 25 °C. The compounds were prepared in 2% EtOH. The cichorine was chosen as the positive control and 2% EtOH was tested as the negative control.The phytotoxic activities of the cichorine analogues were tested on the cut leaves using a moist chamber technique as described in reference . The leap value of < 0.05 was considered to be statistically significant.Experiments were carried out at least in triplicate, and results were expressed as the mean ± standard deviation (SD). Statistical analysis was performed using the SPSS statistical package for the Social Sciences software. A A. nidulans LO8030 by using pathway inactivation. Three compounds were identified as new natural products with an isoindolinone skeleton and exhibited better phytotoxicity than cichorine on plant leaves, indicating that cichorine derivatives would be promising in the course of developing novel herbicides.In summary, we reported the isolation, purification, structural elucidation, and bioassay of six cichorine-derived metabolites from the fungus


Yarrowia lipolytica, the last step of erythritol synthesis involves the reduction of erythrose by specific erythrose reductase(s). In the earlier report, an erythrose reductase gene (YALI0F18590g) from erythritol-producing yeast Y. lipolytica MK1 was identified  might exist in the genome of Y. lipolytica.Erythritol is a four-carbon sugar alcohol with sweetening properties that is used by the agro-food industry as a food additive. In the yeast YALI0D07634g) and g3023.t1 (YALI0C13508g) encoding two novel erythrose reductases (ER). The biochemical characterization of the purified enzymes showed that they have a strong affinity for erythrose. Deletion of the two ER genes plus g801.t1 (YALI0F18590g) did not prevent erythritol synthesis, suggesting that other ER or ER-like enzymes remain to be discovered in this yeast. Overexpression of the newly isolated two genes (ER10 or ER25) led to an average 14.7% higher erythritol yield and 31.2% higher productivity compared to the wild-type strain. Finally, engineering NADPH cofactor metabolism by overexpression of genes ZWF1 and GND1 encoding glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, respectively, allowed a 23.5% higher erythritol yield and 50% higher productivity compared to the wild-type strain. The best of our constructed strains produced an erythritol titer of 190 g/L in baffled flasks using glucose as main carbon source.In this study, we have isolated genes g141.t1  contains supplementary material, which is available to authorized users. Torula corallina [Candida magnoliae [Pseudozyma tsukubaensis [Trichosporonoides megachiliensis [YP/S conversion yield ranging from 0.43 to 0.61 g/g. In yeast, erythritol is synthesized via the pentose phosphate pathway  is a four-carbon sugar alcohol with sweetening properties and applications in the agro-food and pharmaceutical industries [orallina , Candidaagnoliae , 7, Pseuubaensis , and Triiliensis  with YP/ay PPP, , 11) as  as TorulYarrowia lipolytica has also been found to be an efficient erythritol producer [TKL1, YALI0E06479g), transaldolase  and erythrose reductase , allowed the erythritol productivity to increase with various magnitudes (from 16 to 200%) [Recently, the yeast producer . Severalproducer –14, but to 200%) , 15, 16.to 200%) , 16 or cto 200%)  have alsCandida magnoliae [Trichosporonoides megachiliensis  with concurrent NAD(P)H oxidation , 17. Sevisozymes , and twosozymes, ). Howevepolytica . OverexpY. lipolytica and with the aim to further increase the erythritol production, we identified and characterized two additional ER (namely ER10 and ER27) in Y. lipolytica strain CGMCC7326, a strain able to produce erythritol with very high titer  and GND1 (YALI0B15598g) that allow NADPH to be replenished, yielded significantly improved erythritol productivity.In order to get more insights on ER in 150 g/L, , 20). OvYALI0F18590p) in Y. lipolytica based on sequence similarity with erythrose reductase from Candida magnoliae [T. megachiliensis [Moniliella sp. [Y. lipolytica strain CGMCC 7326 for enzymes with reductase and NADPH as keywords. Then only enzymes with NADPH dependent reductase were obtained from the genome annotation.In erythritol producing yeast, the final step of erythritol synthesis consists in the reduction of erythrose by specific erythrose reductase , 11. Recagnoliae . These Eagnoliae , 22. Difella sp. , and theella sp. , 22. We g141.t1 (ER10), g3023.t1 (ER25) and g801.t1 (ER27) (Table E. coli BL21(DE3). Assay for reductase activity on cell extract of IPTG-induced cells using d-erythrose as a substrate and NADPH as a co-factor led to the identification of strain HCE102 , HCE110  and HCE111  with NADPH-dependent erythrose reductase activity. Indeed, reduction of d-erythrose into erythritol could be detected with the above-mentioned cell extract as shown in Fig. E. coli in those experimental conditions.This led to the identification of 12 putative genes, including g141.t1, g3023.t1, and g801.t1 as a query, highlighted in Y. lipolytica strain CLIB122 genes YALI0D07634g, YALI0C13508g, and YALI0F18590g, respectively, with 100% identity. Therefore, gene g801.t1 corresponds to the gene YALI0F18590g identified as encoding ylER in Y. lipolytica strain MK1 [C. magnoliae ER (ACT78580.1), T. megachiliensis ER1 (BAD90687), Tilletiaria anomala ER3 (XP_013243550.1) and Moniliella sp. ER3 (AGB07593.1)  and is relatively distant from Y. lipolytica ER25 and ER27 . This in silico analysis suggests that ER10 has evolved differently from ER25 and ER27 in the ALR family.Protein BLAST search using a translated sequence of genes rain MK1 . Amino aER10, ER25 and ER27), were disrupted in Y. lipolytica strain CGMCC7326. Disruption cassettes, namely pINA-UPER10-DWER10, pINA-UPER25-DWER25, pINA-UPER27-DWER27, were constructed as described in “ER25, strain HCY101) and g801.t1  resulted in a lower erythritol production , a lower productivity  and a lower yield   led to an increase in erythritol titer and yield . Disruption of the three ER encoding genes in strain HCY103 led to a lower erythritol production (decreased by 36.8%), a lower productivity (decreased by 61.2%) and a lower yield (decreased by 37.2%). From this, it could be concluded that the three identified genes encode proteins involved in the erythritol biosynthetic pathway in Y. lipolytica CGMCC7326, deletion of ER10 gene increased erythritol biosynthetic capacity, while deletion of ER25 and ER27 gene decreased erythritol biosynthetic capacity. However, the ability of the multiple disrupted strain HCY103 to synthesize erythritol highlights that alternative biochemical pathway for erythritol synthesis or other unknown ER gene(s) remain to be identified.To confirm that the three putative identified ERs are indeed involved in erythritol biosynthesis, the corresponding encoding gene (i.e. E. coli and purified. After 10 h of induction of the corresponding E. coli BL21(DE3) strains, namely HCE102, HCE110 and HCE111, cells were collected and proteins were purified by Ni–NTA and gel filtration chromatography  and Kcat/Km (1.09 mM−1 s−1) demonstrating a higher ability to catalyze the reduction of erythrose into erythritol than ER10  and ER27 (15.8 s−1 and 0.61 mM−1 s−1) respectively.The catalytic constants were deduced from the non-linear regression curves (Table + was used as the cofactor (2 mM). As shown in Table +, however with a lower efficiency than for the forward reaction . Surprisingly, the reaction product, analyzed by high-performance liquid chromatography, was found to correspond to erythrulose and not to erythrose .We also assessed the ability of the purified ER to catalyze the backward reaction (i.e. the oxidation of erythritol) at pH8.0. For this purpose, erythritol was used as a substrate at different concentrations (from 20 to 1500 mM) and NADPose Fig. . It has F01650g, ). Howevenditions . Based oY. lipolytica strain MK1, the erythrose reductase encoded by gene YALI0F18590g (namely ylER) displays the highest activity at pH 3 in the presence of 0.25 mM of Zn2+ [ylER (pH 3.0) by Janek et al. [Y. lipolytica prefer acidic conditions for maximal activity, which is a typical characteristic of aldo–ketoreducatse .Fig. 4EylER from Y. lipolytica strain MK1, the addition of Zn2+ increases the activity of both ER10 and ER25 . The substrate specificity of ER10 was consistent with the erythrose reductase from C. magnoliae, which also has enzymatic activity against d-erythrose, d-xylose, and d-ribose [The -glucose . Similard-ribose .Table 4SylER is dependent on NADH as a co-factor. To investigate the cofactor preference of ER10 and ER25, reduction of d-erythrose was measured in the presence of both NADPH and NADH. Reduction of erythrose was only detected when NADPH was used as a cofactor . Erythrose reduction could not be measured in the presence of NADH even at high concentration (10 mM). Similarly, oxidation of erythritol by ER10 could only be detected in the presence of NADP (0.65 U/mg of protein). The specific motif IPKSXXXR has been reported as a common catalytic feature found in both aldo–keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) implicated in cofactor binding. Indeed, it has been demonstrated that the lysine residue binds the 2′-phosphate of NADPH [Yeast ALRs can be classified into two groups according to their strict dependency to NADPH or their dual coenzyme (i.e. NADPH and NADH) specificity , 26. Janof NADPH , 28. Prog141.t1), ER25 (g3023.t1) and ER27 (g801.t1) were overexpressed