All the substances showed antioxidant activity and riparins C, D, E, F presented cell growth inhibition rates greater than 70%, suggesting that these molecules have antitumor properties. These substances also caused greater than 80% releases of cytoplasmic lactate dehydrogenase enzyme (LDH). Although the antioxidant and antitumor properties presented herein require further assessment, the outcomes indicate that these novel riparins are promising biologically active compounds.This study aimed to study the  Lauraceae family contains an expressive number of species with great diversity of medicinal and industrial uses and a high commercial value, which has led to its increasing exploitation, making this family vulnerable to extinction benzamide . Melting point: 90 °C . 1H and 1H and 13C-NMR date: see Methyl salicylate (3.3 mmol) and phenylethylamine (6.5 mmol) were shaken for 8 h at room temperature to give 2-hydroxy-N-phenethylbenzamide . Melting point: 95 °C . 1H and Methyl salicylate (3.3 mmol) and 3,4-dimethoxyphenethylamine (6.6 mmol) were mixed at room temperature under magnetic stirring for 6 hours to give 2-hydroxy-N-[2-(4-methoxyphenyl)ethyl]-benzamide . Melting point: 96 °C . 1H and Phenylethylamine (5.9 mmol) was added to a solution of 2,6-dihydroxybenzoic acid ester (2.9 mmol) in dichloromethane (30 mL) containing triethylamine (7.0 mmol) and shaken for 5 h. The mixture then was neutralized with 2% HCl. Next, three portions of 20 mL each were extracted with ethyl acetate (100 mL). The organic phases were combined, dried with anhydrous sodium sulfate, filtered and concentrated under vacuum to afford 6-didyhroxy-N-phenethylbenzamide . Melting point: 155 °C . 1H and N-[2-ethyl]-2,6-dihydroxybenzamide . Melting point: 167 °C. 1H and 13C-NMR date: see 3,4-Dimethoxyphenethylamine (5.9 mmol) was added to a solution of methyl 2,6-dihydroxy-benzoate (2.9 mmol) in dichloromethane (30 mL) containing triethylamine  and shaken for 5 h. The mixture was neutralized with 2% HCl and extracted with three portions of ethyl acetate. The organic phases were combined, dried with anhydrous sodium sulfate, filtered and concentrated on a rotary evaporator, to give in vitro antioxidant tests the samples were emulsified in 0.05% Tween 80 dissolved in saline 0.9% (used as the vehicle) and tested at concentrations of 1, 5, 10, 25 and 100 µg/mL. The samples for the cytotoxicity tests were diluted in pure and sterile DMSO and tested at concentrations of 25 µg/mL.For the 2+  to solutions containing 5 mM 2-deoxyribose, 100 mM H2O2 and 20 mM phosphate buffer (pH 7.2). In order to determine the antioxidant activity of the riparins against the hydroxyl radical, individual tests were performed on each one of the six substances, in which different concentrations of the riparins were added to the system before Fe2+ addition . Reactions were carried out for 15 min at room temperature and were quenched by the addition of phosphoric acid 4% (v/v), followed by TBA 1% . The solutions were boiled for 15 min to 95 °C and then cooled at room temperature. The absorbance was measured at 532 nm and the results were expressed as MDA equivalents formed by Fe2+ and H2O2 [The formation of OH radicals from Fenton reagents was quantified using the condensation product of the 2-deoxyribose oxidative degradation product, malonaldehyde, with 2-thiobarbituric acid (TBA) . Brieflyand H2O2 .w/v) in 20 mM phosphate buffer (pH 7.4), 1 mL of homogenate was sonicated and then homogenized with 0.1 mL of the samples at concentrations of 1, 5, 10, 25 and 100 µg/mL. Lipid peroxidation was induced by the addition of 0.1 mL of 2,2'-azobis(2-methylpropionamidine) dihydrochloride solution . Control was evaluated with only the vehicle. Reactions were carried out for 15 minutes at 37 °C. After cooling, samples (0.5 mL) were centrifuged with 0.5 mL of trichloroacetic acid (15%) at 1,200 ×g for 10 min. An aliquot of 0.5 mL from supernatant was mixed with 0.5 mL TBA (0.67%) and heated at 95 °C for 30 min. After cooling, sample absorbance was measured using a spectrophotometer at 532 nm. The results were expressed as percentage of TBARS formed by AAPH alone (induced control).The TBARS assay was employed to quantify lipid peroxidation and an adapted TBARS method was used to measure the antioxidant capacity of the riparins  using egg yolk homogenate as lipid rich substrate . Egg yolNitric oxide was generated from spontaneous decomposition of sodium nitroprusside in 20 mM phosphate buffer (pH 7.4). Once generated, NO interacts with oxygen to produce nitrite ions, which are measured by the Griess reaction . The rea2 in RPMI 1640  or DMEM medium  supplemented with 2 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin. The antiproliferative activities of riparins were evaluated in the following human cancer cells lineage: HL-60 (pro-myelocytic leukemia), NCIH-292 (lung carcinoma), and HEP-2  obtained from the Rio de Janeiro Cell Bank (RJ-Brazil). For all experiments, 100 µL of tumor cells were plated in 96-well plates (6 × 104 cells/cm2 for adherent cells or 9 × 104 cells/cm2 for leukemia). Tested compounds (0.1–25 μg/mL) dissolved in 1% DMSO (100 µL) were added to each well and incubated for 72 h. Control groups received the same amount of DMSO. After 69 h of treatment, 25 μL of MTT (5 mg/mL) were added. Three hours later, the MTT formazan product was dissolved in 100 μL of DMSO, and absorbance was measured at 595 nm using a multiplate reader  [Cells were maintained at 37 °C with 5% CO Brasil) .2 at 5% in DMEM-F12 supplemented with 10% fetal bovine serum  and gentamicin (50 μg/mL). RAW 264.7 cells (5 × 105 cells/mL) were cultured and incubated for 24 h with the riparins A, B, C, D, E and F at the final concentration of 25 µg/mL for 48 h [Murine peritoneal macrophages of the cell line RAW 264.7 were obtained from the American Type Culture Collection , and cultured at 37 °C with COfor 48 h . The cytfor 48 h .post hoc test. The data concerning release percentage of cytoplasmatic enzyme lactate dehydrogenase (LDH) were reported by mean ± standard deviation and evaluated by one-way analysis of variance (ANOVA) followed by a Tukey post hoc test. In all cases differences were considered significant if p < 0.05, the means and respective standard errors were analyzed in the Graph Pad Prism version 5.0 for Windows software . The IC50 values and their 95% confidence intervals for two different experiments were obtained by nonlinear regression using GraphPad Prism version 5.0.The obtained data were evaluated by one-way analysis of variance (ANOVA) followed by a Student Newman Keuls in vitro tests suggest that because of the removal capacity of hydroxyl free radical and nitric oxide, as well as by avoiding TBARS formation, the tested substances are promising biologically active compounds with antioxidant properties. Additional chemical, pharmacological and biotechnological studies must be performed in order to elucidate the viability of using these substances in the production of possible medicines, destined to combat free radicals, particularly in cancer. In cytotoxicity tests the samples of riparins C, D, E and F were active, which suggests a need for further investigation, as a significant cell growth inhibition potential for these substances is indicated. The mechanism of that process still needs to be elucidated in future tests.The results of


We investigated the mechanism underlying 5FU-resistance, focusing on the changes in the 5FU metabolisms.MKN45 and 5FU-resistant MKN45/F2R cells were treated with 5FU or fluoro-deoxyuridine (FdU) in combination with hydroxyurea (HU) or tipiracil (TPI). The amount of FdUMP was determined by the density of the upper band of thymidylate synthase on Western blotting.The MKN45/F2R cells exhibited 5FU resistance (37.1-fold) and showed decreased OPRT and increased TP levels. In both cells, the FdUMP after treatment with 5FU was decreased when RR was inhibited by HU but not when TP was inhibited by TPI. A metabolome analysis revealed the loss of intracellular deoxyribose 1-phosphate (dR1P) in both cells, indicating that FdUMP was synthesized from 5FU only through the OPRT–RR pathway because of the loss of dR1P. After the knockdown of TK, the FdUMP after treatment with FdU was decreased in MKN45 cells. However, it was not changed in MKN45/F2R cells. Furthermore, TP inhibition caused an increase in FdUMP after treatment with 5FU or FdU and reversed the 5FU resistance in MKN45/F2R cells, indicating that FdUMP was reduced through the TP–TK pathway in MKN45/F2R cells.In MKN45/F2R cells, the reduction of FdUMP through the TP–TK pathway caused 5FU resistance, and the inhibition of TP reversed the resistance to 5FU, suggesting that the combination of 5FU and TPI is a promising cancer therapy. Gastric cancer remains a major cause of cancer death worldwide , as many5FU is currently a key drug for both adjuvant therapy after curative operation –4 and fo5FU is converted to fluoro-deoxyuridine monophosphate (FdUMP) in cancer cells and inhibits TS by forming a ternary complex composed of TS, 5,10-methylenetetrahydrofolate (CH2THF), and FdUMP . TherefoAs mentioned above, decreased levels of OPRT, decreased TP and increased levels of TS can be considered as major factors involved in the development of 5FU resistance . HoweverThese findings suggest that there may be unknown mechanisms of action involved in the development of 5FU resistance. Therefore, we investigated the mechanism of 5FU resistance using 5FU-resistant gastric cancer cell lines focusing on the changes of metabolisms for 5FU.5FU was kindly provided by Kyowa Hakko . FdU, hydrochloride (TPI), and hydroxyurea (HU) were purchased from Sigma-Aldrich .2 at 37 °C.MKN45 cells  were cultured in RPMI-1640 with 5% fetal bovine serum  and sodium pyruvate . MKN45/F2R cells are a 5FU-resistant cell line that was established by continuously exposing MKN45 cells to increasing concentrations (0.1–2 µM) of 5FU over a year, as previously described . These cThe cells were lysed in RIPA buffer  for 15 min on ice. The protein concentration of the lysates was measured using a Bio-Rad Protein Assay Dye Reagent Concentrate . The cell lysates were boiled in sample buffer solution . Total cell protein extracts (10 µg/lane) were separated by 10% SDS-PAGE using SuperSep™ ACE  and electrophoretically transferred onto polyvinyl difluoride (PVDF) membranes . The membranes were blocked with PVDF blocking reagent  for 1 h. The membranes were then incubated with primary antibodies, such as β-actin (13E5) Rabbit mAb #4970 , RRM1 (D12F12) XP Rabbit mAb #8637 , Anti-Thymidine Kinase 1 [EPR3193] antibody (ab76495) , Rabbit polyclonal