In the last decades, the aquaculture industry has introduced plant-based ingredients as a source of protein in aquafeeds. This has led to mycotoxin contaminations, representing an ecological, health and economic problem. The aim of this study was to determine in the RTgill-W1 fish cell line the toxicity of fifteen mycotoxins of common occurrence in aquafeeds. To identify the most sensitive endpoint of toxicity, the triple assay was used. It consisted of three assays: alamarBlue, Neutral Red Uptake and CFDA-AM, which revealed the mitochondrial activity, the lysosomal integrity and the plasma membrane integrity, respectively. Most of the assayed mycotoxins were toxic predominantly at lysosomal level  and its acetylated metabolites 15-O-acetyl-DON and 3-acetyl-DON). Aflatoxins B1 and B2 exerted the greatest effects at mitochondrial level, while fumonisins B1 and B2 and nivalenol were not toxic up to 100 µg/mL. In general, low toxicity was observed at plasma membrane level. The vast majority of the mycotoxins assayed exerted a pronounced acute effect in the fish RTgill-W1 cell line, emphasizing the need for further studies to ascertain the impact of mycotoxin contamination of fish feeds in the aquaculture industry and to establish safe limits in aquafeeds. The worldwide human population has experienced significant growth in the last decades, leading to an increasing demand for fish as an accessible protein source for human consumption ,2,3. SimThe increased use of aquafeeds that are manufactured from vegetal raw materials has given rise to new concerns regarding aquaculture. Mycotoxin contamination has gained attention in this field due to its presence in both starting materials and finished feedstuffs ,6,7. MycAspergillus, Fusarium and Penicillium [Fusarium species produce a wide range of mycotoxins, such as enniatins (ENNs), fumonisins (FBs), zearalenone (ZEN) and trichothecenes, a family of mycotoxins that include deoxynivalenol (DON), nivalenol (NIV) and T-2 and HT-2 toxins. Ochratoxin A (OTA), another prevalent mycotoxin in feeds and foods, is produced by different Aspergillus and Penicillium species, while aflatoxins (AFs) are mainly produced by Aspergillus. AFs are considered among the most dangerous mycotoxins from the point of view of food security due to their potent genotoxic and carcinogenic effects, with the liver as the main organ affected [https://www.biomin.net/solutions/mycotoxin-survey/, (accessed on 19 August 2021)), the most prevalent mycotoxins globally in 2020 were DON (65%), followed by FBs (64%) and ZEN (48%). Other mycotoxins that were found in this mycotoxin survey were the so-called emerging mycotoxins, such as moniliformin (56%), beauvericin , enniatin B1 , enniatin A1 , enniatin A  and enniatin B . Co-occurrence of different mycotoxins in crops and feeds is also common, raising additional concerns about potential combined or even synergistic toxic effects, as evidenced in vitro and in vivo [Mycotoxins are most commonly produced by species of the genera icillium . Fusariuaffected . Mycotoxaffected ,11. As raffected ,11. Regaaffected , FB1 was in vivo . In factThe observed incidence and distribution of mycotoxins in aquafeeds indicate the need to assess their toxic effects in fish. In addition, mycotoxins present in fish feed might pass through the food chain and reach the consumer, also representing a health risk. In the aquaculture field, economic losses related to fungal and mycotoxin contamination were reported, and were due to an increase in the mortality of farmed fish as well as to a decrease in growth and reproductive output . The toxIn the present study, a cell line from rainbow trout gill (RTgill-W1) was exposed over 24 h to 15 different mycotoxins, including some of the mentioned emerging mycotoxins, in order to assess their cytotoxic effects. These mycotoxins were selected because they have been reported as common aquafeed contaminants. The mycotoxins selected consist of four types of ENN , two types of FBs (FB1 and FB2), two types of AFs (AFB1 and AFB2), as well as ZEN, OTA, BEA, NIV and DON and two of its acetylated derivatives, 15-O-acetyl-4-DON and 3-acetyl-DON. This fish cell line was chosen as an alternative system to animal models to predict acute toxicity in fish, given that various studies support its suitability for this purpose ,15. Inde50) were calculated for each assay . AFs were more toxic for the mitochondrial activity of the cells with EC50 values of 11.35 µg/mL and 30.52 µg/mL for AFB1 and AFB2, respectively. OTA was similarly toxic at the mitochondria and lysosomal levels, with EC50 values of 42.85 µg/mL and 43.95 µg/mL, respectively. DON and its metabolites presented a higher toxicity at the lysosomal level, with EC50 ranging from 43.95 to 61.61 µg/mL. Nivalenol and FBs were non-toxic for the fish cell line.The cytotoxic effects associated with different mycotoxins were determined in the RTgill-W1 cell line through the assessment of toxicity at mitochondrial level by the alamarBlue assay, at plasma membrane level by the carboxyfluorescein diacetate-acetoxymethyl ester (CFDA-AM) assay, and at lysosomal level by the neutral red uptake assay (NRU). The three assays were conducted to identify the most sensitive endpoint of toxicity in order to avoid subestimations of toxicity and to explore possible mechanisms of cellular toxicity. Cell viability was determined after 24 h of treatment with a range of mycotoxin concentrations. The effective concentrations that decreased mitochondrial activity or plasma or lysosomal membrane integrities in 50% of the cells  and 12.50 µg/mL (p < 0.05) for ENNB and ENNB1. In the CFDA-AM assay, a LOEC value of 0.78 µg/mL (p < 0.001) was obtained for ENNA and ENNA1, and of 50 µg/mL for ENNB and ENNB1 . In the NRU assay, a LOEC value of 0.78 µg/mL (p < 0.001) was observed for all the tested ENNs. For aflatoxins (p < 0.001) and of 3.13 µg/mL for AFB2 (p < 0.01). Regarding the CFDA-AM assay, significant differences (p < 0.001) were only observed for AFB1, with LOEC corresponding to the highest concentration tested (50 µg/mL). Both AFB1 and AFB2 showed significant toxicity at the lysosomal level at 25 µg/mL and 12.5 µg/mL, respectively. With respect to the toxicity of DON and its metabolites 3-acetyl-DON and 15-O-acetyl-4-DON, the NRU assay was the most sensitive, with LOEC values of 1.56 µg/mL, 6.25 µg/mL and 3.13 µg/mL (p < 0.01), respectively (p < 0.01) and 25 µg/mL (p < 0.05) were observed for DON with the alamarBlue and CFDA-AM assays, respectively. In the case of 3-acetyl-DON, LOEC values of 50 µg/mL (p < 0.01) and 25 µg/mL (p < 0.05) were recorded for the alamarBlue and CFDA-AM assays, respectively. For the metabolite 15-O-acetyl-4-DON, LOEC values of 25 µg/mL (p < 0.05) and 50 µg/mL (p < 0.01) were found with the alamarBlue and CFDA-AM test, respectively. FBs were the least toxic of all mycotoxins tested (p < 0.001) related to the alamarBlue assay, while FB2 only showed a significant effect at lysosomal level with a LOEC value of 50 µg/mL (p < 0.01) evidenced by the NRU assay. NIV only showed toxicity at lysosomal membrane integrity level (NRU) with LOEC values of 6.25 µg/mL (p < 0.05) (p < 0.01) was found for ZEN by the alamarBlue assay, while a LOEC of 6.25 µg/mL (p < 0.001) was observed in both CFDA-AM and NRU assays. OTA showed a significant effect with the alamarBlue assay with a LOEC value of 25 µg/mL (p < 0.01) and also with the CFDA-AM (p < 0.01) and NRU assays, with a LOEC value of 6.25 µg/mL . Finally, the observed toxicity for BEA was significant in all three assays, with LOECs of 0.78 µg/mL (p < 0.05) for alamarBlue, 3.13 µg/mL (p < 0.05) for CFDA-AM and 0.78 µg/mL (p < 0.01) for NRU.latoxins , the alaectively . LOEC vas tested . FB1 sho < 0.05) . In cont < 0.05) . A LOEC 50 values derived from the RTgill-W1 cell line assay were demonstrated to be in excellent agreement with lethal concentrations  determined in acute toxicity tests in fish for a wide range of chemicals. Some exceptions were described. These exceptions were reported for neurotoxic chemicals and for some chemicals of which the transformation products were more toxic, despite a proven ability of the cell line to biotransform chemicals [The growing use of plant-based material in the manufacture of aquafeeds has led to new concerns in aquaculture regarding mycotoxin contaminations. This study was designed to investigate the cytotoxic effects produced by fifteen common mycotoxins, reported as aquafeed contaminants, in the fish cell line RTgill-W1 which is recommended in the prediction of the acute toxicity of chemical compounds in fish ,15,16. Themicals ,16. The hemicals ,18 indic50 > 100 µg/mL, indicating they did not induce an acute effect in the cells. We observed that lysosomal membrane integrity, assessed by NRU assay, was the most sensitive endpoint for predicting cytotoxic effects of mycotoxins in vitro. Only AFBs were more toxic at the mitochondrial level.Our results showed that the RTgill-W1 fish cell line was sensitive to most of the mycotoxins tested at the concentrations used. A ranking of toxicities could be established: ENNA = ENNA1 = BEA > ENNB1 > ZEA > AFB1 > ENNB > AFB2 > OTA = DON = 15-O-ac-DON > 3-ac-DON. NIV and FBs showed EC50 values lower than 5 µg/mL for ENNA, ENNA1 and ENNB1, and of 17 µg/mL for ENNB. Our results accorded with the toxicity reported for ENNs in zebrafish larvae and embryos, where effects were observed at low concentrations, and stronger effects were observed for ENNA than for ENNB [The ENN mycotoxins were shown to be highly toxic at the lysosomal level for the RTgill-W1 fish cell line, with ECfor ENNB . In a refor ENNB . Olleik for ENNB  observedfor ENNB  on the t50 values between 3 and 13 µg/mL. The toxic effect of BEA was mainly exerted at the lysosomal level. The cytotoxicity of BEA was also reported in the fish cell lines PLHC-1 and RTH-149 and in the mammalian cell line H4IIE [50 values ranging from 1.7 to 13.6 µg/mL. At plasma and lysosomal membrane levels, rat cells were shown to be more sensitive than fish cells whereas at mitochondrial level the H4IIE and PLHC-1 cell lines showed no significant differences. The toxicity of BEA was also studied by Olleik et al. [50 values ranging from 5.7 to 43.7 µM after 48 h exposure as evidenced by the alamarBlue assay. An MTT assay was also used in SH-SY5Y human neuronal cells [50 value of 2.5 µM  could only be determined after 72 h of exposure. The cytotoxic effect of BEA in Vero cells using MTT and NRU assays over 24, 48 and 72 h was studied by Ruiz et al. [50 values ranging from 6.25 µM to 10.02 µM  and from 6.77 µM to 11.08 µM  for the MTT and NRU assays, respectively, were reported.BEA showed a high toxicity for the three endpoints of toxicity studied, with ECne H4IIE . In thisk et al.  in diffeal cells  to assesz et al. . EC50 va50 of 8 µg/mL). The EC50 values at the plasma membrane and mitochondrial levels were of 23 and 43 µg/mL, respectively. These results are in agreement with the results obtained by Pietsch et al. [50 values of between 2.8 and 8.9 µg/mL. Similar results were also reported in the study of Zhou et al. [Lepomis macrochirus) where viability curves were determined by the resazurin assay. In this study, the EC50 value was 170.24 µM after 48 h of exposure . These authors also studied the effect of ZEA in zebrafish larvae after a 48 h exposure. The LD50 was 13.83 µM  which indicated a higher sensitivity of this organism to ZEA. The toxic effects and accumulation of ZEA were also studied in different fish species, revealing accumulation mainly in the ovaries [The effects of ZEA after 24 h exposure showed a high cytotoxicity mainly at the lysosomal level  of AFB1 exert high toxic effects in the primary hepatocytes of Cyprinus carpio and sea bream fish [50 values of 6.34 µM (1.98 µg/mL) and 5.15 µM (1.60 µg/mL) were obtained after 48 h and 72 h exposure to AFB1 [50 values of 6.18 µg/mL after 24 h exposure [The toxic effect of AFs was produced mainly at the mitochondrial level. AFB1 was three times more toxic than AFB2, with an ECeam fish ,30. AFB1 to AFB1 . AFB1 waexposure . A recenexposure  showed aexposure ,36,37,38exposure ,39. Rainexposure ,41.50 values of around 43 µg/mL at mitochondrial and lysosomal level. No cytotoxicity was observed at plasma membrane level. It is important to be aware of the high affinity of OTA for proteins, particularly serum albumins [50 values higher than 40.4 µg/mL were reported at the three endpoints studied in the RTH-149 fish cell line. In contrast, OTA was toxic at the lysosomal level in the PLHC-1 and H4IIE cell lines, with EC50 values of 5.5 and 21.6 µg/mL, respectively. EC50 values higher than 100 µM  were found in LLC-PK1  at metabolic and lysosomal levels