under the control of the strong constitutive promoter hp4d, either separately or simultaneously, in the Y. lipolytica strain CGMCC7326. Gene overexpression was confirmed by qPCR , demonstrating that the expression cassettes were functional in transformed strains. The engineered strains were grown in EPMG medium until the carbon source (glucose 300 g/L) was totally consumed. As shown in Table YALI0F18590g (ER27 gene in this study) in the starting strain Y. lipolytica MK1 resulted in an enhanced erythritol production of 44.44 g/L, 20% higher than that of the control strain Y. lipolytica MK1, and 4% higher than the strain HCY106 (13% higher than that of the control strain CGMCC7326). However, the productivity of the HCY106 strain was higher relative to the engineered strain AJD pAD-ylER (2.2 g/L h vs 0.77 g/L h). The time for glucose consumption was also reduced by 13% for strain overexpressing ERs encoding gene as compared to the parental strain. Overexpressing strains also exhibited a slightly higher glucose uptake rate , YALI0C13508g (ER25), and YALI0F18590g (ER27) were increased by 5.3, 15.7 and 16.4-fold, respectively, as compared to the parental strain (data not shown). However in contrast to our expectation, erythritol productivity and yield for strain HCY107 were not further increased as compared to that of strain HCY104, HCY105, and HCY106, respectively  and GND1 (YALI0B15598g), respectively [php4d-ER10-25-27-pTEF-ZWF1-GND1), erythritol yield and productivity were further improved by 6.7% and 14.3%, respectively, as compared to strain HCY107, and 23.5% higher erythritol yield and 50% higher productivity compared to the wild-type strain CGMCC7326 (Table ER genes plus zwf1 and gnd1 genes) than that of HCY107 (overexpression of the three ER genes), the average 6–7% improvement was also important to increase its economy. The titer could be further improved by overexpression of other key genes related to erythritol synthesis such as transketolase or ribose 5-phosphate epimerase genes. This demonstrates that overexpression of genes ZWF1 and GND1 have some positive impact on erythritol synthesis.As demonstrated above, the reduction of erythrose by ER needs NADPH as a cofactor. Therefore, regenerating this cofactor from NADP is a key factor to increase erythritol productivity in strain HCY107. The first reaction of the PPP pathway, catalyzed by the glucose-6P dehydrogenase (G6PD), generates NADPH from glucose-6P and NADPoduction . The 6-pectively . Therefo1 YALI0B1598g, resZWF1 and GND1 could push glucose metabolism into phosphate pentose pathway leading to synthesize more erythritol . Overexpression of genes encoding these ER together with the engineering of NADPH metabolism allowed significantly increased erythritol production titers.We have isolated, purified and characterized two novel ER enzymes of d-erythrose, l-erythrulose, d-erythritol, d-arabitol, l-arabinose, d-xylose, d-ribose, and d-xylulose were obtained from Sigma-Aldrich . Coomassie brilliant blue R-250, cofactor NADP, NAD, NADH, NADPH, and antibiotics were purchased from Sangon Biotech . The chromatographic media were procured from GE Healthcare Life Sciences (Sweden). All other chemicals were of analytical grade and used as such without any purification.Escherichia coli and Y. lipolytica strains used in this study are listed in Additional file E. coli strains were grown at 37 °C in Luria–Bertani medium supplemented with kanamycin sulfate (50 mg/L). The Y. lipolytica strains were grown at 28 °C in YPD  or YNB medium  supplemented sucrose , lactose  or xylitol . Hygromycin (200 μg/mL) was added in YPD when necessary to screen transformants. For erythritol synthesis, the EPMG medium was employed (per L): 300 g glucose, 8 g yeast extract, 8 g corn syrup, 2 g ammonium citrate, 0.05 g MnSO4·H2O, 0.05 g ZnSO4·7H2O and 0.01 g CuSO4·5H2O, initial pH 6.5. For solid media, agar (15 g/L) was added. Shake-flask cultures for erythritol production were performed in triplicate using 2 L baffled flasks containing 500 mL EPMG medium, at 30 °C and 200 rpm. Cultures were performed until glucose was depleted. All the baffled Erlenmeyer flasks containing culture medium were weighed both at the start of fermentation and then prior to taking samples aliquots for analysis. Sterile water was supplemented to compensate for the medium evaporation during fermentation. At the time of sampling, the resulting reduced weight was replenished by the addition of same weight of distilled water. At the end of the fermentation, the final weight was essentially the same as the initial weight.The l-erythrulose, mannitol, arabitol, arabinose, arabitol, xylose, xylitol, ribose, ribitol, and glucose were quantified by HPLC using a refractive index detector (Shodex RI101) and a Shodex SP0810 ion exclusion column (300 × 8 mm). Elution was performed at 70 °C using pure water at a flow rate of 1 mL/min. The concentrations of eluted compounds were calculated by internal standard methods [4, flow rate 1 mL/min, detector was VIS at 430 nm.Erythritol, erythrose, n column 0 × 8 mm.YERY) was expressed in g/g from glucose and was calculated from the equation YERY = P/S. The volumetric productivity (QERY) was expressed in g/L h and was calculated from QERY= P/V·t, where P is the amount of erythritol in the culture liquid at the end of fermentation (g); S is the total amount of glucose consumed (g); V is the initial volume of culture liquid (L), and t is the culture time (h). Glucose consumption rate (rGLU) was calculated as the amount of glucose consumed per hour and per liter of culture medium.The mass yield of erythritol . Yeast transformation was as described in An et al. [Standard media and techniques were used for E. coli , and thelsewhere . The resg et al. . PCR wasn et al. .http://blast.ncbi.nlm.nih.gov/Blast). The phylogenetic tree of putative ER genes was constructed with MEGA7 software [Protein BLAST searches were performed on National Center for Biotechnology Information web server  was then transformed with the different constructs. The correctness of the resulting vectors was verified by DNA sequencing using a T7 primer  disruption plasmid pINA-UPER27-DWER27 was constructed similarly with primers pairs ER27-F1/ER27-R1 and ER27-F2/ER27-R2. P and T fragments were digested with NotI-SalI and EcoRI-NotI, respectively, and cloned in two steps in plasmid pHP4d-Pir1-A.oryGal [NotI digested and the purified ER disruption cassettes were used to transform Y. lipolytica strain CGMCC7326. Transformants were selected using sucrose, lactose or hygromycin B as a selectable marker. The correctness of genes disruption in the resulting strains HCY100, HCY101, and HCY102 was verified by analytical PCR using primer pair ER10-F/ER10-R, ER25-F/ER25-R and ER27-F/ER27-R  and lactose (YNBL), the correctness of ER10 and ER27 gene disruption was verified by analytical PCR using primer pairs ER10-F/ER10-R and ER27-F/ER27-R, respectively. The strain deleted for the three ER encoding genes was named as HCY103  and downstream (T) of gene g141.t1 were PCR amplified using genomic DNA of strain CGMCC7326 as a template, and primers pairs ER10-F1/ER10-R1 and ER10-F2/ER10-R2, respectively. After purification, P fragment was digested with oryFTase , to yiel-PdSIase  to yieldA.oryGal . The aboHindIII and BamHI site at, respectively, 5′ and 3′ end of each ER encoding gene . The resulting strains were named HCY104 (php4d-ER10), HCY105 (php4d-ER25), HCY106 (php4d-ER27). To construct strain HCY107 , g3023.t1 (ER25) and g801.t1 (ER27) were amplified with primer pairs ER10-F11/ER10-R11, ER25-F11/ER25-R11, and ER27-F11/ER27-R11, respectively, and genomic DNA of strain CGMCC7326 as a template. Primers F and R were designed to introduce a -PdSIase , a deriv-PdSIase  that allER10 (HCY104) and we transformed it with ER25 expression cassette. We then selected the corresponding transformant (genotype ER10-ER25) on medium containing 800 μg/mL of hygromycin. This strain was then transformed with ER27 expression cassette and transformants were selected and 2000 μg/mL of hygromycin. In brief, transformants were selected on YPD supplemented with hydromycin B at a concentration of 200 μg/mL of hygromycin for mono-integration, 800 μg/mL for double-integration and 2000 μg/mL for triple-integration.We started from a strain overexpressing ZWF1 (YALI0E22649g) and GND1 (YALI0B15598g) from Y. lipolytica were also overexpressed in the strain HCY107. A DNA fragment containing genes ZWF1 and GND1 under the control of the constitutive hp8d promoter and XDH gene from Scheffersomyces stipitis CBS 6054 used as a selectable marker was synthesized by GENEWIZ , and cloned at EcoRI site of pUC57 to yield pUC57-zwf-gnd . The final strain that overexpresses genes ER10, ER25, ER27, ZWF1 and GND1 was designated as HCY108.Genes 600 of 2.0 and stored at − 80 °C in Trizol solution. Total RNAs were extracted using the TRNzol kit from TianGen Biotech , and cDNA was obtained using PrimeScript™ RT reagent Kit with gDNA Eraser . The qRT-PCRs were performed using SYBR® Premix ExTaq™ II (TliRNaseH Plus), Takara, Dalian, China) and ABI7500 Real-Time PCR system (Applied Biosystems). Primer pairs for qRT-PCRs were, ER10-Ffluo/ER10-Rfluo, ER25-Ffluo/ER25-Rfluo, ER27-Ffluo/ER27-Rfluo for gene ER10, ER25 and ER27, respectively  was grown in 5 mL of Luria–Bertani medium (LB) with 100 μg/mL kanamycin in 30-mL universal tubes at 37 °C for 3 h with shaking at 220 rpm. Thereafter, the 5 mL of cells were transferred to 495 mL of LB