to Thymidine Phosphorylase (ab69120) , dNT-1 (C-10): sc-390041 , Anti-Thymidylate Synthase, clone TS106 (MAB4130) , or Anti-OPRT antibody  for 2 h at room temperature. The primary antibodies were diluted with Can Get Signal Solution 1 . The membranes were then washed with Dako Washing Buffer  and incubated with Goat anti-Mouse IgG, Peroxidase Conjugated, heavy chain + light chain (AP124P) (EMD Millipore) or Goat anti-Rabbit IgG, Peroxidase Conjugate (AP132P) (EMD Millipore) diluted to 1:25,000 with Can Get Signal Solution 2  for 1 h at room temperature. Immunoreactive proteins were visualized with the ImmunoStar LD reagent , and images were captured using a GeneGnome HR system .3 cells were seeded into each well of 96-well plates and cultured for 24 h at 37 °C. The cells were treated with 5FU, FdU or TPI for 72 h, after which the culture medium was removed and 100 µl of a 0.5 mg/ml solution of MTT  was added to each well. The plates were then incubated for 4 h at 37 °C. The MTT solution was replaced with 100 µl of dimethyl sulfoxide  per well, and the absorbance at 540 nm was measured using a Sunrise Rainbow RC-R . Each assay was repeated eight times.A total of 5 × 10Metabolomics analyses were conducted by Human Metabolome Technologies . Therefore, the samples were prepared according to their instructions. In brief, MKN45 cells and MKN45/F2R cells were disseminated to six 10 cm dishes and cultured for 24 h. Half of them were then treated with 2 µM of 5FU for 24 h. Culture media were removed and washed by PBS buffer and treated with supplied reagents. The supernatant was ultrafiltrated using the supplied filters, and frozen at − 80 °C. These samples were then transferred on dry ice.MKN45 cells were cultured in RPMI-1640 with 5% FBS without antibiotics for 24 h to 50%–70% confluence prior to transfection. Cells were then transfected with siRNA oligonucleotides using Lipofectamine RNAiMAX  and serum-free Opti-MEM  at a final concentration of 10 nmol/l siRNA for 48 h. siRNA oligonucleotides for OPRT , TK1  and negative control oligonucleotides  were purchased from Invitrogen .t test.The mean half maximal inhibitory concentration (IC50) values were calculated based on each result of the MTT assays using the Microsoft Excel 2010 software program  and presented as mean ± standard error (SE). The significance of differences in the results of metabolomics analyses was determined by Human Metabolome Technologies using Student’s MKN45/F2R cells that had been previously established as 5FU-resistant cells showed an IC50 of 50.4 ± 2.52 µM, which represented a 37.1-fold increased resistance compared with parental MKN45 (IC50: 1.36 ± 0.24 µM). MKN45/F2R cells also exhibited resistance to FdU (IC50: 28.8 ± 11.0 µM). Furthermore, parental MKN45 cells were very sensitive to FdU (IC50: 0.004 ± 0.000 µM). These results are shown in Fig. Hydroxyurea is the inhibitor for RR, and TPI is the inhibitor for TP. We investigated the changes in the amount of FdUMP after treatment with 5FU combined with HU or TPI to clarify which pathway is important for the synthesis of FdUMP.As shown in Fig. Next, we focused on the amounts of metabolomes, such as phosphoribosyl pyrophosphate (PRPP) and deoxyribose 1-phosphate (dR1P), to clarify the cause of no progression in the TP–TK pathway. PRPP is the metabolome required for the synthesis of fluorouridine monophosphate (FUMP) from 5FU by OPRT, and dR1P is required for the synthesis of FdU from 5FU by TP. These pathways are illustrated in Fig. Reduced OPRT has been said to be a predictive factor for resistance to 5FU , 17, andAs expected, the knockdown of OPRT by siRNA1 (OPRT) (59.6%) and siRNA2 (OPRT) 35.6%) caused a reduction in FdUMP after treatment with 5FU  in parental MKN45 cells  and siRNA2 (TK), the expression of TK almost disappeared on Western blotting, and the amount of FdUMP after treatment with FdU decreased  in parental MKN45, indicating that FdU had been converted to FdUMP by TK in parental MKN45 cells. However, the amount of FdUMP was not decreased as much in 5FU-resistant MKN45/F2R cells after the knockdown of TK as in parental MKN45 cells . However, parental MKN45 cells were not affected by the combination of TPI, as shown in Fig. These results suggested that, in MKN45/F2R cells, intracellular FdUMP was reduced through the TP–TK pathway, which led to resistance to 5FU.In the present study, we clarified that 5FU was converted to FdUMP only through the OPRT–RR pathway. Furthermore, intracellular FdUMP was reduced through the TP–TK pathway in 5FU-resistant MKN45/F2R cells, and the inhibition of TP led to the reversal of the resistance to 5FU in 5FU-resistant MKN45/F2R cells. These hypotheses are illustrated in Fig. In the present study, we mainly used common techniques, such as Western blot analyses, MTT assays and transfection of siRNA, and the only expensive technique used was a metabolomics analysis, which was conducted by an outside party. Therefore, it is easy for other researchers to repeat our study, and we believe that our results can be reproduced. However, to our knowledge, no study has investigated the mechanisms underlying 5FU resistance focusing on the changes in the metabolism of 5FU.Thymidine phosphorylase has been believed to be an enzyme metabolizing 5FU in the first step, and several review articles have described the conversion of 5FU to FdU by TP . TP is aOrotate phosphoribosyl transferase has been reported as an important factor for 5FU resistance in cell lines, and there are many reports of the association between 5FU resistance and decreased OPRT levels , 15, 17.We previously reported the reversal of 5FU resistance by the combination of 5FU with deoxyuridine (dU) . In the TPI used in the present study has been already applied as a component of TAS-102, an anticancer drug used to treat metastatic colorectal cancer . TherefoSeveral limitations associated with the present study warrant mention. In this study, the reversal of 5FU resistance by TPI was found only in MKN45/F2R cells. In addition, predictive markers for the reversal of 5FU resistance by TPI must be developed to apply combination therapy with 5FU and TPI in daily practice.In conclusion, we observed the reversal of 5FU resistance in 5FU-resistant cells by TPI. Further investigations regarding the reversal of 5FU resistance by TPI in other 5FU-resistant cell lines as well as in vivo studies and the application of this combination therapy in clinical practice will be required in the future.


Neat1 and its partner p54nrb are required for CARM1’s association with paraspeckles and for H3R26 methylation. Conversely, CARM1 also influences paraspeckle organization. Depletion of Neat1 or p54nrb results in arrest at the 16- to 32-cell stage, with elevated expression of transcription factor Cdx2, promoting differentiation into the extra-embryonic lineage. This developmental arrest occurs at an earlier stage than following CARM1 depletion, indicating that paraspeckles act upstream of CARM1 but also have additional earlier roles in fate choice.Nuclear architecture has never been carefully examined during early mammalian development at the stages leading to establishment of the embryonic and extra-embryonic lineages. Heterogeneous activity of the methyltransferase CARM1 during these stages results in differential methylation of histone H3R26 to modulate establishment of these two lineages. Here we show that CARM1 accumulates in nuclear granules at the 2- to 4-cell stage transition in the mouse embryo, with the majority corresponding to paraspeckles. The paraspeckle component   •CARM1 accumulates in paraspeckles in the 2- to 4-cell stage mouse embryo•Paraspeckles correlate with heterogeneity of H3R26 methylation•Neat1 and p54nrb are required for CARM1’s association with paraspeckles•Neat1 or p54nrb elevates differentiation marker Cdx2Depletion of  The association of the methyltransferase CARM1 with paraspeckles in 2- to 4-cell stage mouse embryos impacts proper lineage allocation and pre-implantation development. Nanog and Sox2 and are destined to contribute to embryonic rather than extra-embryonic tissues  proteins: PSPC1, p54nrb (NonO), and SFPQ (PSF)  . These aPQ (PSF) .Neat1). Neat1 and its ongoing transcription are required for the structural integrity of paraspeckles (Paraspeckles are built around scaffolds of a specific long noncoding RNA (lncRNA) known as nuclear paraspeckle assembly transcript 1  targeting ll stage A. The efy RT-PCR B. We fouy RT-PCR C–3E. Dety RT-PCR D and 3E.y RT-PCR A and S3By RT-PCR C–S3E. A The above findings raised the question of whether p54nrb might act in partnership with CARM1 to regulate CARM1 expression and/or activity. Because one function of CARM1 is to methylate histone H3 at R26, we asked whether depletion of p54nrb from the zygote stage onward would also reduce H3R26me2 levels. Indeed, we found that downregulation of p54nrb led to a reduction of CARM1 speckles and a reduction in H3R26me2 at distinct nuclear sites F and 3G.Neat1, suggesting that paraspeckles are linked to Neat1 biogenesis  against Neat1 and examined the structure of paraspeckles by analyzing their components, p54nrb and CARM1, at the 4-cell stage  and 32-cell stages   , and S3.GFP mRNA D. Depletst stage E. We con (p54nrb E and 5F. (p54nrb G. To addo arrest E and 5H.Cdx2) and ICM . We found that when we depleted either p54nrb or Neat1, the levels of Sox2, Oct4, or Nanog were not significantly affected at the 16-cell stage of development. However, Cdx2 mRNA levels increased by 4.0- to 5.0-fold  at the 4-cell stage  siblings   . Thus, opression . This sue number . Second,e number . This poe number .Figure 7Neat1 or p54nrb. This suggests that the aggregate of p54nrb seen around the nucleolus, when CARM1 levels are perturbed, might retain some functionality. Paraspeckles are increasingly viewed as liquid droplets within the nucleus that permit sequestering of RNAs and proteins through phase separation .