using the MTT and NRU assay, respectively [50 values of 30.28 µg/mL and 14.24 µg/mL in the human cell line HepG2 at the mitochondrial level [50 (96 h) of 0.28 mg/kg reported in adult sea bass (Dicentrarchus labrax L.). The loss of sensibility of the fish cell lines could be explained by the low availability of OTA to the cells due to its binding to the albumin.OTA was shown to be less toxic than the previous mycotoxins, with ECalbumins . In our albumins . EC50 vaal level ,45. Acco50 ranging from 44 to 62 µg/mL. There are several studies on the cytotoxicity of DON in different mammalian and human cell lines, but only a few studies have been conducted in fish cell lines [50 values at all endpoints assayed and in all the tested cells. In the study carried out by Mayer et al. [50 values of 1.52 µg/mL at the lysosomal level in the PLHC-1 cell lines, whereas EC50 values for RTH-149 could not be calculated at the highest concentration tested (29.60 µg/mL). Mayer et al. [50 values ranging from 5 to 11 µM  [50 values of 0.60 µg/mL and 0.11µg/mL were reported in HepG2 and RAW 264.7, respectively [DON and its metabolites only produced effects at the lysosomal level, ECll lines ,23,46,47ectively . The acu50 was 1746 μM . These authors also reported EC50 values of 919 µM  in zebrafish embryos. In other mammalian and human cell lines, FB1 did not produce toxic effects at concentrations up to 100 µg/mL [50 values below 1 µM .NIV and FBs did not present an acute effect in the fish cell line tested in our study. This result is in agreement with the results reported in a study on the fish cell line RTL-W1 for FB1 . In this00 µg/mL ,54,55. T00 µg/mL  on cytotThus, there is clearly a need for greater understanding of the consequences of the occurrence of mycotoxins in fish feeds, such as their toxicity and bioaccumulation in fish. Further research in this field is essential to find new ways to avoid and remove these toxic compounds, and to establish regulatory limits for acceptable levels of each type of mycotoxin in fish feeds.50 values lower than 10 µg/mL, followed by ENNB and AFs. OTA and DON and its metabolites 3-ac-DON and 15-O-ac-DON were less toxic (EC50 around 50 µg/mL), whereas FBs and NIV were not toxic. The results indicated that most of the mycotoxins tested exert a highly acute effect in RTgill-W1 fish, emphasizing that mycotoxin occurrence in aquafeeds is a concern for both the aquaculture industry and for the consumers. The results point to the need for further assessments of the toxicity of mycotoxins ingested by fish through feed. Moreover, further research is needed to understand the bioaccumulation profile of mycotoxins in fish, and to find ways to remove these toxic compounds from feeds.For most of the mycotoxins tested , the most sensitive assay to evaluate cytotoxicity was the NRU, which indicated cell damage at lysosomal level. However, for AFBs the alamarBlue was the most sensitive assay, suggesting that these mycotoxins exert their toxic effects preferentially at mitochondrial level. Among the studied mycotoxins, ENNA, ENNA1, ENNB1, BEA and ZEA were the most highly toxic with ECDimethyl sulfoxide (DMSO), methanol (≥99.9% purity), neutral red (NR), glacial acetic acid and all mycotoxins  were purchased from Sigma-Aldrich . Ethanol was obtained from Panreac . 7-Hydroxy-3H-phenoxazin-3-one-10-oxide  and 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) were purchased from Invitrogen . Leibovitz’s L-15 Medium (Cat. #11415-049) was obtained from Gibco . Fetal bovine sera (FBS), penicillin/streptomycin , ethylenediaminetetraacetic acid (EDTA) and 0.5% trypsin/0.02% EDTA were obtained from Lonza .The stock solutions of mycotoxins were prepared as follows: ENNA, ENNA1, AFB1 and AFB2 in DMSO at 2 mg/mL ; FB1 and FB2 in DMSO at 5 mg/mL (6934.81 µM and 7092.20 µM); ENNB and ENNB1 in DMSO at 8 mg/mL (12503.52 µM and 12235.22 µM); ZEN, NIV and OTA in DMSO at 10 mg/mL ; 15-O-acetyl-4-DON and 3-acetyl-DON in methanol at 2.85 mg/mL (8423.23 µM and 8348.71 µM); DON in methanol at 11.1 mg/mL (37459.50 µM); and BEA in methanol at 5.5 mg/mL (7015.75 µM).2 flasks at 20 °C, under a humidified atmosphere without CO2. Cells were grown in Leibovitz’s L-15 medium which contained L-glutamine and L-amino acids and which was supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were split twice each week, using PBS/EDTA to wash and trypsin/EDTA mixture to detach the cells. To expose the cells to the mycotoxins, cells were seeded into transparent, flat-bottomed 96-well plates  by adding 100 µL of cell suspension (2.5 × 104 cells/mL) per well. These were left overnight for cell attachment. Cells were then exposed for 24 h to serial dilutions of mycotoxins at the following concentration ranges: 0.012–50 µg/mL (0.019–78 µM) for ENNs, 0.012–50 µg/mL (0.01564 µM) for BEA, 0.012–100 µg/mL (0.03–248 µM) for OTA, 0.78–100 µg/mL (1.11–148 µM) for FB1 and FB2, 0.78–100 µg/mL (2.63–337 µM) for DON, 0.78–100 µg/mL (2.45–314 µM) for ZEN, 0.78–100 µg/mL (2.50–320 µM) for NIV, 0.39–50 µg/mL (1.25–160 µM) for AFB1 and AFB2 and 0.39–50 µg/mL (1.15–148 µM) for 3-acetyl-DON and 15-O-acetyl-4-DON. Cells were also exposed to serial dilutions of SDS as a positive control, and to the highest concentrations of solvent, either methanol or DMSO (0.08–10% v/v), to eliminate cytotoxic effects exerted by the vehicle. In addition, cells receiving only the medium were used as negative controls. Every mycotoxin concentration and the negative and solvent controls were tested in triplicate in each plate, and at least three independent experiments were carried out for each mycotoxin.An RTgill-W1 cell line was used to evaluate the cytotoxic effects associated with different mycotoxins. This cell line was obtained from the American Type Culture Collection  and cultured in 75 cmv/v) alamarBlue and 4 µM CFDA-AM to each well. Cells were incubated in this solution for 30 min in darkness under the culture conditions for the cell line. Afterwards, fluorescence intensity was measured at excitation/emission wavelengths of 532/590 nm and 485/535 nm for the alamarBlue and the CFDA-AM assay respectively. Cells were then washed with PBS and incubated in NR solution (0.03 mg/mL) for 1h. After a wash with PBS to remove excess of NR, the dye retained in viable cells was extracted with a solution of absolute ethanol:glacial acetic acid 1:1 (v/v). NR fluorescence was measured at excitation/emission wavelengths of 532/680 nm. All fluorescence measurements were performed in a Tecan Spark20 microplate reader . Fluorescence readouts were corrected by subtracting the fluorescence intensity measured in cell-free wells, and normalized to the vehicle control and/or the cells receiving only the medium.A triple assay consisting of the alamarBlue, CFDA-AM and NRU assays was performed on the RTgill-W1 (cell passages number 4–21) following the method described in Lammel et al. . After mp < 0.05) followed by a Dunnett’s post-hoc test. The concentration needed to cause a 50% reduction in the effect with respect to the control group (EC50) was calculated from each dose–response curve obtained in each independent experiment using the Excel macro: REGTOX  and the mean ± SEM (n = 3) was provided.All data were represented as the mean ± standard error of the mean (SEM) of three independent experiments performed in triplicate. All statistical analyses were performed using Sigma Plot . The normality and homoscedasticity of all data were automatically checked with the program. The normality of the distribution and homogeneity of variance were confirmed with the Shapiro–Wilk test and Brown–Forsythe test, respectively. Significant differences among the groups treated with mycotoxins and the lowest concentration treated group, for which no effects were observed, were determined using a one-way repeated measures analysis of variance (RMANOVA,


Dendronotus sp., Tritonia tetraquetra, and Colga pacifica collected from deep waters were analyzed and compared with those of Aeolidia papillosa and Coryphella verrucosa from the offshore zone. The high level of FATM such as 22:5n-6 and C20 monounsaturated FAs indicated that Dendronotus sp. preys on sea anemones and/or anthoathecates hydroids similarly to that of shallow-water species A. papillosa and C. verrucosa. The high percentage of tetracosapolyenoic acids and the ratio 24:6n-3/24:5n-6 indicated that T. tetraquetra preys on soft corals such as Gersemia and/or Acanella at a depth of 250 m, but soft corals of the family Primnoidae may be the main item in the diet of T. tetraquetra at a depth of 500 m. The high content of Δ 7,13-22:2 and 22:6n-3 shows that C. pacifica can feed on bryozoans. In C. pacifica, 22:5n-6 may be synthesized intrinsically by the mollusks, whereas odd-chain and branched saturated FAs originate from associated bacteria.Gastropod molluscs such as nudibranchs are important members of deep-sea benthic ecosystems. However, data on the trophic ecology and feeding specialization of these animals are limited to date. The method of fatty acid trophic markers (FATM) was applied to determine the dietary preferences of nudibranchs off the Kuril Islands. Fatty acid (FA) compositions of  Nudibranchs are a group of marine soft-bodied gastropod mollusks . The greatest diversity of nudibranchs is observed in warm shallow waters, although nudibranchs occur worldwide, from Arctic to Antarctic regions, with some species discovered at a depth near 2,500 m . IdentifTritonia tetraquetra , Dendronotus sp., and D. robustus A.E. Verril, 1870 collected in the Kurile Islands region  and inhabiting the same waters was detected by using FATM. The detection of large amounts of dietary FAs in T. tetraquetra (Fatty acids (FAs) have been used as biochemical markers to trace predator–prey relationships in marine ecosystems for more than 40 years . The mets region . FATM shryozoans . The difraquetra  showed tColga pacifica , Tritonia tetraquetra, and Dendronotus sp.) collected from deep waters (up to 500 m) were analyzed and compared with those of two nudibranch species  and Coryphella verrucosa ) from the offshore zone (about 20 m) of the Kurile Islands. Dietary preferences of these five species were studied using the method of FATM. A possible influence of depth on nudibranch feeding specialization was discussed.Waters around the Kuril Islands, with their significant depth differences, are one of the world’s most productive marine ecosystem . NudibraC. verrucosa and 2 specimens of A. papillosa were collected at a depth of 20 m by SCUBA and referred as a shallow-water group. Three specimens of C. pacifica, 3 specimens of Dendronotus sp., and 5 specimens of T. tetraquetra were collected at the depth of 250–500 m by dredging and referred as a deep-sea group. The nudibranch Dendronotus sp. was different from the one already reported ; another piece of foot tissue was frozen at −80 °C for lipid analysis. Unfortunately, two frozen samples (one for A. papillosa and one for T. tetraquetra) were lost in transit.Sampling was conducted aboard the R/V Akademik Oparin near Simushir Island  in July 2019. In total, 18 nudibranchs were sampled. Three specimens of reported . Each spreported ; one pieTo study the radular morphology of each species, the buccal mass was extracted and soaked in proteinase K solution for 2 h at 60 °C. When connective and muscle tissues were dissolved, samples were rinsed in distilled water, air-dried, mounted on an aluminium stub, and sputter-coated with gold for visualization under a JEOL JSM 6380 scanning electron microscope. The radular morphology of each species is shown in Bligh & Dyer (1959). FA methyl ethers (FAME) were prepared using the method of Carreau & Dubacq (1978) and were purified by preparative thin-layer chromatography in benzene. The 4,4-dimethyloxazoline (DMOX) derivatives of FA were prepared according to the method of Svetashev (2011). A gas chromatography analysis of FAME was conducted with a GC-2010 chromatograph  with a flame ionization detector. A Supelcowax 10  capillary column  was held for 2 min at 170 °C, then heated with a 2 °C min−1 ramp to 240 °C that was held for 5 min. A sample volume of 1 µL (about 1 mg mL−1) was injected. The injector (250 °C) and detector (260 °C) temperatures were constant. Helium was used as the carrier gas at a linear velocity of 30 cm s−1. FAME peaks were analyzed by comparing their retention time with those of the standards . The concentrations of individual FAs were calculated from the integrated area . Identification of FAs was confirmed by gas chromatography–mass spectrometry (GC–MS) of their methyl esters and DMOX derivatives on a GCMS-2010 Ultra instrument  (electron impact at 70 eV) and a MDN-5s  capillary column (30 m × 0.25 mm ID). Carrier gas