medium with 100 μg/mL kanamycin in a 2-L Erlenmeyer flask. The cells were incubated at 37 °C with shaking at 220 rpm until the OD600 reached 1.0. Then, isopropyl-β-d-thiogalactopyranoside (IPTG) was added at a final concentration of 1 mM and cultures were further incubated at 25 °C for 10 h (until OD600 reached 4.5). Two hundred and fifty milliliters of cells were harvested by centrifugation at 12,000g and 4 °C for 10 min, before being resuspended in 100 mL binding buffer . Cells were sonicated at 4 °C for twenty cycles of 5 s at 5 s interval. Cell debris was removed by centrifugation at 10,000g for 10 min at 4 °C. The cell extract was finally filtered through a 0.45 μm membrane filter (Millipore).A single colony of recombinant 2PO4-150 mM NaCl-200 mM imidazole (pH 8.0). The Ni–NTA purified erythrose reductases were further applied to gel filtration on Sephadex G-25 and eluted with 50 mM Tris–Cl, 200 mM NaCl, pH 8.0 following the manufacturer’s instructions. SDS-PAGE was performed in a 12% (w/v) acrylamide gel at a constant current of 200 mA. Low molecular weight protein standard was used as size marker . After electrophoresis, gels were stained with Coomassie brilliant blue G-250 via standard procedures. Scanning densitometry of SDS-PAGE gel was performed using Phoretics 1D Non-linear Dynamics  software.Erythrose reductase from cell lysates was purified using Ni–nitrilotriacetate agarose (Ni–NTA) according to the manufacturer recommendation. Proteins were eluted with a buffer containing 25 mM NaHKm values were problematic as commercial d-erythrose  tested were only 75% purity. In order to evaluate more accurately its activity and Km values, we purified it by semi-preparative HPLC to obtain d-erythrose with 95% purity.The accurate assays of erythrose reductase activity or its Erythrose reductase activity was determined by monitoring the reduction or oxidation of cofactor NADP(H) by absorbance measurement at 340 nm , 7. The + (2 mM). Kinetic parameters were calculated by non-linear regression of velocities against substrate concentrations, and processed using OriginPro 8.0 software.The kinetic parameters of the purified ER enzymes were calculated. For this purpose, initial velocity studies were performed with erythrose as a variable substrate concentration ranged from 2 to 200 mM in the presence of a fixed concentration of cofactor . When using erythritol as a substrate, the concentration varied from 20 to 1500 mM in the presence of NADP4, CuSO4, MnSO4, NiSO4 and CaCl2 concentrations (from 0.1 to 5 mM) at 30 °C in 25 mM Mcilvaine’s buffer (pH 6.0) using erythrose as a substrate.The influence of pH on the activity of ERs was carried out at 30 °C in the Mcilvaine’s buffer (from pH 3.0 to 8.0), containing 10 mM erythrose and 2 mM NADPH and 50 μL of purified ER enzymes (1 mg/mL). The assays were performed in triplicate at each pH point. The effect of metal ions on ER activity was studied by assaying different ZnSOd-erythrose, d-fructose, d-glucose, d-galactose, d-arabinose, l-arabinose, xylose, ribose, xylulose as substrate. The reaction mixture contained  50 mM substrate, 2 mM NADPH, 50 μL of purified ER enzymes and 25 mM potassium phosphate buffer (pH 6.0). Specificity was expressed as U/mg of protein and normalized to that obtained for d-erythrose. For cofactor specificity, ER activity on erythrose (10 mM) was determined as described above using cofactor (NADH or NADPH) at a concentration of 2 mM and 10 mM. Oxidation of erythritol was measured in the same conditions in the presence of NADP or NAD.The substrate specificity assays were performed using erythritol, Additional file 1: Table S1. Primers, gene cassettes, and strains used in this study. Table S2. Identity of YALI0D07634p (ER10), YALI0C13508p (ER25), YALI0F18590p (ER27) with ER from Candida magnoliae ER (ACT78580.1), Trichosporonoides megachiliensis ER1 (BAD90687), Tilletiaria anomala ER3 (XP_013243550.1) and Moniliella sp. ER3 (AGB07593.1). Table S3. Effect of divalent metal ions on the activities of ER10 and ER25. Figure S1. SDS-PAGE analysis of the ER10, ER25, and ER27 overexpressed in E. coli BL21(DE3). Lane M: protein standards; lane 1: crude extract of non-induced cells; lane 2: crude extract of IPTG-induced cells; lane 3: purified ER protein by Ni2+ affinity resin; lane 4: purified ER protein by gel-filtration. Figure S2. The non-linear regression plots of initial-velocity against D-erythrose  or erythritol (B) using ER10 , ER25 (C) and ER27 (D) as enzymes. Figure S3. Amino acid sequences of ER27, ER25, and ER10. The specific motif IPKSXXXXR is highlighted in bold. Figure S4. Comparison of HPLC spectrum of culture supernatant of the Y. lipolytica wild-type strain CGMCC7326 and engineered strains, namely HCY104 (php4d-ER10), HCY105 (php4d-ER25), HCY106 (php4d-ER27), HCY108 php4d-ER10-25-27, php8d-ZWF1-GND1). Figure S5. pH or OD600 change during fermentation of strains CGMCC7326, HCY104, HCY107, HCY108, at the starting pH 6.5 , and pH 3.0 . For the control strain CGMCC7326, pH was decreased to 3.2 in 48 h and retained around 2.9±0.2 during fermentation until the depletion of glucose, and pH increased to 3.4 after glucose was completely exhausted, when the starting pH was 6.5 (A); For the engineered strains , pH was decreased to 4.6±0.05 in 24 h and maintained around 4.2±0.2 during fermentation until the depletion of glucose, and pH increased to 4.6±0.1 after glucose was depleted, when the starting pH was 6.5 (A); For the control strain CGMCC7326, the pH was maintained around 2.9±0.1 when fermented at starting pH 3 buffered with citrate until glucose was depleted (B). For the engineered strain, the pH was maintained around 3.2±0.2 when fermented at starting pH 3 buffered with citrate until glucose was depleted (B). The control strain produced more cell biomass and citrate than the engineered strains whenever at starting pH 6.5 or 3.0 . Figure S6. Schematic representation of the DNA fragment used to overexpress ZWF1 and GND1 genes in strain HCY108.


Some food ingredients, such as vitamins and I–, prompt d-amino acids to form uric acid. d-amino acids in beer are one of the key factors responsible for the increase in uric acid levels. The biological response of d-amino acids could explain gout occurrence in beer drinkers.Excessive intake of beer could increase serum uric acid levels, leading to high risk of gout, which was previously attributed to high purine content in beer. Recent reports that purine-rich vegetables and bean products do not cause higher uric acid levels do not support this theory. Why excessive intake of beer could increase a high risk of gout has been unclear. Other factors affecting the accumulation of uric acid in the blood have been explored. Beer contains relatively high levels of  Ser)  to form NH4+, α-keto acid and H2O2  , Fe3+ (364–1388 μg/g) and Cu2+ (17.1–143.9 μg/g) . l/d-isomers of Cys, Asn, Phe, Leu, His, Thr, Pro and Ser were from J&K Scientific Ltd. . Sodium 3,5-dichloro-2-hydroxybenzenesulfonate (DHBS), adenosine deaminase , phenyl isothiocyanate (PITC) and 4-aminoantipyine  were also from J&K Scientific Ltd. d-amino acid oxidase , purine nucleoside phosphorylase  and horseradish peroxidase  were purchased from Sigma-Aldrich, Merck . Calf thymus DNA, riboflavin, xanthine oxidase , vitamin C, vitamin E and catalase (CAT) were obtained from Solarbio Co., Ltd. . Phosphate buffered saline (PBS) and Tris-acetate-EDTA (TAE) buffers were from Sangon Biotech Co., Ltd. . Methanol (chromatographic purity) was purchased from Siyou Co., Ltd. . Agarose was obtained from Tiangen Co., Ltd. . Triethylamine (TEA) was purchased from Shanghai Titan Co., Ltd. . Acetonitrile (ACN) was obtained from Sinopharm Chemical Reagent Co., Ltd. . Acetic acid was obtained from Beijing Chemical Works . Yanjing beer , Tsingtao beer  and Harbin beer  were purchased in a supermarket . We confirm that experimental test was conducted within the warranty period. Ultrapure water was prepared using double distilled water (18 MΩ·cm).All chemicals used are of analytical grade (>99.0%). l- and d-Ala on the formation of H2O2 catalyzed by DAAO was studied. The l- and d-Ala solutions at concentrations of 10, 1.0, 0.1, 0.01 and 0.001 mM were prepared with PBS buffer . Then, the chromogenic agent containing 228 U/L DAAO, 0.4 mM 4-AAP, 2.0 mM DHBS and 1500 U/L HRP was added into each of the above 3.0-mL solutions, and incubated at 37 °C for 30 min in digital constant temperature water bath pot . DAA was oxidized by DAAO to generate H2O2, which was quantified by enzymatic coupling method and UV-Vis spectroscopy . DNA damage was detected using DNA band diffusion assay on agarose gel (1%). The samples containing 0.1 mM Fe2+, 0.75 U/mL DAAO, 50 μg/mL DNA and different concentrations  of d-Ala were parallelly incubated at 37 °C for 30 min, and the degree of DNA damage was detected by agarose gel (1%) using electrophoresis system . The reaction mixtures were terminated by the addition of 2 μL of loading buffer, and 10-μL aliquots were analysed by gel electrophoresis at 120 V for 1.5 h in the presence of a 1×TAE buffer using 1% agarose gel.The effect of 2U was investigated. Samples containing DNA (50 μg/mL), Fe2+ (0.10 mM), different concentrations of d-Ala  and DAAO with different activities  were prepared to simulate the formation of uric acid. The samples were incubated at 37 °C for 30 min, then CAT was added at 37 °C and kept at constant temperature for 30 min to remove H2O2 residues. Afterwards, the samples were