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Author, Magdalena Zernicka-Goetz  Act 1986 Amendment Regulations 2012 following ethical review by the University of Cambridge Animal Welfare and Ethical Review Body (AWERB).Carm1, females heterozygous for a floxed Carm1 gene and a Carm1 deletion (Carm1Δ), and carrying a ZP3-Cre transgene were used . These females were mated with Carm1loxP/Carm1Δ males. The Carm1loxP line was a kind gift from Mark Bedford  k-outs and the 50% Carm1Δ/Carm1loxP embryos that have a wild-type (but floxed) paternal Carm1 allele are heterozygous maternal k-outs. The following set of primers was used to genotype Carm1 knockout (KO) embryos: #1: 5′AGTTGGTGACCCTTGTGTCC3′; #2: 5′AGCTGCCAGGACCTCTGATA3′; #3: 5′CCTGAGGCAGAAAACAGTATGA3′; #4: 5′GCAGAACCTGAAGATGTTCGC3′; #5: 5′AGGTATCTCTGACCAGAGTCA3′. The combination of #1 and #2 primers detects two bands of 188 bp and 322 bp sizes corresponding to wild-type and floxed allel respectively. Combination of #1 and #3 primers was used to detect a deletion in Carm1 floxed allele (the expected size of band after deletion 280 bp). Primers #4 and #5 were used in a combination to detect Cre recombinase.To obtain oocytes depleted of maternal  Bedford . CompletEmbryos were collected from 4-6-week-old F1 (C57Bl6xCBA) superovulated female mice with 7.5 IU of pregnant mares’ serum gonadotropin  and 7.5 IU human chorionic gonadotropin  48 hours later, and crossed with F1 males. Embryos were isolated in M2 medium supplemented with 4% BSA and cultured in KSOM medium and fixed at the following times post hCG injection: early one-cell stage (19 h post hCG), late one-cell stage (30 h post hCG), early two-cell stage (39 h post hCG), late two-cell stage (48 h post hCG), early four-cell stage (54 h post hCG), late four-cell stage (62 h post hCG), early eight-cell stage (68 h post hCG), late eight-cell stage (74 h post hCG), and early blastocyst stage (98 h post hCG). The zona pellucida was removed using Tyrode’s solution. The following CARM1 inhibitor was used: 1-bezyl-3,5-bis(3-bromo-4-hydroxybenzylidene) piperidin-4-one (7g) . InhibitMouse embryonic stem cells (mESC) E14 were cultured on mitomycin C-treated mouse embryonic fibroblasts (MEFs) in Fc medium comprising DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 15% fetal bovine serum, 2mM GlutaMAX, MEM non-essential amino acids, sodium pyruvate and 100 μM β-mercaptoethanol.−1, 100 ng μl−1 or 400 ng μl−1) into the cytoplasm between 24 and 27 h after hCG injection, using an Eppendorf micromanipulator on a Leica inverted microscope. As marker of injection either Gap43-GFP or Gap43-RFP mRNA (200 ng μl−1) were used. Embryos were fixed at indicated times and assessed by immunofluorescence. To deplete p54nrb, CARM1 or Neat1, embryos were injected at the zygote stage with a combination of three siRNAs at a total concentration of 12 μM (for p54nrb depletion) 200nM stealth siRNA (for CARM1 depletion) or 200 nM antisense oligonucleotides ASO (for Neat1 depletion). Controls were injected with 12 μM AllStars Negative Control siRNA or antisense LNA negative control and all embryos were co-injected with 200 ng μl−1Gap43-GFP mRNA as an injection control. The embryos were fixed at indicated time points and assessed by immunofluorescence or subjected to RT-qPCR. See Zygotes from superovulated and mated F1 females were isolated 20 h post-hCG injection. To overexpress p54nrb or CARM1-WT embryos were microinjected with a synthetic mRNA . All transfections were performed according to the manufacturers’ instructions and analyzed after 48h. Plasmids: the CARM1 encoding plasmid was previously described . The p54After removal of the zona pellucida with acidic Tyrode’s solution, mouse embryos were either fixed in 3% PFA for 40 min at RT, followed by permeabilization in 0.5% Triton X-100 for 20 min or fixed in ice-cold 100% methanol for 20 min at −20°C.Embryos were blocked overnight at 4°C in 3% BSA in PBS-T (PBS containing 0.1% Tween) and incubated with primary antibodies in blocking solution overnight at 4°C. The embryos were then washed twice in PBS-T and incubated with secondary antibodies  for 2 h before final washes in PBS-T and imaging in drops of PBS on glass-bottomed dishes, covered by paraffin oil. Blastomeres of 4-cell stage embryo were separated before imaging.The proximity ligation assay (PLA) was performed using Duolink assay reagents. The stages prior to recognition with PLA probes were performed as for the immunofluorescence staining. Recognition with PLA probes was performed using PLA probes diluted in 3% BSA in 1x PBS for 1h at 37°C. The PLA-probes combinations were selected corresponding to the antibodies used depending on the host species of the antibodies used. Ligation of the short oligonucleotide sequences attached to the PLA probes was performed for 30 min at 37°C followed by amplification of the circular DNA formed after the ligation step for 3 h at 37°C in the dark. After the PLA procedure and a series of washes, embryos were counterstained with DAPI and imaged in drops of PBS on glass-bottomed dishes, covered by paraffin oil.Cells were extracted in ice-cold lysis buffer  supplemented with 5 μg/ml DNase and protease and phosphatase inhibitor cocktails. Protein concentration was measured with the BCA Protein Assay Kit and samples of 25 μg total proteins were subjected to SDS-PAGE. Resolved proteins were transferred to a nitrocellulose membrane, which was blocked with 5% BSA, and then incubated overnight at 4°C with the specific primary antibodies diluted in a blocking solution. The membrane was washed three times in PBS-T and incubated with secondary antibodies for 1 hour before final washes in PBS-T and detection with enhanced chemiluminescence (ECL).Total RNA was extracted from embryos at different stages using the Arcturus PicoPure RNA isolation kit and qRT-PCR was carried out using a Power SYBR Green RNA-to-CT 1-Step Kit and a StepOne Plus Real-time PCR machine (Applied Biosystems). Relative levels of transcript expression were calculated using the ddCT method with Gapdh or Histone H3 as endogenous controls. The following primers were used: see After removal of the zona pellucida with acidic Tyrode’s solution, mouse embryos were incubated in PBS containing 6 mg/ml BSA for 15 min. Then, embryos were transferred on coverslips coated with Denhardt’s solution and dried for 30 min at room temperature . EmbryosMulti-channel images were acquired for multiple sections using a Leica SP5 or Leica SP8 confocal microscope (Leica Microsystems). A Plan-Apochromat lens with magnification of 63X, NA = 1.518, and a pixel size of 0,27 mm in the x- and y-direction and 1 mm in the z-direction were used. The gain and offset were adjusted to prevent over-saturation or under-saturation of an image. Laser power and detector gain were maintained constant to quantitatively compare different experimental conditions within a single experiment. The dimension of each 2D slice was 1024x1024 pixels and the gray level dynamic range of each dataset is 8 bits per pixel. For the quantification of the number of speckles and intensity a sum of slices per each image was used. In the pre-processing step the cell nucleus segmentation was carried out using the DAPI channel. We applied conventional techniques for nucleus segmentation (ImageJ software). After completing nucleus segmentation in the DAPI channel, a nucleus mask was applied to indicate which pixels belonged to nucleus and which pixels were background.http://icy.bioimageanalysis.org). We used the UnDecimated Wavelet Transform detector to detect spots. We indicated spots brighter than the background to be detected and specified for the detection the size of the spot between 4 to 6 pixels diameter. In those images a Wavelet Adaptive Threshold (WAT) was computed what allowed removal of the background and the remaining noise. We set a Threshold scale for 80 value  and accepted a size of a spot minimum larger than the largest debris particle. The accuracy of the automated spot detection was checked manually with the first 35 nuclei.The speckle channels include some noise due to incidental and nonspecific staining. Therefore, we applied pre-processing steps through ImageJ software to suppress background noise and smooth the regions within the spots without affecting their edges. To remove the noise and smooth the foreground regions we applied anisotropic diffusion smoothing filter (2 and 5% CO2 on a spinning disc (3i) at intervals of 30 min with a z step of 5 μm. Image processing and analysis were performed with Fiji and Icy software. Image assembly was done in Illustrator CS5.Following injection with siRNAs, embryos were observed in a humidified chamber with 21% On) and precision measurements used (s.e.m.) is reported in the corresponding figure legends.Plots and statistics were generated in GraphPad Prism, version 7.0a. Quantitative data are presented as mean ± standard error of the mean (s.e.m.) and were analyzed by using two-tailed Student’s t-test (Mann Whitney test) or ANOVA test. The p values relative to controls were marked with asterisks on the charts. For all microscopy measurements, the exact value of the number of cells used (


Iron deficiency chlorosis (IDC) is an abiotic stress often experienced by soybean, owing to the low solubility of iron in alkaline soils. Here, soybean lines with contrasting Fe efficiencies were analyzed to test the hypothesis that the Fe efficiency trait is linked to antioxidative stress signaling via proper management of tissue Fe accumulation and transport, which in turn influences the regulation of heme and non heme containing enzymes involved in Fe uptake and ROS scavenging. Inefficient plants displayed higher oxidative stress and lower ferric reductase activity, whereas root and leaf catalase activity were nine-fold and three-fold higher, respectively. Efficient plants do not activate their antioxidant system because there is no formation of ROS under iron deficiency; while inefficient plants are not able to deal with ROS produced under iron deficiency because ascorbate peroxidase and superoxide dismutase are not activated because of the lack of iron as a cofactor, and of heme as a constituent of those enzymes. Superoxide dismutase and peroxidase isoenzymatic regulation may play a determinant role: 10 superoxide dismutase isoenzymes were observed in both cultivars, but iron superoxide dismutase activity was only detected in efficient plants; 15 peroxidase isoenzymes were observed in the roots and trifoliate leaves of efficient and inefficient cultivars and peroxidase activity levels were only increased in roots of efficient plants. Iron (Fe) is an essential micronutrient required for the plant growth, being involved in several metabolic processes, including photosynthesis, respiration, nitrogen fixation, DNA synthesis, hormone production, and chlorophyll biosynthesis . AlthougGlycine max L.) is the most important legume crop with an estimated world production of more than 350 million tons in 2017 ) or Fe deficiency .One Fe efficient (PI437929 / VIR 316) and one Fe inefficient (PI378676A / Primorskaja 500) ccession  Seeds we−1 m−2 of photosynthetic photon flux density at plant level, supplied by a mixture of incandescent bulbs and fluorescent lights. Temperatures were set to 25 °C during the light period and to 20 °C during the dark