was He at 30 cm s−1. The G–MS analysis of FAME was performed at 160 °C with a 2 °C min−1 ramp to 240 °C that was held for 20 min. The injector and detector temperatures were 250 °C. GC–MS of DMOX derivatives was performed at 210 °C with a 3 °C min−1 ramp to 270 °C that was held for 40 min. The injector and detector temperatures were 270 °C. Spectra were compared with the NIST library and the online FA mass spectra archive website  were investigated using PERMANOVA . The PERsampling  availablA. papillosa and C. pacifica .The full FA composition of total lipids in the five nudibranch species from different depths is summarized in Supplement T. tetraquetra. The lowest level of 22:6n-3  and considerable amounts  of very-long-chain tetracosapolyenoic acids (TPA), 24:5n-6 and 24:6n-3, were found in T. tetraquetra. The ratio 24:6n-3/24:5n-6 in Tritonia specimens from a depth of 450–516 m (7.0 ±  2.2) was higher than that in Tritonia specimens from a depth of 210–247 m (1.3 ± 0.1). The level of 20:5n-3 was significantly lower  in the deep-sea C. pacifica than that in the shallow-water C. verrucosa. Unusually high percentages of 22:5n-6 were detected in two specimens of C. pacifica . Individuals of A. papillosa contained the highest level of 22:5n-3 . Several non-methylene-interrupted FAs (NMI FAs) were present in total FAs of all mollusk species. The highest level  of Δ5,11-20:2 in T. tetraquetra specimens distinguished them from other nudibranchs. All species (except for C. verrucosa) contained noticeable amounts of Δ7,13-22:2.Acids 20:4n-6, 20:5n-3, and 22:6n-3 dominated polyunsaturated FAs (PUFAs) of the nudibranchs studied except for p < 0.001) between species. The pairwise comparison has shown significant (p < 0.05) differences for all pairs of species  was lower than that in Tritonia specimens from a depth of 210–247 m (18.5–20.0%)  .p < 0.05) levels of 20:4n-6 and Δ7,15-22:2, but significantly lower (p < 0.05) levels of 14:0, 16:1n-7, 20:1n-11, 20:1n-7, 20:5n-3, 22:4n-6, and 22:5n-3. No differences (p > 0.05) were found for other FAs listed in Analyses of the FA composition data by ANOVA identified certain FAs that were mainly responsible for the difference between species from deep and shallow waters . CompareT. tetraquetra and C. pacifica, respectively, and the third group combined specimens of the deep-sea Dendronotus sp. with the shallow-water species A. papillosa and C. verrucosa.Results of a cluster analysis of the FA composition data  for the T. tetraquetra is clearly separated from all other nudibranch species along the first PCA component, linking positively with 20:4n-6, 24:5n-6, and 24:6n-3, and negatively with 22:4n-6 and 22:6n-3  in the group of Dendronotus sp., A. papillosa, and C. verrucosa than that in T. tetraquetra and C. pacifica. The PCA results  and Leptothecata (the genus Obelia)  . C24 PUF studies . Very-lot corals . Trace aDendronotus sp. , scyphistomaes, and anemones  has shown an intensive transfer of a soft coral FATM (24:5n-6 and 24:6n-3) from prey to predator (T. tetraquetra (1.1 ± 0.2) and several soft coral species. As a result, the soft corals Gersemia rubiformis and Acanella sp. were suggested as the probable food sources of T. tetraquetra  is characteristic of soft corals within the family Primnoidae   , which aColga can feed on members of phylum Bryozoa (C. pacifica (Dendrobeania flustroides from the Sea of Okhotsk (C. pacifica confirms that this deep-sea species likely feeds on bryozoans. The low intraspecific similarity in the FA composition revealed for C. pacifica may indicate a lack of food specialization towards any particular species of bryozoan.Species of genus  Bryozoa . At leaspacifica . A notic Okhotsk . The higC. pacifica such as 22:5n-6 and odd-chain/branched SFAs may originate from own biosynthesis and associated microorganisms, respectively. The unexpectedly high content of 22:5n-6 found in C. pacifica may be a result of high activity of C2 elongase and Δ4 desaturase that convert 20:4n-6 into 22:5n-6. Such activity has been hypothesized in the hydrocoral Millepora to explain the extremely high levels of 22:5n-6 and 22:6n-3 (C. pacifica can be due to either conversion of 20:5n-3 to 22:6n-3 or a deficiency on dietary 20:5n-3 in deep waters (C. pacifica.Other characteristic FAs of  22:6n-3 . The relp waters . Odd-chap waters . Variousp waters . An abunT. tetraquetra appear to change with increasing depth. Deep-sea nudibranchs of genus Colga are most promising objects for future studies, as the proportion between dietary and self-synthesize PUFAs that they feature should be assessed. Future studies employing molecular barcodes to identify nudibranchs gut content can confirm our assumptions on the feeding regimes of the deep-sea species here reported, as FATM provide indirect evidence of trophic interactions and often impair the identification of prey at genus or species level.FA profiles of five nudibranch mollusk species belonging to families Polyceridae, Tritoniidae, Dendronotidae, Coryphellidae, and Aeolidiidae were determined. The feeding specialization of deep-sea and shallow-water species were compared on the base of FATM present in their body tissues. Different species originating from different depths, but with similar food sources, showed similar FATM profiles. Species composition of soft corals consumed by 10.7717/peerj.12336/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.12336/supp-2Supplemental Information 2Tritonia tetraquetra; CP, Colga pacifica; CV, Coryphella verrucosa; DS, Dendronotus sp.; AP, Aeolidia papillosa.Values show the contribution of individual FAs to the percent similarity or dissimilarity of the group. Total similarity or dissimilarity between groups is given in bold. TT, Click here for additional data file.10.7717/peerj.12336/supp-3Supplemental Information 3Aeolidia papillosa; CV, C. verrucosa; CP, C. pacifica; DS, Dendronotus sp.; TS1–TS3, T. tetraquetra specimens from a depth of 450–516 m; TS4 and TS5, T. tetraquetra specimens from a depth of 210–247 m.AP, Click here for additional data file.10.7717/peerj.12336/supp-4Supplemental Information 4Heteropolypus rylovi and Corallimorphus pilatus, the Bering Sea, depth 381 m. The mollusks are pointed by cycles.A, the mollusk on the Primnoa corals, the Sea of Japan, depth 676 m; B, the mollusk on the Primnoa corals, the Sea of Japan, depth 676 m; C, the mollusk near Click here for additional data file.


Two members of the AB5 toxin family were described in Shiga toxin-producing Escherichia coli (STEC), namely Shiga toxin (Stx) and subtilase cytotoxin (SubAB). The functional paradigm of AB toxins includes the B-subunit being mandatory for the uptake of the toxin into its target cells. Recent studies have shown that this paradigm cannot be maintained for SubAB, since SubA alone was demonstrated to intoxicate human epithelial cells in vitro. In the current study, we raised the hypothesis that this may also be true for the A-subunit of the most clinically relevant Stx-variant, Stx2a. After separate expression and purification, the recombinant Stx2a subunits StxA2a-His and StxB2a-His were applied either alone or in combination in a 1:5 molar ratio to Vero B4, HeLa, and HCT-116 cells. For all cell lines, a cytotoxic effect of StxA2a-His alone was detected. Competition experiments with Stx and SubAB subunits in combination revealed that the intoxication of StxA2a-His was reduced by addition of SubB1-His. This study showed that the enzymatic subunit StxA2a alone was active on different cells and might therefore play a yet unknown role in STEC disease development.AB Escherichia coli (STEC) strains produce, in addition to Stx, another AB5 toxin, the subtilase cytotoxin (SubAB)  tryptone, 1% [w/v] yeast extract, pH 7.0), 8 mL of 50× M , 8 mL of 50× 5052 -glucose, 1.0% [w/v] lactose), and 0.8 mL of 1 mol/L MgSO4), respectively. Initial growth temperatures of 37, 25, and 20 °C until induction and 4 h or overnight expression at 25 and 20 °C were applied to increase the subunit expression in the soluble fraction of the bacterial cell pellet or the culture supernatant.Expression tests for both constructs in E. coli C43 (DE3)/pKR09 in auto-induction medium ZYM-5052 without trace elements. The expression cultures were supplemented with 150 µg/mL ampicillin and inoculated 1:50 with an LB overnight culture containing 150 µg/mL ampicillin  of the expression strain. After an initial incubation at 37 °C and 180 rpm until an optical density at 600 nm (OD600) of 1.3–1.5, the temperature was decreased to 20 °C and the culture further incubated for 20 h. After expression, the cells were harvested at 5000× g, 4 °C for 15 min and washed with 30 mL phosphate-buffered saline (PBS). Cell lysis was conducted by resuspending the cell pellet in His-binding buffer  using 5 mL buffer per 1 g cells, the addition of 1 mg/mL lysozyme, and incubation for 1 h on ice. Cell disruption was carried out by ultrasonication using 12 cycles of ultrasonic steps for 10 s each with 20 s cooling on ice in between . The insoluble fraction was separated by centrifugation 25,000× g, 4 °C for 45 min . For the purification of StxA2a-His, the cleared lysate was loaded on a 5 mL HisTrapTM HP column  equilibrated in His-binding buffer. To remove all unbound components, the column was washed with 6 column volumes (CV) His-binding buffer. Elution was performed with a linear gradient from 0 to 100% of elution buffer  over 12 CV. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) as described previously [® stain solution , as well as western blots, were used to identify StxA2a-His containing fractions. Therefore, SDS-PAGE gels were blotted on a polyvinylidene fluoride (PVDF) immunoblot membrane . Stx2a subunits were detected by synthesized specific primary peptide antibodies Anti-StxA2 and anti-StxB2 , and a horseradish conjugated secondary antibody (goat anti-rabbit IgG (H+L) secondary antibody, goat/IgG; Thermo Fisher Scientific, Waltham, MA, USA). Detection was performed by chemiluminescence (ECL system) and a BioRad Chemidoc XRS+ device . Positive fractions were subsequently pooled and concentrated using an Amicon Ultra Centrifugal Filter  following the manufacturer’s recommendations. Remaining impurities were removed by a size exclusion chromatography step performed on a Superdex® 75 pg 16/600  in PBS supplemented with 10% (v/v) glycerol. After sample application the isocratic elution was collected in 2 mL fractions. Again, StxA2a-His-containing fractions were identified with SDS-PAGE followed by western blot and pooled. After concentration and detection of the protein concentration, StxA2a-His was aliquoted in 100 µL portions and stored at −70 °C until further usage.The recombinant StxA2a-His subunit was expressed using eviously  and staiE. coli C41 (DE3)/pKR10 in LB broth supplemented with 150 µg/mL ampicillin. A 400 mL expression culture was inoculated with an overnight culture as described above and incubated at 37 °C and 180 rpm. At an OD600 of 0.4 the expression was induced by the addition of 250 µmol/L isopropyl β-D-1-thiogalactopyranoside (IPTG) and the temperature subsequently reduced to 20 °C. After 20 h further incubation the cells were harvested as described above and the supernatant used for further processing. The culture supernatant was filtered with a 0.2 µm Nalgene filter  and concentrated to less than 50 mL by cross flow using a Vivaflow200 ultrafiltration module . StxB2a-His was purified in a two-column approach consisting of an affinity chromatography and a size-exclusion chromatography step operated on an ÄKTA pure system. First, the concentrated supernatant was loaded on a HisTrapTM HP column. After washing of the column with 6 CV His-binding buffer, impurities were removed by a second wash step of 10 CV with 40% elution buffer. StxB2a-His was eluted with a linear gradient from 40 to 100% elution buffer in 10 CV and 3 mL fractionation. Fractions containing StxB2a-His were identified via SDS-PAGE and western blot as described above and subjected to a Superdex® 75 pg running in PBS supplemented with 10% (v/v) glycerol as described before. Pure StxB2a-His fractions were pooled, concentrated, and stored in 100 µL aliquots at −70 °C.The recombinant expression of StxB2a-His was conducted with Purified Stx subunits were identified after tryptic digestion of protein bands excised from SDS-PAGE using Nano-LC-ESI-MS/MS performed on an Ultimate 3000 RSLCnano system  coupled to a Q-Exactive HF-X mass spectrometer  using an EASY-Nano Flex ion source  from the mass spectrometry core facility of the University of Hohenheim.The SubAB subunits were recombinantly expressed and purified as previously described .