heated at 70 °C for 15 min to inactivate CAT. To catalyze adenine and adenosine produced after DNA breaking into H2U and H2O2, PNPase (9.4 U/L), ADA (10.0 U/L) and XOD (5.0 U/L) were added and incubated at 37 °C for 30 min. All samples were incubated at 37 °C for 30 min in 2.7-mL reaction mixtures containing 1500 U/L HRP, 0.4 mM 4-AAP and 2.0 mM DHBS. The absorbance of H2U was indirectly detected at 512 nm by UV-Vis spectroscopy .The effect of DAA concentration on the formation of Hd- and l-amino acids were determined based on the method of Cohen et al. , 10−11 min mobile phase B 12.5%  and 11–30 min mobile phase A 90%. Injection volume was 5 μL, the column was maintained at 25 °C, detection wavelength was 254 nm. The flow rate of the mobile phase was 0.8 mL/min, the control samples were prepared by dissolving dry powders in 100 μL water without DAAO. The total DAA content in beer samples was calculated by comparing the peak areas of standard amino acid and DAA in the beer at same retention time. Each test was done in triplicate and the content of DAA in the beer was expressed as mean value±standard deviation.Beer samples of 50 μL were dried in vacuum drying oven . The obtained dry powders were mixed with 0.75 U/mL DAAO and dissolved in 100 μL water, and then incubated at 37 °C for 30 min, followed by drying in vacuum drying oven. The 2O2, and then the obtained solution was maintained at 70 °C for 15 min to inactivate CAT, followed by drying again at 45 °C. Control samples containing 50 μg/mL DNA without adenine and adenosine and test group containing 0.75 U/mL DAAO, 0.10 mM Fe2+ and 50 μg/mL DNA were prepared. These samples were incubated at 37 °C for 30 min, and then CAT was added at 37 °C and kept at constant temperature for 30 min to remove H2O2 residues. Afterwards, the samples were heated at 70 °C for 15 min to inactivate CAT, and then PNPase, ADA and XOD with final activities of 9.4, 10.0 and 5.0 U/L respectively were added and the samples were incubated for 30 min at 37 °C. All samples were kept at 37 °C for 30 min in 2.7-mL reaction mixtures containing 1500 U/L HRP, 0.4 mM 4-AAP and 2.0 mM DHBS. The absorbance was read at 512 nm using UV-Vis spectrophotometer .The samples of 0.50 and 1.0 mL of Yanjing beer were pipetted into individual tubes and dried in vacuum drying oven  at 45 °C to remove the ethanol. The dry powers were dissolved in 1.0 mL water, and then the PNPase, ADA and XOD were added to the two samples to remove the adenine and adenosine, thereby eliminating their interference. The interferences were detected by enzymatic coupling method and UV-Vis  and DAAO  were mixed with different concentrations  of vitamin B2 in 3.0 mL of PBS buffer. The samples were incubated at 37 °C for 1 h, and then the absorbance of the pyruvic acid was measured at 260 nm by UV-Vis spectrophotometer  to evaluate the effect of vitamin B2 on the activity of DAAO.The effect of vitamin B2U were studied. The samples containing 0.1 mM Fe3+, 0.75 mg/L vitamin E or vitamin C, 50 μg/mL DNA, 0.1 mM DAA and 0.75 U/mL DAAO were incubated at 37 °C for 40 min, followed by gel electrophoresis  at 120 V for 1.5 h on 1% agarose gel.The effects of vitamins C and E on the formation of H– and Fe3+ on the formation of H2U were estimated by the determination of ˙OH. The highly reactive ˙OH reacts with Phe and produces o-, m-, and p-Tyr (3+ (Fe3+–EDTA complex), 0.1 mM DAA and 0.75 U/mL DAAO was incubated for 40 min at 37 °C. The concentration of ˙OH in the mixture was determined by HPLC , using isocratic elution consisting of 250 mM KH2PO4/H3PO4 (pH=3.01) with 5% methanol (by volume) at the flow rate of 0.8 mL/min. The absorbance of the samples was evaluated at 274 nm by UV- -2550 spectrophotometer (Shimadzu Corporation).The effects of I– (0.10 mM), Fe3+–EDTA (0.10 mM), d-Ala  and DAAO . The degree of DNA damage was determined using DNA band diffusion assay on agarose gel (1%).DNA samples (50 μg/mL) were treated with I2U was studied. As we know, the seafood contains abundance of metal ions (e.g. Fe3+ and Cu2+)  was prepared and incubated at 100 °C for 3 h. The Cu2+ to l-Ala ratios were 0, 1:8, 1:4 and 1:2. The racemization of l-Ala were detected by circular dichroism spectrophotometer  in the 190–240 nm wavelength region at room temperature.The effect of the racemization of LAA on the formation of H2O2 was one of metabolites of d-isomeric amino acids in the presence of d-amino acid oxidase (DAAO) due to stereospecificity of the deamination of amino acids . Our results draw the same conclusions as our previous research  produces ˙OH, leading to DNA damage , indicating that DAA can act as an effective disruptor for DNA in the presence of DAAO and Fe2+. Other research on DNA damage with salmon sperm DNA and pBluescript K+ plasmid had proven that the presence of hydroxyl radicals leads to DNA breaks  fluorescence, which was proportional to uric acid concentration  was determined by HPLC and compared with standard amino acids . PITC reacted with amino acids to form stable phenylthiocarbamyl derivatives . Erbe and Brückner  of the human body  in the presence of Fe3+ were 18054, 5854 and 9944, respectively  by I– promotes the formation of ˙OH. In addition, DNA damage induced by 0.1 mM d-Ala in PBS containing different concentrations of I– and 0.75 U/mL DAAO in the presence of Fe3+ (lanes 1–5) or Fe3+–EDTA (lanes 6–10) was further investigated, as shown in Fig. S4. Lanes 1–5 show DNA damage levels of 30.0, 47.0, 50.6, 51.1 and 51.1%, respectively. However, when Fe3+ was chelated by EDTA, lanes 6–10 showed the DNA damage levels of 33.3, 33.4, 33.4, 33.5 and 33.6%, respectively. This confirms that the complex of Fe3+–EDTA can prevent production of reduction product (Fe2+) by I–, thereby inhibiting the conversion of H2O2 into ˙OH and reducing DNA damage.Actually, chelation by Fe2U accumulation in the blood, caused by purine metabolism disorder. Exogenous (about 20%) and endogenous (about 80%) purines are present in the body. The former originate from daily diet, and the latter are formed by the oxidation of nucleic acids, which mainly lead to the formation of purine. Previous surveys have shown that excessive intake of beer can increase the risk of gout, which could be attributed to high level of purines in beer  in beer on the formation of serum uric acid (H2U) and found that DAA could be oxidized to H2O2 by d-amino acid oxidase (DAAO). Further oxidization gave highly active hydroxyl radicals, which would accelerate the nucleic acid metabolism to purines and elevate H2U level, resulting in gout. We conclude that DAAs in beer have a key role in inducing the increase of H2U, and some food ingredients, such as vitamins and iodide ions, intensify the effect.Here we discussed the effect of


Bacillus amyloliquefaciens RWL-1, was isolated from the seeds of Oryza sativa. Initially, the crude extract of RWL-1 was assessed for potential biological effects of enzyme inhibition and cytotoxicity and was found to exhibit a broad spectrum inhibition for α-glucosidase (37 ± 0.09%) and urease (49.4 ± 0.53%). The screening results were followed by bioassay-guided isolation of secondary metabolite(s) from RWL-1. Extensive chromatographic and spectrophotometry analyses revealed the presence of compound 1 (S)-2-hydroxy-N-((S)-1-((S)-8-hydroxy-1-oxoisochroman-3-yl)-3-methylbutyl)-2-((S)-5-oxo-2,5-dihydrofuran-2-yl)acetamide. Further bioassays of compound 1 showed significant inhibition of α-glucosidase (52.98 ± 0.8%) and urease (51.27 ± 1.0%), compared with positive control values of 79.14 ± 1.9% and 88.24 ± 2.2%, and negative controls (0.08 ± 0.1% and 0.05 ± 0.01%), respectively. The current study suggests that bacterial endophytes are a rich source of novel bioactive compounds with high therapeutic value.Endophytic bacteria are known to produce a wide array of bioactive secondary metabolites with beneficial effects on human health. In the current study, a novel endophytic bacterial strain,  The term “endophyte” refers to microorganisms  that live inside plant tissues without causing apparent diseases and have the ability to colonize the internal tissues of the plant ,2. EndopBacillus amyloliquefaciens afforded the discovery of an antitumor exopolysaccharide derived from the Bacillus genus [Bacillus genus produce a variety of secondary metabolites; for example, B. thuringiensis produces the anti-infective compound anthracene [B. subtilis produces the antimicrobial protein YbdN [Bacillus genus. Owing to their involvement in phytostimulation, disease suppression, and other biological activities, Bacillus sp. are preferred because their long-term viability can facilitate the development of natural products suitable for commercial use [In addition to plant-microbe interactions, endophytes display many important biological activities ,10. Amonus genus . In addithracene  and B. sein YbdN . These rcial use .B. amyloliquefaciens strains have attracted considerable interest because they are able to produce a wide range of active antimicrobial compounds, macrolactins, lipopeptides, hydrolytic enzymes, and certain volatile compounds. For example, B. amyloliquefaciens FZB42 has 8.5% of its genome dedicated to the synthesis of secondary metabolites [abolites , allowinabolites ,21.B. amyloliquefaciens produce a variety of secondary metabolites, and previously, RWL-1 was isolated from rice seeds and was identified as B. amyloliquefaciens. RWL-1 was submitted to NCBI under the accession number (KR677384) and was reported to stimulate