period, and relative humidity was maintained at 75% throughout the day and night. The standard solution for hydroponic growth of G. max included: 1.2 mM KNO3; 0.8 mM Ca(NO3)2; 0.3 mM MgSO4.7H2O; 0.2 mM NH4H2PO4; 25 μM CaCl2; 25 μM H3BO3; 0.5 μM MnSO4; 2 μM ZnSO4·H2O; 0.5 μM CuSO4.H2O; 0.5 μM MoO3; 0.1 μM NiSO4. Hydroponic solution was buffered with 1mM MES [2-(N-morpholino)ethanesulfonic acid], pH 5.5 and, during the experimental time, pH was measured and solutions were changed every three days. The experiment ended 14 days after transferring the plants to the climate chamber, when soybean plants usually show the most contrasting symptoms of IDC [The vessels were placed in a climate chamber  with 16-h day photoperiod providing 325 μmol ss of IDC . Plants 3 in a Teflon reaction vessel and heated in a SpeedwaveTM MWS-3+  microwave system. Each plant organ from all the treatments (n = 5) was ground and five independent digestions were carried out.A total of 100 mg of dried plant tissue (root and trifoliate leaves) was mixed with 5 mL of 65% HNOThe digestion procedure was conducted in five steps, consisting of different temperature and time sets: 130 °C/10 min, 160 °C/15 min, 170 °C/12 min, 100 °C/7 min, and 100 °C/3 min. The resulting solutions of the digestion procedure were then brought to 20 mL with ultrapure water and filtered for further analysis. Mineral concentration determination was performed using inductively coupled plasma optical emission spectrometer (ICP-OES) Optima 7000 DV  with radial configuration.n = 5) were ground with liquid nitrogen, using a mortar and pestle, and 200 mg of ground sample was suspended in 1.5 mL of 20 mM potassium phosphate buffer (pH = 6.8). After centrifuging for 10 min at 16,000g, 400 µL of the supernatant were mixed with 100 µL of acetylacetone. The mixture was incubated for 10 min at 100 °C and then transferred to room temperature (RT), until cool. At this point, 500 µL of modified Ehrlich’s reagent were added to each sample, the mixture was let to stand for 5 min, and then centrifuged for another 5 min at 16,000g. Absorbance was read at 553 nm and ALA concentration was calculated according to a standard calibration curve of 5-aminolevulinic acid hydrochloride .Protocols for ALA quantification in the leaves were optimized based on protocols previously published . In shorn = 5) was performed by measuring the oxidized version of this protein, hemin, using an enzymatic assay kit  following the manufacturer instructions.Estimated heme protein content in leaves and roots (n = 5). The referred compounds were extracted and quantified according to a modified protocol [a and b, and carotenoids were determined according to previously published equations [Chlorophyll, anthocyanin, and carotenoid concentrations were measured on the last fully expanded trifoliate leaf of plants grown in the previously described conditions (protocol . Brieflyquations .n = 5) were homogenized in 10 mL of 0.5% thiobarbituric acid in 20% trichloroacetic acid (w/v) and incubated at 100 °C for 30 min. The reaction was stopped on ice and samples were centrifuged at 5000 rpm for 10 min. The supernatant was filtered, absorption was read at 450, 532, and 600 nm, and MDA concentration (µmol g−1) was calculated from: 6.45 × (A532 − A600) − 0.56A450.To evaluate the lipid peroxidation, thiobarbituric acid reactive substances (TBARS) were measured using a colorimetric adapted method . In shorn = 5) via the spectrophotometric determination of Fe2+ chelated to BPDS (bathophenanthroline disulfonic acid). Roots of each plant were submerged in assay solution containing: 1.5 mM KNO3, 1 mM Ca(NO3)2, 3.75 mM NH4H2PO4, 0.25 mM MgSO4, 25 µM CaCl2, 25 µM H3BO3, 2 µM MnSO4, 2 µM ZnSO4, 0.5 µM CuSO4, 0.5 µM H2MoO4, 0.1 µM NiSO4, 100 µM Fe(III)-EDTA (ethylenediaminetetraacetic acid), and 300 µM BPDS. All nutrients were buffered with 1 mM MES, pH = 5.5. The assays were conducted under dim light conditions at 20 °C and were terminated after 45 min by removal of the roots from the assay solution. Absorbance values were obtained spectrophotometrically at 535 nm, and an aliquot of the solution that had no roots during the assay was used as a blank. Rates of reduction were determined using the molar extinction coefficient of 22.14 mM−1cm−1.Root FRO activity was quantified as previously described by . The mea2O2 with 0.6% final concentration. Change in absorbance at 465 nm was followed for 1 min. A unit of POX activity was defined as µmol H2O2 decomposed ml−1 min−1.Enzyme extraction was performed at 4 °C as described in . SOD and2O2 to inhibit Cu/ZnSODs and FeSODs (MnSOD activity is resistant to both). For staining of POX activity, gels were incubated for 30 min in 200 mM Na-acetate buffer (pH = 5.0) containing1.3 mM 3,3-diaminobenzidine and 3% H2O2 [For separation of SOD and POX isoenzymes 12.5% and 10% native separating gels were used respectively. Equal amounts of proteins (50 µg) were loaded to each lane. SOD activity in gels were detected by photochemical staining in the presence of riboflavin and NBT. The different types of SOD were differentiated by incubating gels in inhibitors of SOD prior to NBT staining, such as 2 mM KCN to inhibit Cu/ZnSODs and 3 mM H 3% H2O2 . Gels stn = 5) and 100 mg of ground tissue were homogenized with 1.5 mL of extraction buffer composed of 0.1 M potassium phosphate buffer (pH = 7.0), 0.1 mM EDTA, and 1% polyvinylpyrrolidone. Samples were vortexed for 2 min and then centrifuged for 10 min at 5000 rpm at 4 °C. The supernatant was collected and diluted three-fold. CAT was measured using 666 µL of the diluted supernatant, to which 334 µL of 73 mM H2O2 in 0.5 M Tris-HCl buffer (pH 7.0) was added. Absorbance was read for 3 min at 240 nm and CAT activity was calculated according to [2O2 in 0.5 M Tris-HCl buffer (pH = 7.0) were added. Absorbance was measured for 3 min at 290 nm and APX activity was calculated according to [For the evaluation of CAT and APX activity, an enzymatic extraction was performed according to . Roots arding to . APX wasrding to .n = 5) was homogenized with 1.5 mL of extraction buffer containing 50 mM Tris-HCl (pH = 7.5) and 1 mM EDTA. The mixture was vortexed for 2 min and centrifuged for 10 min at 5000 rpm at 4 °C. To 100 µL of the supernatant, 1 mL of a solution containing 1 mM EDTA, 0.5 mM GSSSG, 0.15 mM NADPH, 50 mM Tris-HCl buffer (pH = 7.5), and 3 mM MgCl2 was added to each sample. Absorbances were read for 1 min at 340 nm and GR activity was calculated according to [For GR, 100 mg of ground roots and trifoliate leaf tissue (rding to .www.graphpad.com). Differences among all groups (treatments and cultivars) were tested with two-way ANOVA corrected for multiple comparisons using Tukey method. Statistical significance was considered at p < 0.05.Data were analyzed with GraphPad Prism version 6.00 for Mac OS X  was performed to establish the relationships among the different variables. The data set included 16 continuous variables, namely, the concentration of anthocyanins, total chlorophylls, carotenoids, leaf ∂-aminolevulinic acid, root ∂-aminolevulinic acid, leaf hemin, root hemin, leaf MDA and root MDA; and the activity of leaf APX, root APX, leaf GR, root GR, root FRO, leaf CAT and root CAT. This analysis was performed using Tanagra data mining software, version 1.4.5  .Efficient plants under Fe sufficiency do not over accumulate Fe in the roots, and are able to transport adequate levels of Fe to the shoots, thus avoiding IDC. They adjust their pigment concentration under Fe stress to lower photoinhibition and consequently ROS levels while inefficient plants are not able to do so. As such, lower stress levels are sensed by the upper organs and MDA concentrations remain unaltered in root and shoot tissues, independent of Fe supply. ALA remains available in the leaves for chlorophyll synthesis and the heme-group continues to be available for enzyme integration. In this way, FRO activity is higher in efficient plants when compared with the inefficient ones and FeSOD activity is maintained, even under Fe deficiency. Efficient plants do not need to activate their antioxidant system because there is no formation of reactive oxygen species under iron deficiency.In


Notably, PtdInsP2 and ML-SA1 cooperate further increasing calcium efflux. Here we report the structures of human TRPML1 at pH 5.0 with PtdInsP2, PtdInsP2, or ML-SA1 and PtdInsP2, revealing a unique lipid-binding site. PtdInsP2 and PtdInsP2 bind to the extended helices of S1, S2, and S3. The phosphate group of PtdInsP2 induces Y355 to form a π-cation interaction with R403, moving the S4–S5 linker, thus allosterically activating the channel. Our structures and electrophysiological characterizations reveal an allosteric site and provide molecular insight into how lipids regulate TRP channels.Transient receptor potential mucolipin 1 (TRPML1), a lysosomal channel, maintains the low pH and calcium levels for lysosomal function. Several small molecules modulate TRPML1 activity. ML-SA1, a synthetic agonist, binds to the pore region and phosphatidylinositol-3,5-bisphosphate P Transient receptor potential mucolipin 1 (TRPML1) is a lysosomal channel which maintains the low pH and calcium levels for lysosomal function. Here authors use structural biology and electrophysiology to show how lipids bind and allosterically activate TRPML1. Of these ligands, the TRP family has demonstrated regulation by more than 50 endogenous lipids2. TRPML1 regulates lysosomal calcium signaling, lipid trafficking, and autophagy-related processes5. Loss-of-function TRPML1 mutants cause a neurodegenerative disorder, Mucolipidosis type IV (MLIV)8. While recent structural determinations provide insight into the regulatory mechanisms of TRPs by small molecules12, there is still limited structural detail showing how specific lipids regulate activity. Determination of the structural regulation of TRP channels by endogenous lipid ligands can provide a wealth of insight into the physiological function of these channels13.TRP channels, which include six sub-families, are widely regulated by various sensations and ligands2, a low-abundance phosphoinositol in late endosomes and lysosomes14, stimulates the opening of TRPML1, specifically at low pH16. In contrast, PtdInsP2, a similarly structured phosphoinositol lipid abundant in the plasma membrane, decreases the TRPML1-mediated calcium release on the cell surface16. Notably, human fibroblasts derived from patients with Niemann-Pick C disease , exhibit decreased TRPML1 activity15, implying a correlation between lysosomal abnormality and TRPML1 behaviors. Therefore, structural insights into TRPML1 