® 2000 device . Therefore, absorption was measured at 280 nm and the concentrations were calculated by using the respective theoretical extinction coefficient and molecular mass ascertained by ProtParam . Prior to the measurements, all samples were dialyzed in standard PBS buffer overnight and the concentration adjusted to 200 µg/mL for each sample. Denaturation curves were recorded at 220 nm for StxA2a-His and 218 nm for StxB2a-His with 1 °C/min of heating rate, 0.1 °C of data pitch, 1 s of response time, and 1 nm of band width. Denaturation temperatures were estimated with simple logistic fits.Far-UV spectra were measured from 260 to 195 nm at 25 °C with 100 nm/min of continuous scanning, 0.1 nm of data pitch, 4 s of response time, 1 nm of band width, and 10 accumulations at a Jasco J715 CD Spectrometer with a PTC-348 WI Peltier Unit using default settings . Raw data was recorded in millidegrees and the signal was converted to the mean residue ellipticity  was calibrated with two sets of calibration proteins, consisting of thyroglobulin, aldolase, ovalbumin, and ribonuclease A and ferritin, conalbumin, carbonic anhydrase, and aprotinin following the manufacture’s recommendation . The void volume was detected with Blue dextran. The molecular weight was calculated based on a logarithmic fit by the determination of the avK value. All measurements were performed in PBS and at least thrice of protein samples with 200 µg/mL concentration.For an estimation of the molecular size and oligomerization of the subunits, a Superdex3 binding was analyzed by a modified ELISA as described by Zumbrun et al. [3  suspended in 96% EtOH p.A. overnight and washed with 100 µL PBST  Tween 20) for 1 h at 4 °C. After blocking with 200 µL PBST + 3% (w/v) BSA for 2 h at 4 °C and washing twice with 150 µL PBST + 0.2% (w/v) BSA (PBST/0.2% BSA), 20 ng protein in 100 µL PBST/0.2% BSA was applied to each well and incubated for 1 h at room temperature. For the detection, each well was incubated with 100 µL 6×-His tag monoclonal antibody  diluted 1:3000 in PBS after washing the wells thrice with 200 µL PBST/0.2% BSA. After another three times washing with 200 µL PBST/0.2% BSA, goat anti-mouse IgG (H+L) conjugated to horseradish peroxidase  was applied in a 1:2000 dilution in 100 µL PBS as secondary antibody and incubated for 1 h at room temperature. The detection was performed at 450 nm with tetramethylbenzidine (TMB) peroxidase substrate solution  according to the manufacturer’s recommendation. The ELISA was performed with StxA2a-His, StxB2a-His, and a 1:5 molar combination of the two subunits (StxAB2a-His). His-tag labeled murine ER-specific chaperone BiP/GRP78 (mBiP) was used as a negative control. The experiment was conducted in three independent biological replicates.Gbn et al. . Briefly®, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS), 1% (v/v) non-essential amino acid solution , 1% (v/v) sodium pyruvate , and 2 mmol/L glutamine . HCT-116 cells were grown in DMEM medium containing sodium pyruvate  supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS), 1% (v/v) non-essential amino acid solution , and 2 mmol/L glutamine . Vero B4 cells were cultivated in RPMI 1640 medium  supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS). All cultures were passaged by trypsinization and incubated at 37 °C, 5.0% CO2. Cells were used in passages 5 to 30. For cytotoxicity assays, the cells were seeded in a 96-well chamber at a concentration of 1.0 × 104 cells/well and incubated at 37 °C, 5.0% CO2 for 24 h. After washing of the cells, subunit solutions were applied in serial dilutions in the respective medium. For intoxication with Stx2a-subunits, incubation times of 48 and 72 h were used when SubAB1-subunits were applied [Cytotoxic activity assays were conducted as described previously . Briefly applied . Analysi® permanox 8-well chambers  were used. Cell cultures were grown and passaged as described above. For analysis, 1.0 × 104 cells/well were seeded in the 8-well chamber at a total volume of 400 µL/well. Cells were incubated at 37 °C, 5.0% CO2 for 24 h. For intoxication, each well was washed with 400 µL DPBS and cell type-dependent fresh medium was added to the cells. Cells were incubated with 0.5 µg/mL total protein concentration and incubated further at 37 °C, 5.0% CO2. After 24 and 48 h cells were analyzed by microscopy using an inverted microscope Axio Vert.A1  equipped with a color camera . Pictures were detected using a 40×/1.25 objective  and processed by the program ZEN light . Experiments were conducted as two technical replicates in at least three biological replicates.To investigate cytotoxic effects of the Stx2a-subunits on HeLa, Vero B4, and HCT-116 cells, Nunct-test. Variances of data sets were analyzed with Students t-test on variances. Welch’s t-test was applied to not-normally distributed data sets and if equal variances were not given. Effects compared between all cell cultures were analyzed using one-way analysis of variance (ANOVA) followed by post-hoc Tukey test. Statistical significance was given at values of p smaller than 0.05.If not stated differently, all data were analyzed using OriginPro2020 from OriginLab, USA. For statistical analysis of cytotoxic effects, data of cell viability of all biological replicates were used. Initially, data were analyzed on a normal distribution. If the data set was drawn from a normally distributed population, two data sets were compared using the two-tailed Student’s


Acute β-adrenergic stimulation contributes to heart failure. Here, we investigated the role of p53 in isoproterenol (ISO)-mediated metabolic and oxidative stress effects on cardiomyocytes and explored the direct protective effects offered by the antioxidant nutraceutical curcumin. Differentiated H9C2 rat cardiomyocytes treated with ISO were assayed for glucose uptake, lactate release, and mitochondrial reactive oxygen species (ROS) generation. Survival was assessed by sulforhodamine B assays. Cardiomyocytes showed significantly decreased glucose uptake and lactate release, as well as increased cellular toxicity by ISO treatment. This was accompanied by marked dose-dependent increases of mitochondria-derived ROS. Scavenging with N-acetyl-L-cysteine (NAC) effectively lowered ROS levels, which completely recovered glycolytic metabolism and survival suppressed by ISO. Mechanistically, ISO reduced extracellular-signal-regulated kinase (ERK) activation, whereas it upregulated p53 expression in an ROS-dependent manner. Silencing of p53 with siRNA blocked the ability of ISO to stimulate mitochondrial ROS and suppress glucose uptake, and partially recovered cell survival. Finally, curcumin completely reversed the metabolic and ROS-stimulating effects of ISO. Furthermore, curcumin improved survival of cardiomyocytes exposed to ISO. Thus, ISO suppresses cardiomyocyte glycolytic metabolism and survival by stimulating mitochondrial ROS in a p53-dependent manner. Furthermore, curcumin can efficiently rescue cardiomyocytes from these adverse effects. Exposure to the β-adrenergic agonist isoproterenol (ISO) is widely used for in vitro and in vivo models to investigate the pathophysiology of cardiomyocyte damage and the beneficial effects of therapeutic agents ,2.Mitochondria play a key role in normal cardiac function, as well as in its disturbance . In cardIn respect to cardiomyocyte protection, a beneficial effect against toxic ISO responses might be provided by curcumin, a nutraceutical compound that possesses strong antioxidant activity. Previous studies in living rats demonstrated the capacity of curcumin to reduce ISO-induced myocardial injury, as evidenced by lowered serum myocardial enzymes and myocardial oxidative stress ,13,14, aIn the present study, we, therefore, used a cultured cardiomyocyte model to provide a more detailed understanding of the molecular mechanisms underlying ISO-mediated cardiomyocyte toxicity and the protective effects offered by curcumin. We tested the hypothesis that ISO directly stimulates ROS production that suppresses glucose metabolism and survival through p53 signaling, and further investigated the capacity of curcumin to reverse these effects.18F]fluoro-2-deoxy-D-glucose (FDG)) to 70.7 ± 8.5% of the untreated level (p < 0.005). This was accompanied by a similar reduction of lactate production to 84.0 ± 1.9% of controls  by 50 μM, 76.6 ± 4.5% (p < 0.005) by 100 μM, and 51.9 ± 3.8% of control level by 200 μM of ISO (p < 0.001). By 72 h of exposure, cell survival was reduced to 73.6 ± 8.5% (p < 0.005), 53.3 ± 6.0% (p < 0.001), and 34.4 ± 3.1% of control level (p < 0.001) by respective doses.Sulforhodamine B (SRB) assays demonstrated that ISO dose-dependently and time-dependently suppressed cardiomyocyte survival C. The cyThese results demonstrate that the cytotoxic effect of ISO is preceded by decreases in glucose uptake and lactate production, consistent with suppression of glycolytic metabolism.p < 0.001). Again, 1 mM of NAC completely blocked the increase of MitoSOX Red-positive cells, which returned to 1.8 ± 0.6-fold that of the control level  A. NAC isol level B. Hence,p < 0.005) and 40.3 ± 4.8% of that of controls (p < 0.0001) was recovered by NAC to 109.2 ± 7.4% and 107.0 ± 11.6% of controls, respectively  by 200 μM ISO was recovered to 102.9 ± 3.3% of controls in the presence of NAC  signaling, a regulator of cell proliferation in response to stress, was dose-dependently decreased by ISO. Hence, the level of phosphorylated ERK was reduced to 52.4 ± 11.8% of the control level after 24-h exposure to 200 μM ISO. However, there was no change in activated ERK when NAC was present A.In contrast to ERK activation, expression of p53 was dose-dependently and potently increased by ISO. Hence, there was a substantial 21.7 ± 2.7-fold increase of β-actin protein-corrected p53 level by 200 μM ISO B. This rp < 0.001), cells silenced of p53 expression no longer showed reduction in glucose uptake by 200 μM ISO  and 21.0 ± 0.4% (p < 0.001), respectively, for cells transfected with control siRNA  but did not increase the positivity of p53 siRNA-transfected cells  and 5.6 ± 0.8-fold (p < 0.01) increases in the proportion of positive-stained H9C2 cells, this ROS stimulating effect was completely reversed in the presence of 2.5 µM curcumin   .p < 0.01, p < 0.001, and p < 0.005 compared to cells exposed to 50, 100, and 200 µM ISO without curcumin) (The dose-dependent lowering of glucose uptake by ISO was significantly recovered by curcumin (urcumin) A.p < 0.01) and 88.1 ± 12.8% (p < 0.001) of controls, respectively. Moreover, treatment with 10 μM curcumin further improved survival following respective ISO exposures to 108.7 ± 17.0% (p < 0.005) and 106.6 ± 14.3% (p < 0.001) of controls, respectively.When cell survival was tested, curcumin dose-dependently rescued cardiomyocytes from the cytotoxic effects of 48 h exposure to ISO B. Hence,This study investigated the direct cellular actions of ISO and the protective effects offered by curcumin using a cultured cardiomyocyte model, free from influences of systemic factors encountered with in vivo models. The results demonstrated that ISO suppressed glycolytic metabolism and survival of H9C2 cells caused by enhanced mitochondrial ROS generation, and further revealed a crucial role of p53 signaling in this response.Lowered glucose uptake and lactate release by ISO, indicating reduced glycolytic flux, was significant by 24 h, when influence on cell survival was minimal. This demonstrates that the metabolic response of ISO preceded its cytotoxic effect that manifested after 48 h of exposure. At 24 h, the metabolic effect of ISO was accompanied by a marked enhancement of mitochondrial ROS, which can directly and indirectly influence cellular glucose metabolism . In our Mechanistically, ISO decreased activated ERK level and increased expression of the transcription factor p53 in a manner completely blocked by NAC. Therefore, high mitochondrial ROS increased p53, a master regulator of cellular response to stress. This is consistent with the notion that high ROS can increase p53 accumulation through its stabilization . Branco In addition to induction of p53 by increased mitochondrial ROS, p53, in turn, was necessary for ISO to stimulate mitochondrial ROS production. Indeed, p53 silencing completely abolished the ability of ISO to stimulate mitochondrial ROS production. In previous studies, p53 was similarly required for drug treatment to increase ROS production in renal cells  and HeLaIn our results, ISO also decreased phospho-ERK levels in a fashion that was completely blocked by NAC, indicating a consequence of increased mitochondrial ROS. Although it is known that MAP kinases can induce p53 activation in response to DNA damage, recent findings suggest a more complex picture of