phytohormone and secondary metabolite production, promote plant growth, and ameliorate stress [B. amyloliquefaciens, the current study aimed to explore the medicinal potential of RWL-1. The ethyl acetate extract of RWL-1 was subjected to biological assays for anticancer, anti-AChE, anti-α-glucosidase, and anti-urease activity . The inoculated RWL-1 was incubated on shaking incubator (120 rpm) for 7 days at 28 °C and centrifuged at 5000× The cell-free culture broth of RWL-1 was adjusted to pH 2.5 and was completely extracted three times with an equal volume of ethyl acetate (EtOAc). The ethyl acetate extract was then completely dried in a rotary evaporator to obtain the crude extract (1.6 g). The ethyl acetate crude extract was subjected to various biological assays for the assessment of its medicinal potential.n-hexane to 85% EtOAc/n-hexane). TLC  experiments were performed to investigate and determine the different fractions of organic extract of bacterial culture broth. The TLC plates were observed under UV light at 254 and 365 nm .Based on the results of the bioassay, the ethyl acetate extract was analyzed by silica gel column chromatography using a solvent gradient  equipped with pumps A and B (LC-10AD). A solvent gradient of Solvent A—100% MeOH and Solvent B—Water with 5% acetic acid with Solvent program: 0–20 min , 20–40 min , 40–60 min  was used at a flow rate of 1 mL/min. The remaining impurities were removed by loading the semi-pure secondary metabolite on preparative TLC plates (as described in “Secondary metabolite isolation”) and compound 1 (4.5 mg) was purified in DCM/n-hexane (95:5).To achieve further purification, the bioactive fractions were subjected to recycling preparative high-performance liquid chromatography (HPLC) using a Shimadzu device CBM-10  coupled with a UV-VIS detector . The sample was loaded onto a C1 was subjected to spectroscopic analyses  for identification and characterization. The optical measurements were conducted by using a polarimeter . A Bruker ATR-Tensor 37 spectrophotometer  was used to record the IR spectra. To obtain the ESI mass spectra, a QSTAR mass spectrometer  with capillary voltage of 5–5.5 kV was used. The NMR spectra (1H and 13C) were obtained by using a Bruker NMR spectrometer  operated at 600 MHz and 150 MHz, respectively.The purified compound 1 on urease activity was measured in accordance with the method of [2, 0.1 mM EDTA, and 0.01 M K2HPO4·3H2O was reacted with 3 units/mL jack bean urease (0.025 mL)  and various concentrations of crude extract (10–100 µg/mL) in a 96-well plate for 15 min at 37 °C. The urease inhibitory activity was evaluated through the measurement of ammonia production by the indophenol blue method. The absorbance was measured at 630 nm and thiourea was used as the standard inhibitor. The inhibition percentage was calculated from the following equation:The inhibitory effects of the ethyl acetate crude extract and compound ethod of . Briefly1 on α-glucosidase activity were measured in accordance with the method of [p-nitrophenyl α-d-glucopyranoside (PNP-G) was used as the substrate. The absorbance of p-nitrophenol released from pNPG was measured at 405 nm every 5 min. The inhibition percentage was calculated from the standard curve.The inhibitory effects of the ethyl acetate crude extract and compound ethod of . Briefly1 on cell viability was evaluated by an MTT assay in accordance with the protocol described by [2. RPM-1640 medium supplemented with 10% fetal bovine serum and 1% (v/v) streptomycin was used for subculturing. HCT-15 cells were subcultured at density of 105 cells/well in 96-well plates with and without different concentrations  of ethyl acetate crude extract of RWL-1 for 24, 48, and 72 h. The medium was removed and 20 µL MTT solution (5 mg/mL in PBS) was added to each well of the 96-well plate and incubated for 2 h at 37 °C. After incubation, the MTT medium was replaced with DMSO (200 µL). The plate was gently shaken for 1 min and the absorbance was measured at 540 nm. The following equation was used for the calculation of cell viability: The effect of the ethyl acetate crude extract and compound ribed by . Briefly2∙6H2O, were added into a 96-well plate. Subsequently, 50 mM Tris-HCl buffer, pH 8.0, containing 0.1% bovine serum albumin (50 µL) and ethyl acetate crude extract (25 µL) were added at different concentration (50 µg/g to 600 µg/g). AChE  was added and the absorbance was measured at 412 nm at 45 s intervals. Galantamine (0.5–5 µg/mL) was used as a standard inhibitor. The inhibition percentage was calculated from the following formula:The AChE inhibition assay was conducted in accordance with the protocol described by  using a The data are presented as the mean ± S.D. of three replicate experiments. Graphical representations was computed by using GraphPad Prism software 6.01 package  and statistical analyses were conducted with Duncan’s multiple range test in SAS version 9.2 .B. amyloliquefaciens RWL-1 was screened for its biological potential.Throughout history, natural products have consistently been the best source for prominent compounds with a potent role in the fields of agriculture, medicine, and pharmacy ,30. AmonThe biological potential of the RWL-1 crude extract was examined through its inhibitory activity on various enzymes and cytotoxicity . The inhThe crude extract showed inhibition of α-glucosidase and urease as the concentration of RWL-1 crude extract increased (10–100 µg/mL). A higher dose (100 µg/mL) significantly inhibited α-glucosidase (37 ± 0.09%) and urease (49.4 ± 0.53%), with the positive control leading to 74.85 ± 0.06% and 90.86 ± 0.08% inhibition, respectively. The cytotoxicity and AChE inhibition of ethyl acetate crude extract of RWL-1 were determined to occur in a dose-dependent manner at relatively high doses (250–750 µg/mL). The inhibition of cell growth was examined after exposure to different concentrations of the ethyl acetate crude extract of RWL-1. The results revealed that the RWL-1 crude extract showed a small cytotoxic effect (25 ± 0.16%) at a higher concentration (750 µg/mL) compared with the control (100%). A similar trend was also observed for AChE inhibition. No significant decreases were observed in the AChE activity in response to different concentrations (250–750 µg/mL) of the RWL-1 crude extract, although the positive control compound significantly inhibited AChE (94.45 ± 0.31%).1 was conducted by the analysis of NMR and MS spectral data in comparison with the data reported in previous studies [J = 7.4 Hz) were also observed in the 1H-NMR spectrum. In the 13C-NMR spectrum, the unsaturated γ-lactone was indicated by the presence of a carbonyl carbon at δ 176.2, in addition to two characteristic sp2 methine signals at δ 153.7 and 132.4. The aromatic methine carbons of the substituted benzene ring appeared at δ 139.3, 129.9, and 125.3. The spectral data of compound 1 (Name: (S)-2-hydroxy-N-((S)-1-((S)-8-hydroxy-1-oxoisochroman-3-yl)-3-methylbutyl)-2-((S)-5-oxo-2,5-dihydrofuran-2-yl)acetamide) were further compared with those reported in literature [1 were found to be identical to the reported antibiotic Al 77F, which was previously isolated from bacterial strains of Bacillus pumilus [Alternaria tenuis [The structure elucidation of compound  studies ,35,36. Tterature ,35,36. Ta tenuis .1 was evaluated. The inhibition patterns of α-glucosidase and urease in response to compound 1 are shown in Based on the biological potential of the crude extract, the enzyme inhibition potential of compound 1 was examined at different doses and the inhibition percentage significantly increased as the concentration increased (10–100 µg/mL) showing 94.37 µg/mL IC50 value. The highest concentration of compound 1 (100 µg/mL) resulted in the strongest α-glucosidase inhibition (52.98 ± 0.8%). However, the standard drug used as the positive control (10–100 µg/mL) resulted in highest inhibition (79.14 ± 1.9%) at 80 µg/mL displaying an IC50 value of 62.03 µg/mL, while the negative control resulted in 0.08 ± 0.1% inhibition.The α-glucosidase inhibition efficiency of compound 1 increased (10–100 µg/mL) exerting an IC50 value of 97.52 µg/mL. The highest dose of compound 1 (100 µg/mL) marked significant inhibition (51.27 ± 1.0%) and the standard drug used as the positive control resulted in 88.24 ± 2.2% at 80 µg/mL with 55.13 µg/mL IC50 value, while the negative control resulted in 0.05 ± 0.01% inhibition.The urease inhibition was also evaluated at different doses and the inhibition percentage increased as the concentration of compound B. amyloliquefaciens is widely reported [B. amyloliquefaciens and the bioactive compounds isolated from this species. Moreover, the biological potential of isolated compound 1 from B. amyloliquefaciens RWL-1 has been reported to have anti-bacterial and antiulcer properties [In recent years, interest has intensified in the isolation and identification of bioactive α-glucosidase and urease inhibitors that can both be utilized as tools to comprehend biochemical processes and function as prospective therapeutic agents ,33. Althreported , this stoperties ,40. The operties ,33.B. amyloliquefaciens RWL-1 and conducted bioassay-guided isolation of bioactive metabolites responsible for enzymes inhibition (1 (S)-2-hydroxy-N-((S)-1-((S)-8-hydroxy-1-oxoisochroman-3-yl)-3-methylbutyl)-2-((S)-5-oxo-2,5-dihydrofuran-2-yl)acetamide supported the observed effects of B. amyloliquefaciens RWL-1 in α-glucosidase and urease inhibition. B. amyloliquefaciens is considered an ecologically and economically valuable source for the production of bioactive metabolites. Thus, the current study suggests the importance of endophytic microbes in the search for natural bioactive metabolites with high therapeutic potential.The current study evaluated the biological potential of endophytic hibition . The scr