regulation by lipid ligands represent a strong opportunity to study the lipid-mediated regulatory mechanism among TRP channels.In previous studies, PtdInsP20. TRPML1 forms a classic tetramer: each subunit contains six transmembrane helices (S1–S6), two pore helices (PH1 and PH2) and a ~30 kD lumenal/extracellular domain  PtdInsP2, and (3) ML-SA1 with PtdInsP2 at 3.5–3.7-Å resolution, revealing a lipid-binding site to allosterically activate the channel, distinct from the previously reported site in TRPV121. These structures along with electrophysiological characterizations reveal an allosteric site and illuminate how lipids regulate TRP channels.In this manuscript, we report three structures of human TRPML1 with (1) PtdInsP2 and used cryo-EM to detect the conformation of TRPML1 with PtdInsP2. Unfortunately, the particles failed to reconstitute a structure at atomic resolution. To address this, we purified the protein in a low pH digitonin buffer and independently incubated with either PtdInsP2, PtdInsP2, or PtdInsP2 plus ML-SA1 to capture TRPML1 in its various ligand-bound states. The resulting three structures P2, PtdInsP2, and PtdInsP2 with ML-SA1; Fig. To investigate the role of inositides in TRPML1 activation, we first assembled the human TRPML1 protein in nanodiscs containing PtdInsP2 bound TRPML1 is detected in a closed conformation at 3.5 Å resolution P212. Single channel recordings of mouse TRPML1 activity indicated that the open probability of TRPML1 remained quite low when the PtdInsP2 was bound, with less than half of the channel population presented in an open conformation18. This finding suggests that it may be difficult to capture significant populations of PtdInsP2-mediated opening of TRPML1 for cryo-EM structural determinations. K55, R61, and K65 in S1, and R318 and R322 in S2 are involved in PtdInsP2 binding P2. In addition to the residues in S1 and S2, the side chain density of Y355 in the PtdInsP2-bound structure is adjacent to the density of the phosphate group of PtdInsP2, implying a strong binding between these two groups Ping Fig. . Mutatioups Fig. . As R4032 and PtdInsP2, we determined the structure of PtdInsP2 bound TRPML1 at 3.6 Å resolution P2 P2-bound structure P2-bound structure, preventing the strong π-cation interaction with R403 P2 may lie in the 3′ phosphate supporting the formation of a π-cation interaction between Y355 and R403. Previous studies showed that PtdInsP2 can decrease the ion efflux of TRPML1 by competing with PtdInsP2 binding to the channel18. This is consistent with our structural observation that PtdInsP2 and PtdInsP2 bind the same area. PtdInsP2 may serve as an agonist blocker on the cell surface, preventing the other lipid agonist from entering the lipid-binding site, therefore keeping the channel inactive. In normal cells, PtdInsP2 and PtdInsP2 are not abundant on the same membrane, and the activity of TRPML1 cation efflux depends on its cellular location.In order to compare the binding sites of PtdIns3,5)P,5P2 and nd PtdIns,5P2, we )P2 Fig. . Howeverure Fig. . This di403 Fig. . Y355 an218 Fig. . From th22 mutations, TRPML1-Y355A-LA and TRPML1-R403A-L/A. The four leucine-to-alanine mutations in TRPML1-L/A have been reported to increase cell surface localization for subsequent electrophysiological characterization via whole-cell patch clamp22. With PtdInsP2 perfused in the cytoplasmic solution, there is a significant increase in current density for HEK cells expressing WT-L/A at low pH P2 P217, suggest an alternate site for inositide binding separate from ML-SA1 that can allosterically affect channel opening. Notably, since an R403 mutation causes MLIV, it is likely that this mutant cannot be activated by PtdInsP2.Earlier reports describe a significant reduction of PtdInsP)P2 Fig. . All exp2 and ML-SA1 cooperate to stimulate TRPML1 considerably more than either agonist alone15. To investigate whether or not the π-cation interaction is involved in this potentiation, we utilized the ability of lysine to form a stronger π-cation interaction than the endogenous arginine23. Compared with arginine, lysine has a shorter side chain that presents less flexibility and an amine group with a more compressed positive charge, theoretically making it easier to capture the aromatic ring of Y355 and form a stronger π-cation interaction Pion Fig. .2 or ML-SA1. In the presence of 10 μM ML-SA1, current densities of both WT and R403K were greatly enhanced P2.We expressed this mutant and compared the current with WT channels with and without PtdInsPced Fig. . Cytoplaels Fig. . This le2 cooperation, the ML-SA1/PtdInsP2-bound structure was determined at 3.7 Å resolution in an open conformation P2-bound structures shows that ML-SA1 binding causes the S3 and S4 of the PtdInsP2 binding site to move away from the pore center P. 2 Fig.  and S1–Ster Fig. . The movter Fig. . Althoug2 binders using basic amino acids. PtdInsP2 induces Y355 in S3 to form a π-cation interaction with R403 in S4 through the binding of its 3′ phosphate group to the hydroxyl group of Y355. This π-cation interaction affects the movement of the S4–S5 linker, a common allosteric site among TRP channels. Compared with ML-SA1, which directly forces the pore open, this stimulation is indirect and mild, explaining the low proportion of TRPML1 channels that are active with PtdInsP2. The fact that the R403K mutant appears to facilitate ML-SA1 pore opening but does not significantly affect PtdInsP2 mediated channel opening poses an interesting hypothesis. It is possible that the inositide binding site allosterically regulates channel opening in several different ways. As noted by earlier reports, the idea that both PtdInsP2 bind to the same pocket but have significantly different effects on channel opening could represent a way to control channel activity through localization within different lipid enriched compartments. In this manner endogenous TRPML1 is kept relatively inactive at the surface of the cell due to an abundance of PtdInsP2. As TRPML1 is trafficked to the lysosome, the membrane is enriched with PtdInsP2. Concurrently, PtdInsP2 is displaced by PtdInsP2 allowing the formation of the observed π-cation interaction. In this conformation, the channel is more susceptible to pore opening by agonists like ML-SA1. However, this does not rule out the possibility of additional open conformations supported by PtdInsP2 binding. As the R403K mutation does not increase channel activity, it is also possible these conformations may not rely on the π-cation formed between R403 and Y355.In this manuscript, we report three human TRPML1 structures with different ligands. TRPML1 employs its extensions of IS1–IS3 to recognize the PtdInsP17. The structure of TRPV6 in the open conformation reveals a lipid bound to the S4–S5 linker, the allosteric site of the channel, presumably stimulating channel opening10 . In our reported structures, there is no lipid density in the S4–S5 linker implying this site may not be used for the channel activation among TRPML family. Instead of the S4–S5 linker, the allosteric site for TRPML activation resides in the extensions of IS1–IS3, which capture the PtdInsP2 lipid.The structures of other TRP channels have revealed several lipid-binding sites among the different channels. The structure of TRPML3 in the closed conformation with amphipols shows several sterol-like molecules attached near the pore region including our observed ML-SA1-binding pocket P2 due to the high abundance of PtdInsP2 in the lysosomal environment. Thus, our structural observations show an allosteric site among TRP channels that can host lipid ligands, and may reveal one molecular mechanism for lipid-mediated TRPMLs regulation.Previous reports showed that other ion channels, such as GIRK2− cells (ATCC). These cells tested negative for mycoplasma contamination. At 48 h post infection at 37 °C, cells were disrupted by sonication in buffer A  with 1 mM PMSF and 5 μg ml−1 each of leupeptin and aprotinin. After low-speed centrifugation at 3470 g, the resulting supernatant was incubated in buffer A with 1% (w/v) lauryl maltose neopentyl glycol  for 1 h at 4 °C. The lysate was centrifuged at 34,572 g and the supernatant was loaded onto an anti-Flag M2 affinity column (Sigma). After washing twice, the protein was eluted in 20 mM HEPES, pH 7.0, 150 mM NaCl, 0.1 mg ml−1 Flag peptide and 0.01% MNG, and then concentrated. The protein was purified by Superdex-200 size-exclusion chromatography  in a buffer containing 20 mM sodium acetate, pH 5.0, 150 mM NaCl and 0.06% (w/v) digitonin (Sigma). The peak fractions were collected and concentrated to 5–7 mg/ml for grid preparation. The mutated DNA constructs were generated using QuikChange Mutagenesis Kit (Agilent) . Input resistances for the capillary pipettes were 2–10 MΩ, and the apparent cell resistances were 0.5–2 GΩ. External solutions were adjusted to RT (23–25 °C) in gravity-fed parallel solution lines with an outlet flow velocities of 2–5 mm/s allowing extracellular solution changes within 2–3 s. For TPRML1 current–voltage analysis, a voltage ramp was generated from −150 mV to 20 mV over 1.5 s. Voltage ramps were aligned and statistical analysis was performed at −100 mV. Boroscilicate glass pipettes were fire polished and back-filled with the cytoplasmic solutions containing in mM: 120 cesium methanesulfonate, 4 NaCl, 10 EGTA, 2 MgCl2, 20 HEPES, pH 7.2 with CsOH (~25 mM). A 5 mM stock solution of C8: PtdInsP2 (Echelon) was prepared in ddH2O and aliquots were stored at −80 °C. For PtdInsP2 stimulated recordings, a 1:100 dilution of lipid was added to the cytoplasmic solution and briefly sonicated. Extracellular solutions were set to pH 4.6 and contained in mM: 140 sodium gluconate, 5 KCl, 10 glucose, 10 HEPES, 10 MES, 1 MgCl2, 1 CaCl2, and 8 HCl. For control experiments at pH 7.4, bath solution contained in mM: 140 NaCl, 5 KCl, 10 glucose, 20 HEPES, 1 MgCl2, 1 CaCl2, and 8 NaOH. A total of 10 µM ML-SA1 was added to the appropriate bath solution immediately prior to recording. Low passage HEK293T (ATCC CRL-11268) cells used for electrophysiology were maintained in DMEM (GIBCO) with 10% FBS, penicillin-streptomycin, and l-glutamine (Sigma), and routinely monitored for mycoplasma infection using MycoSensor PCR kits (Agilent). HEK293T cells were transiently transfected with Lipofectamine 3000 (Invitrogen) and used within 48–72 h. The cells were deplated with trypsin (0.25%) and placed in a bath solution on an inverted Nikon TE2000U inverted microscope equipped with a 60X oil immersion, 1.45-NA objective. A Lambda DG-4 xenon epifluorescence power supply with Semrock FITC filters were used