functional p53- MAPK interaction. Indeed, activation of phosphatases by p53 suggests a mechanism by which p53 can negatively regulate MAPK signaling. For instance, p53 can induce Pac1 in response to oxidative stress, which in turn is able to inactivate ERK-mediated MAPK signaling . TherefoFinally, we explored whether curcumin, a biocompatible polyphenol of the dietary spice turmeric, as the capacity to protect cardiomyocytes from the metabolic and cytotoxic effects of ISO. Previous cardiomyocyte experiments have used nanoparticle preparations for more efficient delivery of curcumin that exhibits low aqueous solubility . Althoug2 in Dulbecco’s modified Eagle’s medium containing 1 g/L glucose  supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin. Cells were differentiated over 7 days by incubation in media containing 1% FBS that was changed daily. Experiments were performed 24 to 48 h after seeding of differentiated cells by adding compounds to culture medium. N-Acetyl-L-cysteine (NAC) and curcumin was added 1 h prior to initiation of ISO treatment.H9C2 rat cardiomyoblast cells obtained from the Korean Cell Line Bank  were maintained in a humidified atmosphere at 37 °C and 5% COISO hydrochloride, NAC, and curcumin were from Sigma Aldrich . MitoSOX Red, Lipofectamine RNAi MAX reagent, and GIBCO Opti-MEM media were from Invitrogen Life Technologies . Antibodies against phosphorylated extracellular-signal-regulated kinase-1/2 , total ERK2 (#9108S) and p53 (#9282S), and anti-rabbit and anti-mouse secondary antibodies were from Cell Signaling Technology . Non-targeted small interfering RNA (siRNA), siRNA against p53, and anti-β-actin antibody (#sc-47778) were from Santa Cruz Biotechnology .4 cells per well, as previously described -fluorodeoxyglucose (FDG) for 30 min at 37 °C, as previously described [Cells seeded in 24-well plates were incubated with 175–370 kBq of [Gene silencing was performed with siRNA according to manufacturer’s protocol . BrieflyCells in 96-well black plates were grown to 80% confluence, and medium was replaced with fresh phenol red-free RPMI 1640 with 2% FBS. After treatment, medium was replaced with HBSS buffer containing Ca/Mg, 2% BSA and 5 µM of MitoSOX Red mitochondrial superoxide indicator. After 20 min, cells were washed with HBSS buffer containing Ca/Mg but not BSA, which was then added to the cells. Cell fluorescence was visualized and imaged using a TE2000 fluorescence microscopy with 510/590-nm excitation/emission filters .Cell culture medium was replaced with phenol red-free RPMI 1640 containing 2% FBS; MitoSOX Red was added to a concentration of 5 µM. Following 30 min incubation, cells were washed with PBS, trypsinized, washed with medium and again with HBSS buffer containing Ca/Mg and 1% BSA, and centrifuged. Cells were then resuspended in HBSS buffer, transferred to a sterile strainer tube, and underwent mitochondrial superoxide measurement on a FACSCalibur or FACSVerse flow cytometer (BD Bioscience) using CellQuest or FACSSuite software . Data were collected at FSC, SSC, FL2 and FL3 channels.Immunoblotting was performed as previously described , with 30p < 0.05 was considered statistically significant.Data are mean ± SE of values obtained from two independent experiments or mean ± SD of values obtained from a single representative experiment, as specified. Student’s t-tests were used to evaluate significance between groups; The β-adrenergic agonist ISO significantly suppressed H9C2 cardiomyocyte glycolytic metabolism and substantially increased mitochondrial ROS production, in a manner that preceded its cytotoxic effect. Mechanistically, ISO upregulated p53, which was necessary for the metabolic response, oxidative stress, and cytotoxic effect of ISO. Importantly, curcumin efficiently rescued cardiomyocytes from the metabolic and mitochondrial ROS-stimulating effects and offered protection from ISO toxicity.


The number of porphyrin–alkanethiolate adsorbates per particle was adjusted by a post-synthetic thiol/thiolate exchange reaction on 1-dodecanethiolate–protected gold nanoparticles. Even with a low loading level of adsorbates , the shoulder absorption at the Soret band was intensified, indicating the formation of aggregates of porphyrin adsorbates on the nanoparticles. Steady-state fluorescence quantum yields could be adjusted by the bulkiness of substituents at the  The design of dye molecules with specific photophysical properties is necessary for many applications including optoelectronics, photocatalysis, and bioimaging . Variouset al.–enhanced electromagnetic field  and one with bulkier substituents at three meso-positions of porphyrin  were chosen as adsorbates . The 2.5-nm species (A and B) showed weak LSPR around 510 nm , while tsorbates . Compounsorbates  (Shinohasorbates .c0 = 15–50 mM) under a constant concentration of the AuNPs, and the reaction time extended to obtain samples with different loading levels. The range of loaded porphyrin–alkanethiolate adsorbates per particle (N) were determined to be 1.2–3.1 (1b@A) and 1.5–5.5 (2@A) of 126 total thiolate sites  in toluene are shown in The UV-vis extinction spectra of 1b@A and 2@A  from the porphyrin π plane  to the Soret band (∼420 nm), /εSoret , S4. Thegregates . A signi π plane . Therefocoupling .via hydrogen bonds. Such an electrostatic interaction would provide a dominant cohesive force compared with π–π stacking in the nonpolar environment  . It may seem strange that the porphyrin adsorbates form the aggregates on the AuNPs, despite their low loading level. It is known that the Au–S covalent adsorbates on the AuNPs dynamically migrate. This enables the self-assembly of adsorbates on the AuNPs when the formation of aggregates is thermodynamically favored . The aggtoluene)  and so htoluene) .N, the FWHM only shows a slight expansion according to the increase in N .The broadening of the Soret band ) was also remarkable in 1b@A but not in 2@A. Interestingly, unlike the shoulder growth, which depends on N are shown in λem) were slightly red-shifted on loading (λem: 1b: 654 and 720 nm and 2: 655 and 721 nm) but not affected by N, which is similar to the tendency of the absorption spectra. The fluorescence of the porphyrin adsorbates on the AuNPs is almost quenched (fluorescence quantum yields ΦF = 0.07–0.14% (1b@A), 0.13–0.25% (2@A), ΦF = 11% (1b), 12% (2)). Such strong quenching of the fluorescence by the AuNPs is broadly found in various fluorophores such as pyrene, anthracene, and rhodamine 6G , which decreases exponentially as N increases (ΦF of 2@A (0.15 ± 0.02) is higher than that of 1b@A (0.10 ± 0.02%) upon removing the outlier at N = 2.6 that fell more than two standard deviations (SD) above the mean  and 9–43 , respectively , but it was not in the results. Steric repulsion between the adsorbents is expected to be one of the factors that determine the aggregation formation in the successive adsorptive loading. One particular consideration in the adsorptive loading onto the nanoparticles to form three-dimensional monolayers is the curvature of the substrate surface. At the same curvature, the shorter linkers increase the porphyrin–porphyrin steric repulsion, which is thought to inhibit the formation of aggregates (n = 5) and 1c (n = 10), which is independent of the particle size, was found (n = 2), the universal trend was no longer held. This deviation suggests that the aggregation in 1a on the larger nanoparticles C, due to the π–π stacking and/or the hydrogen bonds between the amide groups, with a small curvature is thermodynamically unfavorable . Note that no significant difference was found between n = 2 and 5 (n = 2 (SD = 0.03–0.04%). Strong aggregation at n = 5 (odd number) is likely to contribute to the reduction in the fluorescence quantum yield, however is not, as evidenced by the lack of dependence on N. This is consistent with previous reports of an increasing donor–acceptor distance reducing quenching efficiency (1a–c@B) and LSPR-active (1a–c@C) conjugates (n = 2–10).The average  2 and 5 , which ificiency . In addificiency . There aficiency , and sinficiency ; howevernjugates . This capO2 = ca. 0.21 atm) before use.All starting materials and reagents were purchased from commercial suppliers and used without further purification. Spectroscopic-grade toluene was saturated with air  at ambient temperature in deuterated chloroform containing ca. 0.03% (v/v) tetramethylsilane as an internal standard (δ = 0.00 ppm). TEM images were obtained using an FEI Tecnai Osiris field emission transmission electron microscope with an accelerating voltage of 200 kV.UV–vis spectra were recorded on a Shimadzu UV-1800 spectrometer equipped with a Peltier temperature controller. Steady-state fluorescence spectra were recorded on a JASCO FP-5300 fluorospectrometer equipped with a temperature controller. Obtained spectra were corrected by referencing to a tungsten sub-reference lamp (JASCO). tert-butylamine complex  in chloroform/ethanol (12.5 ml/5 ml) was added. After 24 h of stirring at ambient temperature, the mixture was concentrated under reduced pressure, and ethanol (40 ml) was added. The precipitate was collected by centrifugation and repeatedly washed with ethanol under sonication to remove starting materials and other byproducts to obtain A as a black waxy solid (61 mg). The mean diameter and size distribution (a standard deviation) were determined to be 2.5 and 0.5 nm, respectively, by transmission electron microscopy (TEM) (The title nanoparticles A were synthesized by one-phase method reported in the literature . To a sopy (TEM) .tert-butylbenzaldehyde  was added, and the solution was stirred for 3 h at ambient temperature. After the removal of the solvent under reduced pressure, the residue was purified by column chromatography , followed by recrystallization (methylene chloride/methanol) to obtain 3 as purple crystals . 1H NMR : δ/ppm −2.70 , 1.51–1.55 , 7.80 , 7.81 , 8.08 , 8.08 , 8.43 , 8.64 , 8.74 , and 8.90–8.97 .Pyrrole , 4-nitrobenzaldehyde , and 3,5-di-aldehyde  : δ/ppm −2.69, , 1.50–1.55 , 4.02 , 7.06 , 7.78 , 7.79 , 8.01 , 8.07  8.09 , 8.82–8.92 , and 8.94 .To a dispersion of 3  in concentrated hydrochloric acid (50 ml), stannous chloride dihydrate  was added, and the suspension was stirred for 12 h at 70°C. The mixture was neutralized by the addition of sodium carbonate and extracted with methylene chloride. The organic layer was washed with water and brine, dried over sodium sulfate, then concentrated under reduced pressure. The residue was purified by column chromatography (silica gel and dichloromethane) and then recrystallized from methylene chloride/methanol to obtain 4 as purple crystals . The majority of 3 was unreacted, apparently due to its low solubility. ε-bromocaproic acid (ca. 10 eq.) and N,N-dimethylformamide (1 drop) in toluene, excess thionyl chloride was added. After 1 h of stirring at ambient temperature, the mixture was concentrated under reduced pressure. The residue was redissolved in toluene and concentrated again to obtain ε-bromocaproyl chloride as yellow oil, which was subjected to the following reaction without purification.To a mixture of ε-bromocaproyl chloride (ca. 10 eq.) in dry methylene chloride was added dropwise. After 1 h of stirring, the mixture was concentrated under reduced pressure to obtain crude 5 as purple solid. This material was subjected to the following reaction without further purification.An ice-cold mixture of 4  and dry pyridine (0.1 ml) in dry methylene chloride (5 ml), N,N-dimethylformamide, and potassium thioacetate  was added at 0°C. After 1 h of stirring at ambient temperature, the mixture was diluted with methylene chloride (40 ml) and washed with water. The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel and methylene chloride) and subsequent reprecipitation from methylene chloride/methanol to obtain 6 as purple solid. 49 mg (83% from 4). 