Acinetobacter baumannii, Acinetobacter spp., Enterococcus faecalis, Enterococcus faecium, Pseudomonas aeruginosa, Staphylococcus epidermidis, Staphylococcus haemolyticus, and Stenotrophomonas maltophilia, were designed and evaluated. The data indicated that meropenem monotherapy administered at a high dose of 2 g q 8 h and as an OTAT achieved a PTA of ≥90% for isolates with an MIC of up to 128 mg/L and a CFR of ≥90% for all of the targeted pathogen populations when 50% f T > MIC  is chosen as the PD target, with Enterococcus faecalis being the sole exception. Even though 50% f T > 5 × MIC is chosen as the PD target, the aforementioned dosage regimen still reached a PTA of ≥90% for isolates with an MIC of up to 32 mg/L and a CFR of ≥90% for Acinetobacter spp., Pseudomonas aeruginosa, and Klebsiella pneumoniae populations. In conclusion, meropenem monotherapy displays potential competency for infections due to such highly resistant bacterial isolates provided that it is administered as a reasonable OTAT but not as the currently widely recommended TSPI.Infections due to meropenem-nonsusceptible bacterial strains (MNBSs) with meropenem minimum inhibitory concentrations (MICs) ≥ 16 mg/L have become an urgent problem. Currently, the optimal treatment strategy for these cases remains uncertain due to some limitations of currently available mono- and combination therapy regimens. Meropenem monotherapy using a high dose of 2 g every 8 h (q 8 h) and a 3-h traditional simple prolonged-infusion (TSPI) has proven to be helpful for the treatment of infections due to MNBSs with MICs of 4–8 mg/L but is limited for cases with higher MICs of ≥16 mg/L. This study demonstrated that optimized two-step-administration therapy  for meropenem, even in monotherapy, can resolve this problem and was thus an important approach of suppressing such highly resistant bacterial isolates. Herein, a pharmacokinetic (PK)/pharmacodynamic (PD) modeling with Monte Carlo simulation was performed to calculate the probabilities of target attainment (PTAs) and the cumulative fractions of response (CFRs) provided by dosage regimens and 39 OTAT regimens in five dosing models targeting eight highly resistant bacterial species with meropenem MICs ≥ 16 mg/L, including  Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter spp., which are defined as any isolate displaying minimum inhibitory concentration (MIC) of 2, 4, and 4 mg/L with meropenem, imipenem, and/or doripenem, respectively  q 8 h for the isolates with MICs of 16 mg/L, 1 g  q 8 h for the isolates with MICs of 32 mg/L, 0.75 g  q 6 h for the isolates with MICs of 64 mg/L, 2 g  q 8 h and 1.5 g  q 6 h for the isolates with MICs of 128 mg/L produced sufficient PK/PD exposures when 50% f T > MIC was used as the PD target. However, the power of these dosage regimens was weakened by nearly half when 50% f T > 2 × MIC was chosen as the PD target and substantially reduced when 50% f T > 5 × MIC was chosen as the PD target. Interestingly, the dosage regimen of 2 g [preferred 1.5 g (5-min IVB) + 0.5 g (5 h)] q 8 h still showed good antibacterial properties for the isolates with MICs of up to 32 mg/L even though the highest PD target of 50% f T > 5 × MIC was used. Of note, as a modification of the dosage regimen of 2 g q 8 h, the dosage regimen of 1.5 g q 6 h produced a PTA of ≥90% for the isolates with MICs of 16 mg/L when 50% f T > 5 × MIC was used as the PD target when it was administered as an OTAT  q 6 h], suggesting a potentially useful dosage regimen for these isolates. PTA vs. MIC profiles for simulations of different dosage regimens are presented in f T > MIC was chosen as the PD target, the dosage regimen of 0.5 g [preferred 0.25 g (5-min IVB) + 0.25 g (6 h)] q 8 h yielded a CFR of ≥90% for only the Acinetobacter spp., S. epidermidis, and S. maltophilia populations; however, the dosage regimens of 1 g [preferred 0.5 g (5-min IVB) + 0.5 g (6 h)] q 8 h, 2 g  q 8 h, 0.75 g [preferred 0.5 g (5-min IVB) + 0.25 g (6 h)] q 6 h, and 1.5 g  q 6 h for all of the targeted pathogen populations achieved the requisite CFR, with E. faecalis being the sole exception. When using a higher PD target of 50% f T > 2 × MIC, the majority of the simulated dosage regimens had decreased coverage of the targeted pathogens population at the condition of achieving ≥90% CFR. However, the dosage regimens of 2 g [preferred 1.5 g (5-min IVB) + 0.5 g (6 h)] q 8 h and 1.5 g [preferred 0.5 g (5-min IVB) + 1 g (6 h) or 1 g (5-min IVB) + 0.5 g (6 h)] q 6 h still covered the A. baumannii, Acinetobacter spp., E. faecium, P. aeruginosa, S. epidermidis, S. haemolyticus, and S. maltophilia populations at this condition. However, when using an aggressive PD target of 50% f T > 5 × MIC, these simulated dosage regimens had a drastically decreased coverages of the targeted pathogen populations at the condition of achieving ≥90% CFR. Surprisingly, however, the dosage regimen of 2 g [1.5 g (5-min IVB) + 0.5 g (5 h)] q 8 h still reached ≥90% CFR for the Acinetobacter spp., P. aeruginosa, S. epidermidis, S. haemolyticus, and S. maltophilia populations. CFR vs. various targeted pathogen populations for simulations of different dosage regimens are displayed in To the best of our knowledge, this study is the first to analyze meropenem as a monotherapy aimed at MNBSs with MICs ≥ 16 mg/L. In this study, we considered the tissue penetration profiles of meropenem for infections at different sites when establishing the PK/PD model associated with clinical response. We believe that this study is worthwhile because the PK/PD outcomes generated from our data could help clinicians treat these isolates more effectively through optimization of dosage regimens, especially when better treatment options are absent. Notably, our results support that meropenem as a monotherapy is still competent for isolates with MICs ≥ 16 mg/L provided that the drug is administered as a reasonable OTAT but not as the currently widely recommended TSPI.P. aeruginosa -producing K. pneumoniae  + 0.5 g (5 h)] q 8 h, as shown in Currently, clinical experience with meropenem monotherapy for MNBSs with MICs ≥ 16 mg/L is indeed limited because from a clinical point of view, determination of meropenem MICs ≥ 8 mg/L for the identified stains by susceptibility tests causes the vast majority of clinicians to switch to other better options. However, previous studies indicated that meropenem using a high dose and TSPI  in monotherapy can provide some therapeutic benefit and therefore be considered to treat infections due to MNBSs with MICs ≤ 4 or even ≤ 8 mg/L based on the therapeutic efficacy of K. pneumoniae. Joint guidelines prepared by the Working Party of the British Society for Antimicrobial Chemotherapy, the Healthcare Infection Society and the British Infection Association  and for situations in which meropenem exposures are not reduced relative to plasma exposures, such as bloodstream infections, skin and soft tissue infections, and urinary tract infections. Meropenem monotherapy, in which it must be administered at a high dose of 2 g q 8 h or 1.5 g q 6 h and as an OTAT, can achieve optimal PK/PD exposures for MNBSs with MICs of up to 128 mg/L and for pathogen populations with a pooled MIC distribution, as demonstrated by achieving a PTA of ≥90% at an MIC of 128 mg/L and a CFR of ≥90% for Acinetobacter spp., P. aeruginosa, and Enterobacteriaceae populations when using the dosage regimen of 2 g [1.5 g (5-min IVB) + 0.5 g (6 h)] q 8 h or 1.5 g [1 g (5-min IVB) + 0.5 g (6 h)] q 6 h and using 50% f T > MIC as the PD target for such types of infection.Inconsistent with these reports, our data supported that even meropenem monotherapy, rather than plazomicin monotherapy, colistin monotherapy, or MCCT, is sufficient for the treatment of such infections due to these pathogens  + 0.5 g (5–6 h)] q 8 h for isolates with MICs of 16 mg/L, the dosage regimen of 2 g [preferred 1.5 g (5-min IVB) + 0.5 g (6 h)] q 8 h, 0.75 g [preferred 0.5 g (5-min IVB) + 0.25 g (6 h)] q 6 h or 1.5 g [preferred 1 g (5-min IVB) + 0.5 g (6 h)] q 6 h for isolates with MICs of up to 64 mg/L, and is thus sufficient. Satisfactorily, for infections occurring in the peritoneum, the dosage regimen of 0.5 g [preferred 0.25 g (5-min IVB) + 0.25 g (5–6 h)] q 8 h would be sufficient for isolates with MICs of 16 mg/L, and the dosage regimen of 1 g [preferred 0.5 g (5-min IVB) + 0.5 g (5–6 h)] q 8 h, 0.75 g [preferred 0.5 g (5-min IVB) + 0.25 g (6 h)] q 6 h, 2 g [preferred 1.5 g (5-min IVB) + 0.5 g (6 h)] q 8 h or 1.5 g [preferred 1 g (5-min IVB) + 0.5 g (6 h)] q 6 h for isolates with MICs of up to 32, 64, and 128 mg/L would be adequate, respectively. Surprisingly, even for infections located in the lung, bronchus, and pleura, meropenem with the preferred regimen of 2 g [1.5 g (5-min IVB) + 0.5 g (5 h)] q 8 h still proved effective for isolates with MICs of up to 32 mg/L, suggesting that for LRTIs and pleural infections, meropenem monotherapy can still perform good bactericidal action on isolates with MICs of up to 32 mg/L.f T > 2 × MIC, these dosage regimens could produce a CFR of ≥90% for all of the tested bacterial