to detect cells expressing surface TRPML1-GFP mutants. Statistical analyses were performed in Matlab and SigmaPlot (SigmaStat) using either a Rank Sum Test or a Student's t-test as determined by the SigmaPlot Normality Test. Figures were created in GraphPad Prism 7.05. Current–voltage relations represent the mean of at least 4 voltage ramps, and agonist-induced current density changes represent the mean and s.e.m. of at least four independent cells per condition.The full-length human TRPML1 (L/A) was subcloned to pEGFP-C1 for electrophysiological assays. Whole-cell patch clamp recordings of cell electrical parameters were performed using Matlab based Capmeter v7.22 or PtdInsP2 bound protein, the protein in a buffer containing 20 mM sodium acetate, pH 5.0, 150 mM NaCl and 0.06% digitonin was incubated with 0.2 mM PtdInsP2 or PtdInsP2  on ice for 1 h before grid preparation and freezing. For PtdInsP2/ML-SA1 bound protein, the protein in a buffer containing 20 mM sodium acetate, pH 5.0, 150 mM NaCl and 0.06% digitonin was incubated with 0.2 mM PtdInsP2 and 0.3 mM ML-SA1  on ice for 1 h before grid preparation and freezing. The grids were imaged with a 300 keV Titan Krios (FEI) with a Gatan K2 Summit direct electron detector (Gatan). The data were collected at 1.07 Å per pixel with a dose rate of eight electrons per physical pixel per second. Images were recorded for 12-s exposure in 30 subframes to give a total dose of 84 electrons per Å2.A protein sample was added to Quantifoil R1.2/1.3 400 mesh Au holey carbon grids (Quantifoil), blotted with Vitrobot Mark IV (FEI), and frozen in liquid ethane. For PtdInsP30. The contrast transfer function (CTF) was estimated using CTFFIND431. After automatic picking and manual micrograph inspection, the particles were extracted for subsequent 2D and 3D classification. Motion correction of individual particles was performed using the program alignparts_lmbfgs32. Using the structure of human TRPML1 (EMD-8840) low-pass filtered to 60 Å as the initial model, 3D classification was carried out in RELION33. The best classes of four structures, containing 50k~60k particles, provided a 5–6 Å map. Refinement was performed in FREALIGN34 using this best class as the initial model and all particles post motion correction to generate the final map for model construction.Dark subtracted images were first normalized by gain reference that resulted in a pixel size of 1.07 Å per pixel. Drift correction was performed using the program Unblur2 bound) and 3.57 Å P2 bound) and 3.7 Å P2 bound), and a B-factor value of −100 Å2. To precisely determine the location of PtdInsP2, we took advantage of the stronger signal phosphate atoms have in the electron density compared to other atoms , and we increased signal to 8–10σ level to determine its head position. The entire model was built in COOT35.To obtain better side-chain densities for model building, we sharpened one half-map using BFACTOR.EXE (author: Nikolaus Grigorieff) with a resolution limit of 3.53 Å P36 and also in reciprocal space using Refmac38. Structural factors were calculated from a half-map (working) using the program SFall38. Fourier shell correlations (FSCs) were calculated between the two half maps, the model against the half1 map, the half2 map, and full map40. Local resolutions were estimated using Blocres41. MolProbity42 was used to validate the geometries of the model. Structure figures were generated using PyMOL (http://www.pymol.org) and Chimera43.The model was refined in real space using PHENIXSupplementary Information


In this paper, the roles of H2O2 and NO on salvianolic acid B  production in salicylic acid (SA)-induced Salvia miltiorrhiza cell cultures were investigated. The results showed that H2O2 could be significantly elicited by SA, even though IMD (an inhibitor of NADPH oxidase) or DMTU (a quencher of H2O2) were employed to inhibit or quench intracellular H2O2. These elicited H2O2 levels significantly increased NO production by 1.6- and 1.46 fold in IMD+SA and DMTU+SA treatments, respectively, and induced 4.58- and 4.85-fold Sal B accumulation, respectively. NO was also markedly elicited by SA, in which L-NNA (an inhibitor of NO synthase) and cPTIO (a quencher of NO) were used to inhibit or quench NO within cells, and the induced NO could significantly enhance H2O2 production by 1.92- and 1.37-fold in L-NNA+SA and cPTIO+SA treatments, respectively, and 3.27- and 1.50-fold for Sal B accumulation, respectively. These results indicate that elicitation of SA for either H2O2 or NO was independent, and the elicited H2O2 or NO could act independently or synergistically to induce Sal B accumulation in SA-elicited cells.Hydrogen peroxide (H Plants provide a wide variety of secondary metabolites useful to mankind, such as pharmaceuticals, food additives, flavors, and other industrial materials. Often high-value secondary metabolites are found in low abundance in Nature . BecauseSalicylic acid (SA), a phenolic compound broadly distributed in plants, has been recognized as a regulatory signal mediating plant response to abiotic stresses such as drought, chilling, heavy metal tolerance, heat and osmotic stress . Most of2O2) and nitric oxide (NO) are key signaling molecules produced in cells in response to various stimuli and involved in a diverse range of plant signal transduction processes .2O  or removed by cPTIO (NO scavenger), the contents of NO within cells decreased slightly but not significantly when compared with H2O . This phoduction ,24,25,26reatment . In thisectively . As prop2 uptake , which mS. miltiorrhiza cell culture and SA elicitation treatment were provided by Dong et al. [2O2, DMTU , CAT (Sigma), IMD , sodium nitroprusside , Nω-nitro-L-arginine  and cPTIO  were prepared in distilled water and then sterilized after filtration through 0.22 μm membrane. To scavenge H2O2 and NO in S. miltiorrhiza cell culture, final concentrations of 500 μmol·L−1 DMTU and 50 μmol·L−1 cPTIO were added separately to the cell culture 30 min before SA treatment. All experiments were performed with three replicates.The detailed protocols of g et al. . Stock s−1 sulphuric acid (5 mL) after removing pigments by using acetone. This solution was used for H2O2 determination and the absorbance was measured at 415 nm. The detection was determined after 8 h SA-induction and this point of time was determined by experiment ahead of detection.The calli (0.2–0.3 g) were homogenized with acetone (5 mL) precooled below 4 °C and then ground to a homogenate on ice. The mixture was centrifuged at 10,000 rpm for 10 min at 4 °C, and 5% titanous sulfate (0.5 mL) and concentrated ammonia water (2 mL) were added to 2 mL of supernatant. After mixing, it was centrifuged at 10,000 rpm for 10 min at 4 °C and the supernatant was removed. The pellet was dissolved by 2 mol·LS. miltiorrhiza cell culture was leached with 0.22 μm membrane and Greiss reagent (1 mL) was added to 1 mL of filtrate. The mixture was incubated for 30 min at room temperature to convert nitrite into a purple azo-dye and the absorbance was determined at 550 nm. The detection was determined after 4 h SA-induction and this point of time was also determined by experiment ahead of detection.The content of NO was determined according to the production of nitrite with Greiss reagent that could oxidize NO to nitrite under acidic conditions . The S. S. miltiorrhiza cells were collected from cell cultures by centrifugation at 1,200 rpm, and then dried at 47.5 °C in an oven to a constant weight. The dried cells (0.05 g) were extracted ultrasonically with methanol-water solution  for 45 min. The extract was filtered through a 0.22 μm membrane and the filtrate was obtained for detection.The −1 and the injection volume was 10 μL. The DAD detection wavelength was 281 nm. The extraction yield of Sal B was calculated based on the integration of the chromatographic peak areas. More details were described in Dong et al. [The content of Sal B was quantified by HPLC analysis that was performed using a Shimadzu  system equipped with a UV/visible absorbance detector (DAD). A Shim-pack VP-ODS column  was used at a column temperature of 30 °C. The flow rate was 1 mL·minS. miltiorrhiza cell cultures. H2O2 could be significantly elicited in SA-induced cells, even though H2O2 was inhibited or quenched. These elicited H2O2 significantly increased NO production and Sal B accumulation. NO was also markedly elicited by SA under the circumstances where NO was inhibited or quenched, and the induced NO could significantly enhance H2O2 production and Sal B accumulation. The elicitation of SA for either H2O2 or NO was independent, and the elicited H2O2 or NO could act independently or synergistically to induce Sal B accumulation in SA-elicited cells.The present work shows SA is an effective elicitor inducing accumulation of Sal B in


Thermus thermophilus is a thermophilic bacterium which is capable of natural transformation, the uptake of external DNA with high efficiency. DNA uptake is thought to be mediated by a competence-associated pilus, which binds the DNA substrate and mediates its transfer across the outer membrane and periplasm. Here, we describe the structural and functional analysis of two pilins which are known to be essential for DNA uptake, ComZ and PilA2. ComZ adopts an unusual structure, incorporating a large β-solenoid domain into the pilin structural framework. We argue on structural grounds that this structure cannot readily be accommodated into the competence pilus fiber unless it is at the tip. We also show that ComZ binds DNA and identify two lysine residues which appear to be important for DNA binding. These results suggest a model in which ComZ and PilA2 form a tip-associated DNA receptor which mediates DNA uptake. Thermus thermophilus, ComZ, consists of a type II secretion pseudopilin-like domain, with a large β-solenoid domain inserted into the β-sheet of the pilin-like fold. ComZ binds with high affinity to another competence-associated pilin, PilA2, which lies adjacent to the comZ gene in the genome. The crystal structure of PilA2 revealed a similar type II secretion pseudopilin-like fold, with a small subdomain; docking simulations predicted that PilA2 binds between the pseudopilin-like and β-solenoid domains of ComZ. Electrophoretic shift analysis and DNase protection studies were used to show that ComZ alone and the ComZ/PilA2 complex are able to bind DNA. Protection against reductive dimethylation was used in combination with mass spectrometry and site-directed mutagenesis to identify two lysine residues in ComZ which are involved in DNA binding. They are located between the two domains