1H NMR : δ/ppm −2.71 , 1.50–1.56 , 1.58 , 1.72 , 1.91 , 2.54 , 2.96 , 7.56 , 7.78 , 7.79 , 7.91 , 8.07 , 8.08 , 8.19 , 8.86 , and 8.87–8.92 .Crude 5 was dissolved in bench 2 was synthesized according to the method reported in the literature : δ/ppm −2.70 , 1.41 , 1.51–1.55 , 1.60 , 1.75 , 1.88 , 2.52 , 2.62 , 7.43 , 7.78 , 7.79 , 7.90 , 8.07 , 8.08 , 8.15–8.19 , 8.85 , and 8.87–8.93 .The title compound terature . To a so400(C12H25S)126 = 1.04 × 105 Da  in toluene (1 ml), was allowed to equilibrate for 24 h at ambient temperature. The mixture was concentrated under reduced pressure at ambient temperature using a centrifugal evaporator (EYELA CVE-2000 equipped with a common oil rotary vacuum pump). The residue was purified twice by size exclusion chromatography  to remove unreacted porphyrin–alkanethiol.The solution of A  and porvide infra).Purified porphyrin–AuNP conjugates were dissolved in toluene to achieve an absorbance at 515 nm of 0.20 ± 0.01 [−], and extinction spectra were recorded. The same solution was subjected to fluorescence measurement (ε) of AuNPs are related to their size and surface dielectric constant =a×A(λ) is the actual extinction spectrum of porphyrin–AuNP conjugates. εPor(λ) and εAuNP(λ) are the separately measured molar absorption coefficients of porphyrin–alkanethiol (1a–c or 2) and 1-dodecanethiolate-protected AuNPs (A–C), respectively. The proportional coefficients a and b were determined by the non-linear least-squares method (300 ≦ λ ≦ 800 nm), and the residue R(λ) was obtained. Finally, to obtain the number of porphyrin–alkanethiolate adsorbates per particle (N), as the quotient of the coefficients a and b, the following equation was used:Here, εPor(λ) was further deconvoluted into two Gaussian functions, and the relative intensity of shoulder to the Soret band  and the area of fluorescence spectra (600–800 nm), respectively.Five milliliters of the solution of porphyrin–AuNP conjugates, used for the extinction spectra measurement (absorbance at 515 nm–0.2), was volumetrically diluted by toluene to make 50 ml. The absorbance of the solution was reduced to below 0.02 (>95% transmittance) in the measurement range, to ensure the elimination of reabsorption of fluorescence by the sample itself. equation :ΦF=ΦF,rf = 0.11 ) as an eH-aggregates soon after the onset of the thiol/thiolate exchange reaction. Fluorescence in the adsorbates was determined to be strongly quenched, due to intraparticle energy transfer from the excited adsorbates to the AuNPs. Contrary to our hypothesis, the aggregation contributes little to the fluorescence quenching. Fluorescence quantum yields are affected by linker chain length but neither by the loading level nor nanoparticle diameter, unlike our previous report on the singlet oxygen quantum yields. Thus, this work has advanced our understanding of the photophysical properties of dye adsorbates on AuNPs.In this work, a series of porphyrin–alkanethiols that have different linker chain lengths and substituents were loaded on the AuNPs by the post-synthetic thiol/thiolate exchange reaction. The conditions of the reaction were optimized to obtain low loading levels of total thiolate sites on the AuNPs. The conjugates retain the unique photoabsorption features of porphyrin (the Soret band and Q band) after loading. Slight red-shift, shoulder growth, and broadening were observed in the Soret band but not in the Q bands. Interestingly, porphyrin adsorbates form


Rubus fraxinifolius leaves and used enzyme inhibition assays to isolate compounds with protective effects against skin aging. Two triterpenoids were isolated from Rubus fraxinifolius Poir. leaves. The structures were characterized by spectroscopic analyses  and comparison to reported data. Compound 1 and 2 were determined as 2,3-O-ethyleneglycol, 19-hydroxyurs-12-en-23,28-dioic acid and 2,3-O-propanediol,19-hydroxyurs-12-en-28-oic acid. Methanol extract and isolates were assessed for their inhibitory effects on elastase and tyrosinase. Compounds 1 and 2 inhibited elastase with IC50 122.199 µg/mL and 98.22 µg/mL, and also inhibited tyrosinase with IC50 207.79 µg/mL and 221.51 µg/mL, respectively. The molecular docking proved that both compounds have affinities toward the enzymes.Numerous therapeutic compounds have been isolated from naturally abundant organic resources, which may offer economical and sustainable sources of compounds with safe and efficacious biological activities. In the cosmetics industry, natural compounds with anti-aging activities are eagerly sought. Thus, we prepared various extracts from  Rubus (Rosaceae) are distributed globally, even though few species are found in the tropics. This genus containing nutrients , secondary metabolites  and have many anti-aging activities 2. In addition, Rubus species also reported containing diverse triterpenes with various biological activities7.Although challenging to achieve, natural anti-aging substances that are safe for humans can be economically and sustainably harvested from abundant natural resources. About 700 species of R. fraxinifolius Poir. (Rosaceae) is distributed in Java, Borneo, etc., and popularly known as ‘arben.’ Moreover, some local farmers harvest these plants, and the berry fruit is usually consumed either fresh or frozen. Our previous study demonstrated that R. fraxinifolius stem extract has the activity to inhibit elastase, tyrosinase, and as an antioxidant8. Some other investigations also showed that the leaf and fruit of R. fraxinifolius have a potent antioxidant activity10. However, the study of R. fraxinifolius has been little researched, and no studies of chemical components have been reported.In Indonesia, 12. Melanogenesis is mediated by tyrosinase and regulates melanin biosynthesis through a two-stage reaction. In the initial step, L-tyrosine is hydroxylated to L-3,4-dihydroxyphenylalanine (L-DOPA). In the second step, L-DOPA is oxidized to the corresponding O-quinone. Therefore, natural compounds with tyrosinase inhibitory activity are commonly used in cosmetics that inhibit hyperpigmentation with melanin and hence favor skin whitening13. The elastase and tyrosinase enzyme inhibitors could be developed as skin whitening, anti-aging, or anti-wrinkle agents to treat dermatological disorders14.Elastase is a serine protease enzyme, which has a crucial role in skin wrinkling or sagging through the degradation of dermal elastic fiber (elastin) and causes loss of skin elasticity. One of these is skin fibroblast-derived elastase. Thus, as moderators of elastin fiber degradation that causes skin aging, elastase inhibitors have attracted attention as agents for cosmetic preparationsR. fraxinifolius leaves and elucidated their structures in spectroscopic analyses using electrospray ionization mass spectroscopy (ESI-MS) and 1H NMR, 13C NMR, and 2D NMR . Subsequently, we performed assays of elastase and tyrosinase inhibitory activities of crude extracts, fractions, and isolated compounds.This study isolated ursane triterpenoids from 16. The macromolecules used in this research were obtained from protein data bank  with identity 2y9x and 3hgp for tyrosinase and elastase, respectively18. The macromolecules were optimized in UCSF Chimera and the molecular docking results were observed with PyMOL20.The activity of two selected compounds was also observed through in silico method in this research. A molecular docking approach was performed with DockThorR. fraxinifolius leaves showed potential as elastase inhibitors with percent inhibitory > 40% in 100 µg/mL, whereas n-hexane extract had no activity , R. compactus, R. robustus, etc.21. Hence, we fractionated the active extracts using vacuum liquid chromatography/VLC and collected 11 fractions from each. Fractions from ethyl acetate extract showed weak elastase inhibitory activities (< 20% in 100 µg/mL), but some methanol fractions showed potential activity in these assays. We choose methanol fraction 8 (M8) for further isolation because it had the largest yield (57%) and also have elastase inhibitory activity (44.82%). M8 was further partitioned and purified over silica gel and through a Sephadex column, and two amorphous powders were produced.The ethyl acetate and methanolic extracts of 1H and 13C NMR, DEPT, heteronuclear single quantum coherence (HSQC), heteronuclear multiple quantum correlation (HMQC), and heteronuclear multiple bond correlation (HMBC). Table Isolates were identified and characterized using liquid chromatography-mass spectroscopy (LCMS), 1: was isolated as an amorphous white powder. LCMS–ESI spectra for this compound displayed a molecular ion peak at m/z 543.32 [M-H]+, suggesting the molecular formula C32H48O7 with nine unsaturation equivalents. IR spectra revealed absorption maxima corresponding with hydroxyl (3.343.4 cm−1) and olefinic (1.684 cm−1) functional groups. Moreover, 1H NMR spectra of compound 1 revealed methine proton (CH) a singlet signal at δH 2.6, a characteristic signal for the H-18 of ursane-type triterpenes with 19-O-substitutions. The triterpene type 19α-hydroxy-ursane also has a typical proton signal in the area around δH 2.6 ppm. This deshielded signal can differ markedly from other ordinal methylene proton signals, with a characteristic shift due to an olefinic proton (H12) at δH 5.28 . Complete and unequivocal 1H and 13C chemical shift assignments were assisted by HMQC (13C × 1H) and HMBC (13C × 1H) spectra. From the HMBC cross peak, the proton at δH 2.60 was confirmed to be H-16 , a methyl doublet at δH 0.92 , and the olefinic proton signal at δH 5.30 . The present 13C NMR spectra show 32 carbon resonances, and with DEPT and HMQC spectra, displayed two carbonyl carbons , an olefinic carbon at δC 127.23, C-12, an olefinic quaternary carbon , an oxygen-bearing quaternary carbon , ten aliphatic methylenes and six methyl carbons. The two olefinic carbons at δC 127.23 (C-12) and 139.49 (C-13) were characteristic of the ursane-triterpenoid type. Another signal indicated the presence of ethylene glycol (-OCH2-CH2O-) was showed at signals at δH 3.62 , 4.05 , 3.52  and 4.06  and supported with δC 61.27 (t) and 63.20 (t). It was located at C-2 and C-3 based on the presence of long-range correlation in the HMBC spectra. Hence, compound 1 was identified as 2,3-O-ethylene glycol, 19-hydroxyurs-12-en-23,28-dioic acid . In LCMS-ESI spectra, a molecular ion peak is present at m/z 527.33 [M-H]+, indicating the molecular formula C33H52O5, which required 8 degrees of unsaturation. The IR spectrum contained absorption maxima corresponding with hydroxyl  and olefinic  functional groups. Moreover, 1H NMR spectra for compound 2 showed a singlet at δH 2.42, which is characteristic of the H18 of a ursane-type triterpene with 19-O-substitution. Other specific characteristic spectra included the presence of six methyl singlets at δH 1.22, 0.76, 0.97, 0.69, 1.30 and 1.17 , a methyl doublet at 0.91 , and the olefinic proton signal at δH 5.37 . Corresponding 13C NMR and DEPT spectra show 33 carbon resonances, and with HSQC and HMQC spectra indicate the presence of a carbonyl carbon , an olefinic carbon , an olefinic quaternary carbon , an oxygen-bearing quaternary carbon , eleven aliphatic methylenes, and seven methyl carbons. These data indicated that compound 2 carries a ursane-triterpenoid skeleton. The other signals are indicated the presence of 1,3-propanediol functional group, at δH 3.22 , 3.42 , 1.38 (m), 1.51 (m) and 4.02  and 3.36 . Its functional group has any long-range correlation with C2-C3. Based on these results, compound 2 was thus identified as 2,3-O-Propanediol, 19-hydroxyurs-12-en-28-oic acid 22, which is a ursane triterpenoid that has been found in several Rubus species25. Tormentic acid had strong similarities with compounds 1 and 2, except that chemical shifts related to C-23 and C31-33 differed. This moiety was also confirmed in DEPT, HMQC, and HMBC experiments  reported having elastase inhibition activity28. The IC50 elastase inhibition value of compound 2 is lower than that of compound 1. Both compounds were less inhibition activity than the positive control oleanolic acid, which had an IC50 value of 90.39 µg/mL. In agreement, previous studies showed an IC50 for oleanolic acid of 76.5 μg/mL and an IC50 value of 31.0 μg/mL for ursolic acid28. Previously reported kinetic analyses of pentacyclic triterpenes showed that these compounds competitively and reversibly inhibit neutrophil elastase. In the same study, molecular docking experiments showed that the molecular scaffolding moiety 28-COOH and double bonds in pentacyclic triterpenes are essential for their inhibitory activities29.Figure 30. As shown in Fig. 1 and 2 demonstrated moderate activities as tyrosinase inhibitors.One of the problems that arise with increasing age is hyperpigmentation. Hence, there is an ongoing search for skin-lightening agents or new depigmenting. Suppression of tyrosinase can act against melanogenesisIn this research, molecular docking was also used to analyze the binding activities of selected compounds to tyrosinase and elastase as their targets. Crystal structures used were 2Y9X, a crystal structure of tyrosinase from Agaricus bisporus with inhibitor tropolone; and 3HGP, a crystal structure of porcine pancreatic elastase complexed with a potent peptidyl inhibitor FR130180. Both were selected because they were obtained from the same organism used for the in vitro assay of this research. The macromolecules are also bound to their respective inhibitors so that the active state of the enzyme conformations could be obtained. The cocrystals were