populations with MICs ≥16 mg/L, including A. baumannii, Acinetobacter spp., E. faecium, P. aeruginosa, S. epidermidis, S. haemolyticus, and S. maltophilia, with the sole exception of E. faecalis. This result suggests that in the empiric therapy of meropenem, these dosage regimens would be competent for the vast majority of infections  due to these pathogens based on the profiles of meropenem tissue penetration. However, for LRTIs and pleural infections, meropenem monotherapy with the most promising dosage regimen of 2 g [1.5 g (5-min IVB) + 0.5 g (5 h)] q 8 h would be sufficient for infections due to only Acinetobacter spp., P. aeruginosa, S. epidermidis, S. haemolyticus, and S. maltophilia. However, the exact MIC values, especially those >16 mg/L, are often unavailable because automated systems such as VITEK-2, which indicate MIC values as high as >16 mg/L, are unable to determine the precise MIC. Regarding this problem, our data provided the CFR for the targeted bacterial population and summarized the treatment options . As a poK. pneumoniae are critically ill and have altered renal function /MIC > 5 recommended by Li et al.  the MIC for the infecting pathogen isolates is ≤ 32 mg/L and (ii) a reasonable OTAT is used to drive the PK/PD profiles to acceptable exposures. However, in the absence of control trials, the continued appraisal of meropenem for use as a monotherapy, along with the optimal dosage regimens in clinical experience, will provide further important information on its utility against MNBSs.All datasets generated for this study are included in the manuscript.XS performed the model simulation and wrote the manuscript. YW, LC, DY, and ML conceptualized and supervised the manuscript. All authors approved the final version of the manuscript.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.


The present work evaluates for the first time the use of urea-crosslinked hyaluronic acid (HA-CL), a novel derivative of native hyaluronic acid (HA), to produce microspheres (MS) by emulsification-solvent evaporation, for dermal delivery of sodium ascorbyl phosphate (SAP). As the term of comparison, HA MS were prepared. A pre-formulation study—investigation of the effects of polymers solutions properties  and working conditions—led to the production of optimized HA-CL MS and HA-CL—SAP MS with: almost unimodal size distributions; mean diameter of 13.0 ± 0.7 and 9.9 ± 0.8 µm, respectively; spherical shape and rough surface; high yield, similar to HA MS and HA–SAP MS (≈ 85%). SAP was more efficiently encapsulated into HA-CL MS (78.8 ± 2.6%) compared to HA MS (69.7 ± 4.6%). Physical state, thermal properties, relative moisture stability of HA-CL MS and HA-CL–SAP MS were comparable to those of HA MS and HA–SAP MS. However, HA-CL–SAP MS exhibited an extended drug release compared to HA–SAP MS, despite the same kinetic mechanism—contemporaneous drug diffusion and polymer swelling/dissolution. Therefore, HA-CL formulation showed a greater potential as microcarrier (for encapsulation efficiency and release kinetic), that could be improved, in future, using suitable excipients. Hyaluronic acid (HA) is a naturally occurring glycosaminoglycan, pervasively diffused in the human body: it is found in the extracellular matrix, skin dermis, eye vitreous, hyaline cartilage, synovial fluid, and umbilical cord. HA is well known for its numerous biological functions ,2 and itPropionibacterium acnes, the major bacterium responsible of acne vulgaris  of HA MS respectively decreased from 117.7 ± 20.0 μm and 246.0 ± 53.6 μm to 71.7 ± 4.2 μm and 121.0 ± 13.4 μm in presence of SAP. Similarly, Dv50 and D of HA-CL MS respectively decreased from 290.0 ± 32.5 μm and 311.0 ± 32.5 μm to 245.5 ± 27.6 μm and 300.5 ± 24.7 μm in presence of SAP. In the same way, after 30 min of emulsification, HA and HA-CL MS mean size decreased when encapsulating SAP, as reported in Concerning the influence of SAP on the properties of HA and HA-CL solutions, it increased the pH, thus causing  a stse of Cf . In factoaded MS , as highp < 0.05) in the case of 10 and 30 min of emulsification. For MS produced after 60 min of mixing, this effect was negligible. Indeed, particles were all comparable in term of size, regardless of polymer type and SAP loading, with Dv50 ranging from 2.5 ± 0.1 to 13.0 ± 0.7 µm, and D ranging from 3.1 ± 0.2 to 21.6 ± 4.0 µm (p < 0.05), and it was in agreement with previously reported results [p < 0.05)  . For exa < 0.05) . p < 0.05) for HA and HA-CL formulations: respectively 69.7 ± 4.6% and 41.3 ± 1.6% for HA–SAP MS, and 78.8 ± 2.6% and 33.6 ± 2.3% for HA-CL–SAP MS (p > 0.05). Moreover, all the optimized formulations showed almost unimodal size distributions . The EE% and DL% were satisfying, even if statistically different  influenced MS surface properties. Indeed, SAP-loaded and unloaded HA MS exhibited a regular and smooth surface, while SAP-loaded and unloaded HA-CL MS were characterized by an irregular and rough surface . Wide angle X-ray diffractometry was performed to investigate the molecular states of MS formulations in comparison to SAP . The difDSC and TGA were used to characterize the thermal behavior and stability of the drug and the formulations, providing information on their hydration properties and their physical state ,59. Figuw/w) between ambient temperature and 223 °C (moisture loss), and a second region (20.5% w/w) from 223 to 400 °C (melting with decomposition and release of volatile degradation products). TGA curve of HA MS (b) consisted of three distinct degradation stages: the first one ; the second (223–269 °C) and the third stages (269–400 °C), typical of a two-stages polysaccharide degradation. In the second stage, the 37.3% w/w of weight was lost due to a partial breakage of the molecular structure. Residues of hyaluronan were then degraded in the third stage, characterized by the 12.0% w/w of weight loss. Similar findings have already been described in the literature for native HA [w/w of weight loss in the first region (20–208 °C), 26.2% w/w in the second (208–258 °C), and 12.0% w/w in the third (258–400 °C). For HA-SAP MS the decomposition of polymer and drug seemed to occur at once. TGA thermograms of HA-CL MS (c) and HA-CL-SAP MS (e) presented the two-stages polysaccharide degradation observed also for HA MS formulations (b and d), with an additive stage for pentylene glycol evaporation and more stages for water loss. The more gradual moisture evaporation was due to the water-binding action of pentylene glycol, humectant contained in HA-CL matrix. In detail, TGA trace of HA-CL MS (c) exhibited the following weight loss regions: three stages for water and then pentylene glycol evaporation , and two stages for HA degradation . TGA thermal profile of HA-CL-SAP MS (e) was characterized by two regions for moisture and then pentylene glycol loss , and two regions for HA-CL and SAP decomposition .TGA thermal profiles of SAP and MS formulations are reported in ative HA ,64,65. AIt is well known that HA is a highly hygroscopic macromolecule, therefore by nature susceptible to moisture sorption and elevated relative humidity (RH) ,66. To fThe solubility of SAP in PBS  at 32 °C was 425.0 ± 0.9 mg/mL. This result displayed that sink conditions were guaranteed during the in vitro drug release studies, as SAP concentration, in the case of complete release, could reach values of 0.033 mg/mL in the dialysis tests, and 0.087 mg/mL in the Franz diffusion cell tests.One of the most important steps in the study of the efficacy of new delivery systems is the in vitro drug release analysis. Topical carriers are an advanced form of powders for which, so far, there are no compendial or standard release techniques and apparatuses ,32. Therf1 < 10 and f2 > 50   . For exaf2 < 50) . A slowe–Peppas model resulted in the highest R2 values when compared to Zero order, First order and Higuchi models, for both the formulations ; mean diameter of 13.0 ± 0.7 and 9.9 ± 0.8 µm, respectively ; spherical shape and rough surface; high yield—similar to that of HA MS and HA–SAP MS (≈ 85%). SAP could be more efficiently encapsulated into HA-CL MS (78.8 ± 2.6%) compared to HA–SAP MS (69.7 ± 4.6%). Physical and molecular state, thermal properties, relative moisture stability of HA-CL MS and HA-CL–SAP MS were comparable to those of HA MS and HA–SAP MS. However, a preliminary Franz diffusion cells test displayed a more extended drug release for HA-CL–SAP MS with respect to HA–SAP MS, despite the same kinetic mechanism (contemporaneous drug diffusion and polymer swelling and dissolution). This underlined that the implemented mechanical properties of the novel HA-CL could result in more efficient microsystems, which could be potentially improved, in future, by the addition of excipients able to further slow down drug release. In vitro studies on skin cells are currently performed to explore SAP release/transport across the cells and the bioactivity of HA-CL MS and HA-CL–SAP MS, in order to understand if they effectively improve hydration and re-epithelialization compared to HA MS and HA–SAP MS. The present study showed, for the first time and with a systematic approach, that HA-CL could be a promising biopolymer to prepare drug-loaded microspheres with a water-in-oil (w/o) emulsification solvent evaporation technique. Appropriate working conditions led to the production of HA-CL MS and HA-CLFallacara, A.; Vertuani, S.; Manfredini, S.; Citernesi, U.R. 2018c. Patent Appl. Filed, n. 102018000008192.