in ComZ, on the opposite side from the predicted PilA2 binding site. These results suggest a model in which PilA2 assists ComZ in forming the competence pilus tip and DNA binds to the side of the fiber. The results demonstrate how a type IV pilin can be adapted to a specific function by domain insertion and provide the first structural insights into a tip-located competence pilin.Natural competence is the term used to describe the uptake of “naked” extracellular DNA by bacteria; it plays a significant role in horizontal genetic exchange. It is associated with type IV pili, and specialized competence pili mediate DNA uptake. Here, we show that the crystal structure of a competence-associated protein from  Haemophilus influenzae, Streptococcus pneumoniae, and Neisseria meningitidis. Natural competence is also found in thermophilic, as well as mesophilic, bacteria, notably Thermus thermophilus, where it could have a role in assisting environmental adaptation  is a process whereby genetic material is transferred between bacterial cells. HGT is mediated by three different mechanisms: conjugation, transduction, and natural transformation (NT); it therefore plays an important role in bacterial adaptive evolution. NT is characterized by the ability of bacteria to take up “naked” DNA from outside the cell: one strand of the DNA molecule is degraded, and the other is transported into the cytoplasm –3. DNA u–aptation .T. thermophilus is mediated by a multiprotein complex, consisting of 16 proteins which span the outer membrane, the periplasm, and the inner membrane , which catalyzes pilus fiber assembly  pili . T4a pilassembly , 13–19. a pili . mophilus . In addig to DNA . Anotherg to DNA .T. thermophilus do not impair natural competence  associated with natural competence (pilA1 to pilA4 [pilA1-4]) were type IVa pilin-like genes, and a fifth, comZ, encoded an unusual and much larger protein that lacked some of the sequence features characteristic of pilin genes  and the beginning of the first β-strand (Asn71). From a comparison of the three chains, the relative orientation of the two domains is invariant . The seg10.1128/mBio.00614-19.1FIG S1FIG S1, DOCX file, 1.0 MB.Superposition of ComZ chains A, B, and C. Chains B and C were superimposed onto chain A using CCP4MG , with roCopyright © 2019 Salleh et al.2019Salleh et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the T. thermophilus minor pilins to type II secretion system pseudopilins (Escherichia coli (ETEC) has parallels with ComZ, in that both are structurally related to GspK and both have additional β-rich domains outside the pilin fold  using CCP4MG  was superimposed onto a single chain from the structure of g CCP4MG . ComZ A Copyright © 2019 Salleh et al.2019Salleh et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Flavobacterium frigoris (PDB accession code 4NU2), as the closest structural relative of the ComZ β-solenoid domain  superposition of PilA2 structure (red) with EpsI (blue), GspI (green), and TTHA1218 (yellow). See the text for other details. Download FIG S3, DOCX file, 0.4 MB.Structure of the competence-associated type IV pilin PilA2. (A) Two orthogonal ribbon plot views of PilA2, colored by secondary structure.  Topology diagram for PilA2. The additional subdomain is indicated by the blue box; (middle) topology diagrams for EpsI from Copyright © 2019 Salleh et al.2019Salleh et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00614-19.9TABLE S2Table S2, DOCX file, 0.01 MB.X-ray data collection and refinement statistics for PilA2. Download Copyright © 2019 Salleh et al.2019Salleh et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the dK) of 0.69 μM  profiles for ComZ and PilA2, ComZ alone, and PilA2 alone are shown in  0.69 μM .Similar studies of binding to ComZ were carried out using the PilA1 and PilA3 pilins. PilA1 failed to show any evidence of binding to ComZ . The elu10.1128/mBio.00614-19.4FIG S4FIG S4, DOCX file, 0.6 MB.Size exclusion chromatography analysis of ComZ interaction with PilA1 and PilA3. (A) ComZ and PilA1. (Upper) Elution profiles (absorption at 280 nm) of ComZ-PilA1 mixture (profiles B and C), ComZ alone (profile A), and PilA1 alone (profile D). Separation was carried out on a HiLoad 16/600 Superdex 200 PG column  at a flow rate of 1 ml/min in buffer of 25 mM Tris-HCl, pH 8.0, 200 mM NaCl, 5% glycerol. Prior to SEC separation, the proteins were incubated in the buffer at 4°C for 30 min. Aliquots of 0.036 μmol of ComZ and 0.027 μmol of PilA1 were loaded. (Lower) SDS-PAGE of eluted peaks from each SEC run. (B) ComZ and PilA3. (Upper) Elution profiles (absorption at 280 nm) of ComZ-PilA3 mixture (profile A), ComZ alone (profile B), and PilA3 alone (profile C). SEC separation was carried out as for panel A. Download Copyright © 2019 Salleh et al.2019Salleh et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00614-19.5FIG S5FIG S5, DOCX file, 0.5 MB.Size exclusion chromatography analysis of ComZ/PilA2 interaction with PilA1 and PilA3. (A) ComZ/PilA2 and PilA1. (Upper) Elution profiles (absorption at 280 nm) of ComZ-PilA2-PilA1 mixture (profile A and D), ComZ alone (profile B), PilA2 alone (profile C), and PilA1 alone (profile E). Separation was carried out on a HiLoad 16/600 Superdex 200 PG column , with a flow rate of 1 ml/min in buffer containing 25 mM Tris-HCl, pH 8.0, 200 mM NaCl, 5% glycerol. Prior to SEC separation, the proteins were incubated in the buffer at 4°C for 30 min. Aliquots of 0.036 μmol of ComZ/PilA2 and 0.027 μmol of PilA1 were loaded. (Lower) SDS-PAGE of eluted peaks from each SEC run. (B) ComZ/PilA2 and PilA3. (Upper) Elution profiles (absorption at 280 nm) of ComZ-PilA2-PilA3 mixture (profile B), ComZ alone (profile A), PilA2 alone (profile C), and PilA3 alone (profile D). SEC separation was carried out as described for panel A. (Lower) SDS-PAGE of eluted peaks from each SEC run. Download Copyright © 2019 Salleh et al.2019Salleh et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00614-19.6FIG S6FIG S6, DOCX file, 0.8 MB.ComZ affinity binding assays. (A) ComZ/PilA2. Both proteins were incubated in 25mM Tris-HCl, pH 8.0, 200 mM NaCl, 5% glycerol at 4°C for about 30 min and applied to a Streptavidin column, and eluted proteins were examined by SDS-PAGE. Three mg of ComZ and 2 mg of PilA2 were loaded. (B) ComZ/PilA3. Both proteins were incubated together and applied to a Streptavidin column, and eluted proteins examined by SDS-PAGE. Three mg of ComZ and 2 mg of PilA3 were loaded. Buffer and temperature were as described for panel A. (C) ComZ/PilA2/PilA3. Both proteins were incubated together and applied to a Ni affinity column, and eluted proteins were examined by SDS-PAGE. Buffer and temperature were as described for panel A. Download Copyright © 2019 Salleh et al.2019Salleh et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the comZ, pilA1, pilA2, and pilA3 Thermus mutants are impaired in natural competence but retain type IV pili ; this is the case for ial ComP . In manyeriplasm , 53. TheVibrio cholerae, the binding of type IV competence pili to extracellular DNA and pilus retraction transporting the bound substrate to the cell surface . An exa10.1128/mBio.00614-19.7FIG S7FIG S7, DOCX file, 0.9 MB.Electrostatic surface of ComZ (chain A) computed using CCP4MG . DownloaCopyright © 2019 Salleh et al.2019Salleh et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the T. thermophilus by NT. ComZ is located at the tip of a competence pilus, supported in position by the minor pilin PilA2. It acts as the initial receptor for DNA binding outside the cell; the assembled pilus is retracted to bring the DNA substrate into the periplasm and into close proximity with ComEA and ComEC. DNA is then transferred to ComEA, which is inner membrane associated, and probably works in concert with the ComEC channel. The role of DNA binding to ComEA has been highlighted by the recent observations of Hepp and Maier in Neisseria  was amplified by PCR using primers CTTCACCATGGCCATAGAGCTCTGGACCACCCGCAACGAC and CGGTGTGACTCGAGGCGGCGCTCATAGGAGAGCACCTG and T. thermophilus HB27 genomic DNA as the template. The amplified gene and the pET-22b vector (Novagen) were digested with restriction enzymes NcoI and XhoI, purified, and ligated. The comZ-22b construct coded for the ComZ protein (residues 31 to 554) with the PelB leader sequence at the N terminus and hexahistidine tag at the C terminus. Synthetic constructs corresponding to the soluble domains PilA1 (residues 30 to 156), PilA2 (residues 33 to 193), and PilA3 (residues 33 to 233) were designed, optimized for expression in E. coli, and synthesized (GeneArt) with a Strep-tag II prior to subcloning into pET-22b vector (BamHI and XhoI sites), which includes the PelB leader at the N terminus. Recombinant plasmids were transformed into T7 Express cells (New England Biolabs [NEB]) for ComZ expression or Lemo21(DE3) cells (NEB) for minor prepilin expression and grown on Luria-Bertani (LB) plates with 100 μg/ml ampicillin (T7 Express) or 100 μg/ml ampicillin and 30 μg/ml chloramphenicol (Lemo21 cells) at 37°C for 14 h. Several colonies were inoculated into 50 ml of starter culture (LB medium with antibiotics) and grown until the optical density at 600 nm (OD600) reached 0.6 to 0.8. Cells were subsequently grown in Terrific broth (TB) medium supplemented with antibiotics and were induced with 0.4 mM isopropyl-β-d-thiogalactopyranoside (IPTG) when the OD600 reached 0.8. Cells were left shaking at 16°C for 16 h to allow protein expression before being harvested by centrifugation at 8,000 rpm  for 30 min at 4°C. Cell pellets were resuspended in buffer A  supplemented with protease inhibitor cocktail (Roche) and DNase I (5 μg/ml). Cells were disrupted by sonication  for 7 min at 30% power. Debris and unbroken cells were removed by centrifugation at 18,000 rpm  for 45 min at 4°C. For ComZ, purification was carried out by using a HisTrap HP column , followed by an elution step using buffer A plus increasing concentrations of imidazole (up to 500 mM). The purification of Strep-tag II-tagged pilins was carried out on a StrepTrap HP column . The streptavidin column was first equilibrated with 5 column volumes (CV) of water and 5 CV of buffer A. Crude cell lysates were applied to the column, which was washed with 6 CV of buffer A, followed by elution with 6 CV of buffer A plus 5% glycerol and 2.5 mM desthiobiotin. Collected fractions from cell lysates, wash, and elution buffer were analyzed by SDS-PAGE. A final purification step by SEC was carried out using a HiLoad 16/600 Superdex 75 PG column  in 25 mM Tris-HCl, pH 8.0, 200 mM NaCl, 5% glycerol at 25°C.The 2PtCl4) for 