used as the center of molecular docking target to narrow down the compound binding probabilities so that the scoring process becoming more efficient.16. The redocking of designated inhibitor tropolone was done with average binding affinities − 7.41 kcal/mol. The average binding affinities of compounds 1 and 2 to tyrosinase were − 7.84 kcal/mol and − 8.37 kcal/mol, respectively  compared to the inhibitor used hence the scores may overlap1H and 13C NMR spectra were recorded at 500 MHz using a JEOL JNM-ECZ500R/S1 instrument. Infrared (IR) spectra were measured with an FTIR, IRPrestige-21, Shimadzu. Other analyses were performed using a Waters UPLC-MS XEVO G2-XS QTof instrument, a VersaMax Microplate reader and a BioTek ELX800 Microplate Reader, pre-coated aluminum sheets TLC-Silica gel 60 GF254 , column chromatography (CC) was performed using Silica gel 60  with 70–230 mesh for open chromatography and 230–400 mesh for vacuum chromatography.R. fraxinifolius leaves were collected from plantation area in mount Pangrango, West Java at the height of 4,343 ft, in December 2018. The specimen was identified by a botanist (Dr. Joni Setijo) at the Research Centre for Biology, Indonesian Institute of Sciences, Indonesia, with specimen number 033/IPH.1.01/If.07. The collection of plant material had received permission from farmer and complied with institutional regulations and guidelines.1 (18 mg) and 2 (31 mg). The purity of all the isolates was evaluated by two-dimensional TLC and visualize the spot using 5% sulphuric acid in methanol, followed by heating the plates at 110 °C for 5 min.Air-dried powdered leaves (2300 g) were extracted using a Soxhlet apparatus with gradient solvent  to provide the respective extracts, then evaporated with a rotary evaporator and vacuum oven. The methanol extract (291 g) and the ethyl acetate extract (65 g) were adsorbed on the silica gel and performed vacuum liquid chromatography/VLC, eluting with a stepwise gradient of EtOAc: MeOH (from 1:0 to 0:1) to produce 11 fractions for each extract (Ea1–Ea11 and M1–M11 and). Similar fractions which had positive reactions with vanillin sulfuric reagents in TLC were combined. Fraction yield: Ea1-3 (2.27 g); Ea4-6 (8.47 g); Ea7-8 (12.9 g); Ea9-11 (22.3 g); and M1-3 (4.89 g); M4-5 (5.1 g); M6-7 (6.02 g); M8 (166.42 g); M9 (10.53 g); M10-11 (2.9 g). All fractions were submitted for inhibitor elastase activity, Fr. M8 gave the greatest yield and had potent activity. Fr. M8 successively further partitioning by CC over silica gel (eluent CH2Cl2/MeOH with a stepwise gradient) and purified using Sephadex LH-20 column  NMR, heteronuclear single quantum coherence (HSQC), heteronuclear multiple quantum correlation (HMQC), and heteronuclear multiple bond correlation (HMBC).The crystals were identified and characterized using liquid chromatography-mass spectroscopy (LCMS), 1H NMR , 13C NMR  spectroscopic data see Table + .White amorphous powder; 1H NMR , 13C NMR  spectroscopic data see Table + .White amorphous powder; 32. Briefly, in Nunc-96 well microtiter plates, 20-µL aliquots of 0.8-units/mL PPE in Trizma® base buffer (pH 8.0) were mixed with 20-µL samples, and the mixtures were then diluted to 180 µL in Trizma® base buffer. Test extracts were preincubated with enzyme for 15 min, and 20-μL aliquots of the substrate N-succinyl-Ala-Ala-Ala-p-nitroanilide  were added and incubated for another 15 min. Positive control and blank wells contained oleanolic acid and water, respectively. Experiments were conducted in triplicate, and inhibition rates were determined according to absorbance at 401 nm using a VersaMax microplate reader.Elastase inhibition assay was performed as described previously with some modifications50 values were determined from a linear graph of percent elastase inhibition against concentration .Percentage inhibition was calculated using the following equation:13. Briefly, in 96-well plates, 20 μL aliquots of DMSO (control) or test compounds at varying concentrations were mixed with 40 μL aliquots of 30 U/mL mushroom tyrosinase (Sigma Aldrich) and 100-μL of 0.1-M phosphate buffer (pH 6.8). They were preincubated for 10 min at room temperature. Reactions were initiated by adding 4 μL aliquots of 10 mM L-DOPA to each well and incubating at 37 °C for 20 min. Tyrosinase activity was then determined by measuring absorbance at 475 nm. Kojic acid was used as a positive control. Experiments were performed in triplicate. Percentage of tyrosinase inhibition was calculated using the following equation:50 values were determined from a linear graph of percent elastase inhibition against concentration .Tyrosinase inhibition was determined using the DOPA-chrome formation method as described previously with slight modificationsIn this research, in silico pharmacological activity was predicted with molecular docking using DockThor. Targeted molecular docking was done by using a cocrystallized ligand as the center of the active site. Structure 2Y9X was used for tyrosinase molecular docking with tropolone, an inhibitor of mushroom tyrosinase, as its cocrystal. The center of the molecular docking site is defined at − 10.032; − 28.769 and − 43.467 as X, Y, Z dimensions respectively. Chain A was separated for use and the Holmium atom was removed from the structure using UCSF Chimera.The structure of 3HGP, a porcine elastase was also used in this research. FR130180, as the cocrystal was used as the center of the molecular docking site. The coordinates were 12.453, 9.237, and 1.199 for X, Y, Z dimensions, respectively. The binding affinity was analyzed from the ten best conformers of each molecular docking calculation.Supplementary Information.


T, a Gram-negative, aerobic, chemoorganotrophic bacterium, motile by polar flagella, was isolated from cultured European seabass, Dicenthrarchus labrax, in Spain. It grows from 5 to 42 ºC, 6–9 pH and 1–12% total salinity. Major cellular fatty acids are C15:0 iso, summed feature 9 (C17:1 iso w9c/C16:0 10-methyl) and C17:0 iso. The genome size is 2.5 Mbp and G + C content is 49.5 mol%. Comparative analysis of the 16S rRNA gene sequence shows that the strain is a member of Pseudidiomarina, with highest similarities with Pseudidiomarina halophila (97.0%) and Pseudidiomarina salinarum (96.9%). Phylogenomic tree based on UBCG program shows P. halophila as its closest relative. ANI and in-silico DDH with other Pseudidiomarina spp. are lower than 87 and 20%, respectively, suggesting that strain CECT 9734 T represents a new species, for which we propose the name Pseudidiomarina piscicola sp. nov. and CECT 9734 T (= LUBLD50 7aT = LMG 31044 T) as type strain.Strain CECT 9734The online version contains supplementary material available at 10.1007/s00203-020-02131-3. Pseudidiomarina is a genus belonging to the family Idiomarinaceae, proposed by Jean et al.Idiomarina. As more species were incorporated, more difficult was their classification, and finally, Taborda et al. , Castellón, Spain (40°08′16.9"N 0°09′56.0"E) on May, 2000. This strain, originally labeled LUBLD50 7a, was obtained from a Marine Agar (MA) plate inoculated with a loop from the liver of a fish specimen and incubated at 26 ºC for 2 days. Once isolated, the strain was maintained as a stab culture in a semisolid Marine Agar screw-capped tube at room temperature in the dark. It was recovered in 2018, and deposited in the Spanish Type Culture Collection (CECT), where it is maintained by freeze-drying under the accession number CECT 9734 T. A partial 16S rRNA sequence suggested the strain belonged to the genus Pseudidiomarina and might be representative of a new species.Strain CECT 9734 T was performed following already described techniques  (see below), and compared with the sequence obtained after amplification, resulting on a 100% coincidence. Phylogenetic analysis using different treeing methods were done using ARB  of the University of Valencia  using an Illumina Miseq technology with 2 × 250 paired-end reads. The reads were analyzed for quality control using FASTQC, a common quality control tool developed by Babraham Bioinformatics to check raw sequencing data. After filtering, the remaining reads were assembled using SPAdes 3.9.0 software  following the standard protocol recommended by the manufacturer. Genome sequencing of strain CECT 9734isDDH) with the Genome-to-Genome Distance Calculator (GGDC 2.0) ; growth at 15% salinity was scarce and no growth was obtained at 0.5% or 18% or higher salinity. The temperature range for growth was 5–42 ºC (26–30 ºC optimum) and pH range was 6–9 (optimum: 7–8). No growth was obtained at pH 5.0 or less.Strain CECT 973415:0 iso (21.6%), Summed Feature 9 (C17:1 iso ω9c/ C16:0 10-methyl) (18.5%) and C17:0 iso (14.8%), with minor amounts of C16:0 (9.0%), Summed Feature 3 (C16:1ω7c/ω6c) (6.9%), C11:0 iso 3OH (5.1%) and Summed Feature 8 (C18:1ω7c/ω6c) (4.9%) (Supplementary Table S1).Major fatty acids were CPseudidiomarina using EzBioCloud identification tool , P. salinarum (96.9%), P. homiensis (96.7%) and P. aquimaris (96.7%). Phylogenetic tree based on 16S rRNA gene sequences, constructed using Neighbor Joining (NJ) method, show strain CECT 9734 T within the species of the genus Pseudidiomarina , the most abundant respiratory quinone in the family Idiomarinaceae. It also contains genes coding for cardiolipin synthase, the enzyme responsible for diphosphatidyl glycerol (DPG) production and phosphatidylserine decarboxylase, which allows synthesis of phosphatidyl ethanolamine (PE). Along with phospahtidyl glycerol (PG), these are the major identified polar lipids of the genus Pseudidiomarina.The annotated genome of CECT 9734 T does not contain any virulence or disease-related genes, and none of the protein secretion systems that have been related to pathogenicity is present in its genome. Siderophores are also absent. Eleven genes of a type IV pilus are present as well as multidrug resistance efflux pump component genes and several genes involved in copper, zinc, cobalt, cadmium and mercury resistance.Although the strain was originally isolated from the liver of a fish, the genome of CECT 9734 T is related to their closer relatives by ANI values of less than 87% and in-silico DDH values lower than 20%  to preclude species level relationships between them.Genome of strain CECT 97340% Table ; both va T as representative of a yet undescribed Pseudidiomarina species.Thus, both phylogenetic and genomic information point to the novelty of CECT 9734Pseudidiomarina  as the type strain of the species.In consequence, we propose to recognize the strain as representative of a new, yet unnamed species of the genus Pseudidiomarina piscicola .2. It does not produce indole. Hydrolyzes gelatin but not urea, esculin, starch, casein, Tween 80 or alginate. Does not assimilate glucose, arabinose, mannose, mannitol, N-acetyl-glucosamine, maltose, gluconate, caprate, adipate, malate, citrate or phenylacetate in API 20NE. Negative for arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase activities. Unable to grow on any of the following sole carbon and energy sources: d-xylose, l-arabinose, d-mannose, d-cellobiose, sucrose, d-melibiose, lactose, d-sorbitol, d-gluconate, d-glucuronate, 2-oxoglutarate, 3-hydroxybutyrate and putrescine. Unable to oxidize or ferment any carbohydrate in API 50CH/E strips. Positive for alkaline phosphatase and leucine arylamidase activities but negative for estearase C4, estearase lipase C8, lipase C14, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, acidic phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl β-glucosaminidase, α-mannosidase and α-fucosidase in API ZYM.Cells are Gram-reaction negative rods, motile by a polar flagellum. Colonies on Marine Agar are regular, convex, with regular border and non-pigmented. May present opaque and transparent colony variants. Cells do not accumulate polyhydroxybutyrate (PHB). Aerobic chemoorganotrophic bacterium, unable to ferment carbohydrates. Oxidase and catalase positive. Grows from 5–42 ºC, but not at 45 ºC (optimum 26–30 ºC) and from 1 to 12% salinity but not at 0.5 or lower, or 18% or higher. Values of pH supporting growth are 6.0–9.0, with optimal growth at 7–8. Reduces nitrates to nitrites but not to N15:0 iso, Summed Feature 9 (C17:1 iso ω9c/C16:0 10-methyl) and C17:0 iso. G + C molar content is 49.5 mol%.Major cellular fatty acids are C T (= LUBLD50 7aT = LMG 31044 T), it was isolated from liver of a cultured seabass (Dicentrarchus labrax). The accession numbers of 16S rRNA gene sequence and draft genome of the type strain are MN809182 and CACRZB01, respectively. The type strain genome size is 2.5 Mbp.Type strain is CECT 9734Supplementary file1 (PDF 351 KB)Below is the link to the electronic supplementary material.