Cyperus rotundus L. collected from two different locations  were obtained by hydrodistillation and analyzed by capillary GC and GC/MS. Forty-one and 43 components were identified, representing 89.9% and 92.0% of sample A and sample B, respectively. α-Cyperone (11.0%), myrtenol (7.9%), caryophyllene oxide (5.4%) and β-pinene (5.3%) were major compounds in the oil of sample A. The main constituents of the oil of sample B were β-pinene (11.3%), α-pinene (10.8%), α- cyperone (7.9%), myrtenol (7.1%) and α-selinene (6.6%).The essential oils from the rhizomes of  Cyperus rotundus L., (family Cyperaceae), also known as purple nutsedge or nutgrass, is a common perennial weed with slender, scaly creeping rhizomes, bulbous at the base and arising singly from the tubers which are about 1-3 cm long. The tubers are externally blackish in colour and reddish white inside, with a characteristic odour. The stems grow to about 25 cm tall and the leaves are linear, dark green and grooved on the upper surface. Inflorescences are small, with 2-4 bracts, consisting of tiny flowers with a red-brown husk. The nut is three-angled, oblong-ovate, yellow in colour and black when ripe. C. rotundus is indigenous to India, but are now found in tropical, subtropical and temperate regions  were deposited at the University of Zululand Herbarium.Fresh plant materials of Finely chopped fresh rhizomes of each sample (500 g) were separately subjected to hydrodistillation in a Clevenger-type apparatus for 4 h in accordance with the British Pharmacopoeia specification . The samn-alkanes were run under the same conditions for determination of retention indices.GC analysis was carried out on a Hewlett Packard HP 6820 Gas Chromatograph equipped with a FID detector and DB-5 column (60 m x 0.25 mm id), film thickness was 0.25 µm and the split ratio was 1:25. The oven temperature was programmed from 50 °C (after 2 min) to 240 °C at 5 °C/min and the final temperature was held for 10 min. Injection and detector temperatures were 200 °C and 240 °C, respectively. Hydrogen was the carrier gas. An aliquot (0.5 µL of the diluted oil) was injected into the GC. Peaks were measured by electronic integration. A homologous series of GC-MS analyses of the oils were performed on a Hewlett Packard HP 6890 Gas Chromatography interfaced with Hewlett Packard 5973 mass spectrometer system equipped with a DB-5 capillary column . The oven temperature was programmed from 70- 240 °C at the rate of 5 °C/min. The ion source was set at 240 °C and electron ionization at 70 eV. Helium was used as the carrier gas at a flow rate of 1 mL/min. Scanning range was 35 to 425 amu. 1.0 µL of diluted oil in hexane was injected into the GC/MS.The components of the oils were identified base on the comparison of their retention indices and mass spectra with those standards, Wiley 275 library mass spectra databased of the GC/MS system and published data ,46,47.


Fasciola hepatica NEJ isolates derived from different snail hosts: Lymnaea viatrix and Pseudosuccinea columella. The data used in the analysis are related to the article ‘A proteomic comparison of excretion/secretion products in Fasciola hepatica newly excysted juveniles (NEJ) derived from Lymnaea viatrix or Pseudosuccinea columella’ Here we present the proteomic profile datasets of two  The complete list of identified proteins and their corresponding peptides have been provided in F. hepatica-derived proteins) and B. taurus-derived proteins). Also, provide a comparison between Bos taurus-derived proteins identified in Pseudosuccinea columella and Lymnea viatrix NEJ ESP samples  from two isolates of  samples .Table 1B22.1Lymnaea viatrix, and the US Pacific North West wild strain derived from cercariae developed in Pseudosuccinea (Lymnaea) columella. NEJ in vitro excystation proceeded at the same time and laboratory conditions for both strains. In brief, metacercariae (n = 500–1000) were activated in vitro and NEJ were allowed to excyst as previously described 2 in 1 mL of sterile culture medium . The ESP-containing supernatants were collected after incubation for 12 h. All ESP supernatant samples were syringe-filtered (0.22 μm), and concentrated using centrifugal filter of 3 kDa molecular weight cut-off. The protein concentrates were quantified by absorbance at 280 nm in a Nanodrop 1000 spectrophotometer , lyophilized, and stored at - 80 °C.The biological materials, metacercariae, were purchased from the laboratory DILAVE “Miguel C. Rubino” in Montevideo, Uruguay (Rubino strain) and from Baldwin Aquatics Inc. in Oregon, USA (US Pacific North West wild strain). The Rubino strain derived from cercariae developed in 2.22 buffer with trypsin . Formic acid  was added to stop the reaction, and samples were centrifuged at 17,000 g for 5 min at 4 °C for debris removal. Reversed-phase pre-columns were prepared in 250 μm ID/360 μm OD capillaries with a Kasil frit at one end. Pre-columns were packed in-house with 2 cm of a reversed-phase resin (5-μm ODS-AQ C18) particle slurry stored in methanol. Analytical reversed-phase columns were prepared by pulling a 100 μm ID/360 μm OD silica capillary into a 5-μm ID tip. Reversed-phase resin (20 cm) was packed directly onto the pulled column. Pre-columns and analytical columns were connected using a zero-dead volume union.Lyophilized ESP samples were diluted in a buffer containing 8 M urea/0.1 M Tris, pH 8.5, reduced with 5 mM Tris (2-carboxyethyl) phosphine hydrochloride, and alkylated with 25 mM iodoaceamide. The ESP samples were hydrolyzed overnight at 37 °C in 2 M urea/0.1M Tris pH 8.5, 1 mM CaCl2.3Nanoflow LC-MS/MS with an Easy NanoLC II coupled to a Q Exactive mass spectrometer  was used to analyze the peptide mixtures. Mobile phases were solution A (5% acetonitrile/0.1% formic acid) and solution B (80% acetonitrile/0.1% formic acid), at a flow rate of 400 nL/min. Sample material (1.5 μg per injection) was applied into the column. LC-MS/MS was performed in 155-min chromatographic runs as follows: 1–10% B for 10 minutes, 10–40% B for 100 minutes, 40–50% B for 10 minutes, and 50–90% B in 10 minutes. The column was flushed with 90% B for 10 minutes, then brought back to 1% B, and re-equilibrated prior to the next injection. Peptides eluted from the analytical column were electrosprayed directly into the mass spectrometer.6. The 10 most abundant ions in each MS scan were selected for MS/MS with AGC target of 2 × 105, and an underfill ratio of 0.1%. Normalized collision energy was set to 25 and maximum fill times were 20 ms and 120 ms for MS and MS/MS scans, respectively, with dynamic exclusion of 15 s. Peptide and protein identification was done with Integrated Proteomics Pipeline–IP2 . Tandem mass spectra were extracted from Thermo RAW files using RawExtract 1.9.9.2 Fasciola hepatica genome Bos taurus Uniprot reference database, in addition to reverse sequences of all entries. The search space included all fully-tryptic and half-tryptic peptide candidates with no missed cleavage restrictions. Carbamidomethylation on cysteine residues was used as a static modification. Data was searched with 50 ppm precursor ion tolerance and 20 ppm fragment ion tolerance. Identified proteins were filtered using DTASelect. Filtering required a minimum of 2 peptides per protein, at least one tryptic terminus for each peptide identification, and less than 1% FDR . Normalized spectral abundance factor (NSAF) of the different samples was calculated according to Zybailov et al. L. viatrix and from P. columella snails, using the PaternLab's TFold module The mass spectrometer was operated in a data-dependent mode, collecting a full MS scan from 400 to 1,200 m/z at 70,000 resolution and AGC target of 1 × 102.4B. taurus proteins found in both samples (F. hepatica NEJ derived from P. columella metacercaria F. hepatica metacercariae batches were obtained, processed and analyzed at the same time.The functional annotation and classification of the matched proteins by BLASTP searches against several databases were performed using a program developed and provided by Dr. José M. C. Ribeiro  samples . Proteom