Breast cancer accounts for 30% of all new cancer diagnoses in the United States. The most common type of breast cancer is invasive breast cancer. A hallmark trait of breast cancer is uncontrolled cell growth due to genetic alterations. TFIIIB-mediated RNA polymerase III transcription is specifically deregulated in human cancers. The TFIIIB BDP1 subunit is not well characterized in human cancer. The objective of this study was to analyze publicly available clinical cancer datasets to determine if BDP1 alterations correlate with clinical outcomes in available breast cancer datasets. BDP1 copy number and expression negatively correlate with breast cancer outcomes, including stage, grade, and mortality. n = 1602; p = 8.03 × 10−9) and mRNA expression  are specifically decreased in patients with invasive ductal carcinoma (IDC). In IDC, BDP1 copy number negatively correlates with high grade  and advanced stage . BDP1 mRNA expression also negatively correlated with high grade  and advanced stage  IDC. Decreased BDP1 expression correlated with poor clinical outcomes (n = 295 samples): a metastatic event at three years (p = 7.79 × 10−7) and cancer reoccurrence at three years (p = 4.81 × 10−7) in IDC. Decreased BDP1 mRNA correlates with patient death at three (p = 9.90 × 10−6) and five (p = 1.02 × 10−6) years. Both BDP1 copy number  and mRNA expression  are altered in triple-negative invasive breast cancer (TNBC). Together, these data suggest a role for BDP1 as potential biomarker in breast cancer and additional studies are warranted.TFIIIB is deregulated in a variety of cancers. However, few studies investigate the TFIIIB subunit BDP1 in cancer. BDP1 has not been studied in breast cancer patients. Herein, we analyzed clinical breast cancer datasets to determine if BDP1 alterations correlate with clinical outcomes. BDP1 copy number ( In the United States (U.S.), breast cancer accounts for 30% of new cancer diagnoses in women . Breast Many cancers, including breast cancer, are driven by genetic and epigenetic alterations leading to deregulated cell proliferation. In eukaryotes, cell proliferation is regulated, in part, by three RNA polymerases (pol) . RNA poln = 98, but clinical outcome data were not reported in the commentary [It is well-established that RNA pol III transcription is deregulated in various human cancers ,7. The immentary . Recentlmmentary . Togethen = 1602; p = 8.03 × 10−9) and mRNA expression  are specifically decreased in patients with invasive ductal carcinoma (IDC). In IDC, BDP1 copy number negatively correlated with high grade  and advanced stage . BDP1 mRNA expression also negatively correlated in high grade  and advanced stage  IDC. Interestingly, decreased BDP1 expression correlated with clinical outcomes (n = 295 samples): a metastatic event at three years (p = 7.79 × 10−7) and cancer reoccurrence at three years (p = 4.81 × 10−7) in patients with invasive breast cancer (IBC). Decreased BDP1 mRNA correlates with patient death at three (p = 9.90 × 10−6) and five (p = 1.02 × 10−6) years. BDP1 copy number decreased in triple-negative invasive breast cancer (TNBC) . Both BDP1 copy number  and mRNA expression  are altered in TNBC. Additionally, BDP1 mRNA expression is increased by the breast cancer chemotherapeutics doxorubicin , etoposide , fluorouracil , and bortezomib  in well-studied breast cancer cell lines [The objective of this study was to analyze publicly available clinical cancer datasets to determine if BDP1 alterations correlate with clinical outcomes in available breast cancer datasets. Herein, we report that BDP1 copy number  [BDP1 copy number and mRNA expression in breast cancer were analyzed using microarray datasets available in the OncominePlatform ,38. The platform ,38 contaplatform ,38. The analysis ,38. For es (FDR) ,38.p-values are indicated in the figure legends. The Oncomine™ Platform  was used for analysis and visualization. Public datasets queried are noted below, with hyperlinks to the available datasets and study descriptions  [We queried all breast cancer datasets housed in the Oncominesamples) ,38 to deTM Research Edition Platform [p = 8.03 × 10−9) in the TCGA breast cancer dataset (n = 1602) [p = 0.002) in the Esserman breast cancer dataset (n = 130) [A query of the 132 breast cancer datasets in the OncominePlatform ,38 for B = 1602) ,46,47, F = 1602) ,46,47. Fn = 130) . The genn = 130) .n = 1992) [p = 2.62 × 10−19. BDP1 copy number is also significantly decreased in advanced stage IDC, p = 0.005, n = 55) [p = 6.81 × 10−4. BDP1 mRNA expression is significantly decreased in the TCGA breast dataset (n = 593) [p-value = 4.66 × 10−4. We further analyzed BDP1 expression and breast cancer substage using the GEIPA platform [Copy number alterations of specific genes at specific chromosomes have been demonstrated to correlate with clinical outcomes in breast cancer, including a chromosomal loss in 5q13.2–q15 . BDP1 ha = 1992)  to deter n = 55) , and corn = 593) ,46,47, pplatform , Supplemn = 295) [n = 295)  identifip = 7.79 × 10−7) in patients having a metastatic event at three years. BDP1 mRNA expression is significantly decreased  in patients with recurring IBC at three years, as shown in p-value = 9.90 × 10−6) in patients who died at three years. BDP1 mRNA expression is significantly decreased  in patients with IBC who died at five years, as shown in p = 1.07 × 10−5) in patients with recurring IDC at three years. Additionally, BDP1 copy number is decreased  in patients with recurring IDC at five years, as shown in p-value, and gene rank are noted in The estrogen receptor (ER) and progesterone receptor (PR) steroid receptors were the first biomarkers classified in breast cancer . ER-negap = 1.04 × 10−21, n = 3785 samples across three datasets [p = 5.23 × 10−6, n = 2434 samples across three datasets [https://epd.epfl.ch, accessed January–February 2022) [p = 0.001, in the BDP1 promoter that may regulate BDP1 expression.BDP1 mRNA expression correlates with ER and PR mRNA expression in the context of clinical outcomes measured in datasets ,44,47, adatasets , and BDPdatasets ,42,43, ary 2022)  and idenp = 1.04× 10−21) and significant gene rank (74) in TNBC, across three studies and n = 3785 samples [In this study, we demonstrate BDP1 copy number and mRNA expression decrease in IDC . BDP1 al samples ,44,47  in the basal-like hTERT-immortalized human mammary epithelial (HME) cell line ME16C cells treated with doxorubicin (DOX), as shown in https://epd.epfl.ch, accessed January–February 2022) [p = 0.001, in the BDP1 promoter, suggesting a potential mechanism for regulation of BDP1 expression in DOX treated ME16C cells. p = 8.15 × 10−4) in etoposide (ET) treated ME16C cells, as shown in p = 0.005. In p = 0.002, as shown in We first queried the Troester  cell linry 2022)  identifiTo the best of our knowledge, this is the first study to correlate BDP1 alterations with clinical outcomes in breast cancer. The functional genomics approach to investigating BDP1 alterations in breast cancer is significant. The BDP1 gene encodes a 2250 amino acid protein with a predicted molecular weight of 250 kilodaltons and characterized in vitro characterization ,9. Earlyn = 1602; p = 8.03 × 10−9) and mRNA expression  are specifically decreased in patients with invasive ductal carcinoma (IDC), as shown in n = 1992; p = 2.62 × 10−19) and advanced stage , as shown in n = 55; p = 6.81 × 10−4) and advanced stage  IDC. Decreased BDP1 expression correlated with clinical outcomes, as shown in n = 295 samples): a metastatic event at three years (p = 7.79 × 10−7) and cancer reoccurrence at three years (p = 4.81 × 10−7) in patients with invasive breast cancer. Decreased BDP1 mRNA correlates with patient death at three (p = 9.90 × 10−6) and five (p = 1.02 × 10−6) years.Herein, we report that BDP1 copy number , etoposide , fluorouracil , and bortezomib  in well-studied breast cancer cell lines [BDP1 mRNA expression is increased by the breast cancer chemotherapeutics doxorubicin  and mRNA expression  are decreased in patients with invasive ductal carcinoma (IDC). In IDC, BDP1 copy number negatively correlated with high grade  and advanced stage . BDP1 mRNA expression negatively correlated in high grade  and advanced stage  IDC. BDP1 mRNA correlates with patient death at three (p = 9.90 × 10−6) and five (p = 1.02 × 10−6) years. Also, BDP1 copy number decreased in TNBC . Both BDP1 copy number  and mRNA expression  are altered in TNBC. Finally, BDP1 mRNA expression is increased by the breast cancer chemotherapeutics doxorubicin , etoposide , fluorouracil , and bortezomib  in well-studied breast cancer cell lines. Together, the data presented suggest a role for BDP1 alterations in invasive breast cancer. Breast cancer accounts for 30% of all new cancer diagnoses in the United States. The most common type of breast cancer is invasive breast cancer. A hallmark trait of breast cancer is uncontrolled cell growth due to genetic alterations. TFIIIB-mediated RNA polymerase III transcription is specifically deregulated in human cancers. The TFIIIB BDP1 subunit is not well characterized in human cancer. The objective of this study was to analyze publicly available clinical cancer datasets to determine if BDP1 alterations correlate with clinical outcomes in available breast cancer datasets. BDP1 copy number and expression negatively correlate with breast cancer outcomes, including stage, grade, and mortality. Specifically, we report that BDP1 copy number (