2 to 3 h. Crystals were cryoprotected with reservoir solution supplemented with 20% glycerol and flash-cooled in liquid nitrogen. The native ComZ data set was processed using the xia2 (2PtCl4) were manually scaled and merged using AIMLESS, as implemented in the CCP4 suite (Purified ComZ (8 to 10 mg/ml) was crystallized by sitting drop vapor diffusion. Equal volumes (200 nl) of protein and a reservoir solution containing 0.2 M potassium thiocyanate, 0.1 M Bis-Tris propane, pH 6.5, and 20% (wt/vol) polyethylene glycol (PEG) 3350 were mixed and incubated at 293K. For phasing, crystals were soaked with reservoir solution supplemented with 10 mM potassium tetrachloroplatinate(II)  and 200 nl of 0.2 M ammonium nitrate, 0.1 M Bis-Tris propane, pH 8.5, and 18% (vol/vol) PEG Smear High using a Mosquito robot (TTP Labtech). For phasing, crystals were soaked with the reservoir solution, supplemented with solid potassium iodide (KI) and 15% PEG 200 for 5 min. Crystals were cryoprotected with the reservoir solution, supplemented with 20% glycerol, and then flash-cooled in liquid nitrogen. The native data set was processed using xia2 , the autFor studies of the interaction of ComZ with PilA1-3, analysis was carried out on a HiLoad 16/600 Superdex 200 PG column . Prior to the chromatography, the target proteins were incubated together in 25 mM Tris-HCl, pH 8.0, 200 mM NaCl, 5% glycerol at 4°C for 30 min. Affinity tag binding assays used the principle that both ComZ and pilin proteins were expressed with different tags. The ComZ reading frame encoded a C-terminal hexahistidine tag, whereas PilA1, PilA2, and PilA3 incorporated a C-terminal Strep-tag II (WSHPQFEK). A HisTrap HP column was used to study the ComZ-PilA2-PilA3 interaction, and a StrepTrap HP column was used for interaction analysis of ComZ-PilA2 and ComZ-PilA3. Three mg of ComZ and 2 mg of each pilin were loaded in each case. Washing and elution from each affinity column was carried out under the same conditions as those used for affinity chromatography purification in the protein purification protocols described above.dK), stoichiometry (n), enthalpy (ΔH), and entropy (ΔS), were calculated using the manufacturer’s software by measuring heat absorbed or released  by titrating 20 2-μl aliquots of PilA2 (314 μM) into the sample cell containing 300 μl of ComZ (33 μM), with rapid stirring at 25°C. Thermodynamic parameters, i.e., binding constant (released .Electrophoretic mobility shift assays (EMSA) were carried out by mixing ComZ and linearized DNA plasmid (pET-22b predigested with BamHI), incubation at 4°C for 1 h, and separation by electrophoresis on a 1.5% agarose gel, supplemented with SafeView nucleic acid stain . Increasing concentrations of ComZ (0 μM to 44 μM) were added to a fixed quantity of linearized pET-22b (200 ng) in 25 mM Tris-HCl, pH 8.5, 100 mM NaCl in a final volume of 10 μl. After electrophoresis at a constant voltage (110 V) for 30 min, the gel was visualized and photographed under UV light using a UVpro transilluminator (UVItec). For the DNase protection assay, linearized pET-22b (200 ng) was added to 8.7 μM ComZ in 25 mM Tris-HCl, pH 8.5, 100 mM NaCl in a final volume of 10 μl and incubated at 55°C for 15 min. Samples were cooled to 37°C, DNase I was added to the specified concentration, and samples were incubated for a further 30 min at 37°C prior to separation by electrophoresis.Protection from reductive dimethylation was used to map surface-exposed lysine residues on ComZ, which bind DNA. The protocol was adapted from a method described by Rayment . Purifie−1, using a 75-mm by 250-μm-inner-diameter 1.7 μM CSH C18 analytical column (Waters). Peptides were selected for fragmentation automatically by data-dependent analysis. The data produced were then analyzed using Progenesis QI for Proteomics software (Waters) to align and quantify the peptide signals, which were then identified using Mascot software (Matrix Sciences) from the Swiss-Prot database of protein sequences (European Bioinformatics Institute) from January 2018, using oxidation on methionines and dimethylation on lysines as variable modifications. The signal intensities of peptides that were identified from ComZ were compared to determine the level of protection from dimethylation.For trypsin digestion, 500 μl of 10.5 μM purified ComZ was incubated at 90°C for 1 h in 20 mM ammonium bicarbonate and 10 mM DTT and cooled to 22°C, and iodoacetamide was added to a final concentration of 10 mM. The reaction mix was incubated in the dark at 22°C for 45 min before addition of trypsin at a mass ratio of 1:75 and incubated for 14 h at 37°C. Samples were desalted by reverse-phase, solid-phase extraction using Oligo R3 beads (Thermo Scientific) with elution in 40% acetonitrile in 0.1% formic acid, followed by evaporation via vacuum centrifugation and subsequent resuspension in 10 μl of 5% acetonitrile in 0.1% formic acid. Samples were analyzed by LC-tandem MS (LC-MS/MS) using an UltiMate 3000 rapid separation LC  coupled to an Orbitrap Elite  mass spectrometer. Peptide mixtures were separated using a gradient from 92% A (0.1% FA in water) and 8% B (0.1% FA in acetonitrile) to 33% B in 44 min at 300 nl min6QVI and 6QVF, respectively.Structure factors and atomic coordinates for ComZ and PilA2 have been deposited in the Protein Data Bank with accession codes


The surface morphology, functional groups, and elemental analysis were done to characterize the synthesized nanoparticles. The cytotoxicity performance of the synthesized nanoparticles was analyzed by exposing nanoparticles to MCF-7 and Hep-G2 cancer cell lines through MTT colorimetric assay and the IC50 value was defined as 100 μg/mL and 200 μg/mL, respectively. The antibacterial performance of synthesized nanoparticles against four different bacterial strains including Staphylococcus aureus, Corynebacterium, Pseudomonas aeruginosa, and Klebsiella pneumoniae were assessed through microdilution broth method. The synthesized Fe3O4 nanoparticles using Malva sylvestris demonstrated higher antibacterial effects against Gram-positive strains with MIC values of 62.5 μg/mL and 125 μg/mL which increase the inhibitory percentage to more than 90%.The biosynthesis of materials using medicinal plants can be a low-cost and eco-friendly approach due to their extraordinary properties. Herein, we reported a facile synthesis of Fe To validate the successful synthesis of Fe3O4 nanoparticles using Malva sylvestris, Fourier transform infrared spectroscopy (FTIR) was applied to determine the functional groups in the nanoparticles , Iron(III) Chloride Six hydrate (FeCl3.6H2O) and Ammonia solution were purchased from Merck Co. (Germany). First, 4.75 g FeCl3.6H2O, 3.89 g FeSO4.7H2O, and 320 mL deionized water were poured into a round-bottom flask and stirred for 1 h while the temperature was set to 80 °C [Malva sylvestris was ultrasonically mixed in 120 mL of deionized water for 30 min and then poured into the previous suspension. The obtained suspension was stirred under 80 °C for about 2 h and after that 40 mL NH3 was gradually added to the suspension [All of the materials utilized in this investigation like Ferrous(ІІ) Sulfate Heptahydrate FeSO4.7HO, Iron, scanning electron microscope , and EDX (Tescan model S Max detector Mira III).The synthesized nanoparticles were characterized using Fourier-transform infrared spectroscopy  in the region of 400–4000 cm2.33) were placed in each well of a sterile 96-well microplate and incubated in a humidified atmosphere of 5% CO2, 95% air at 37 °C to reach about 75–90% confluence. Then, 100 μL of the synthesized nanoparticles in a wide range of concentrations was replaced with previous media. Afterward, 25 μL of the MTT 3--2,5-diphenyltetrazolium stock solution (with 4 mg/mL concentration) was transferred into each well and incubated for 4 h in standard condition. The mitochondrial performance of viable cells led to formation of purple formazan crystals, where for dissolving these crystals we applied 100 μL dimethyl sulfoxide (DMSO). In the final step, the absorption of solution was recorded at 570 nm wavelength using a microplate reader .The cytotoxicity of the synthesized nanoparticles was evaluated on Hep-G2 and MCF-7 cell lines using MTT colorimetric assay. In this method, hydrogen peroxide was considered as the positive control and culture medium was the negative control. Briefly, a certain number of Hep-G2 and MCF-7 cells (10*102.40.5 McFarland (OD600: 0.1–0.2) in a way that the concentration of the compounds was 1000 μg/mL in the first wells. The plates were incubated for 24 h at 37 °C. Later the optical density was measured at 600 nm by a microplate reader . This procedure was done in triplicate.In MICs test, all of the procedures were carried out according to the standards of the Clinical and Laboratory Standards Institute (CLSI) for assessing the antibacterial susceptibility of the synthesized nanoparticles . Briefly2.55–106 CFU/mL were prepared for each one. The total of 90 μL of serially diluted concentrations of compounds (from 1000 μg/mL to 7.8 μg/mL) was added to a 96-well micro-plate consisting of 90 μg/mL BHI, then 10 μg/ml of bacteria were added to each cell. Micro-plates were incubated for 24 h at 37 °C. Then, 10 μL of each bacterial suspension was added to a newly prepared BHI and incubated for another 24 hours at 37 °C to exam bactericidal performance of each compound The lowest concentration of compounds that leads no growth of bacteria was regarded as minimum bactericidal concentration (MBC). This procedure was also repeated three times.All the selected microorganisms were cultured overnight in BHI, and then stocks with the concentration of 10


Mangifera indica L. cv. ‘Fahlun’) (Anacardiaceae) and its major phenolic principle  exhibited dose-dependent inhibitory effects on enzymatic activities of phospholipase A2 (PLA2), hyaluronidase and L-amino acid oxidase (LAAO) of Calloselasma rhodostoma (CR) and Naja naja kaouthia (NK) venoms by in vitro tests. The anti-hemorrhagic and anti-dermonecrotic activities of MSKE against both venoms were clearly supported by in vivo tests. Molecular docking studies indicated that the phenolic molecules of the MSKE could selectively bind to the active sites or their proximity, or modify conserved residues that are critical for the catalysis of PLA2, and selectively bind to the LAAO binding pocket of both CR and NK venoms and thereby inhibit their enzymatic activities. The results imply a potential use of MSKE against snake venoms.The ethanolic extract from seed kernels of Thai mango (MSKE) ( Naja naja kaouthia Lesson (NK)  causes the highest mortality due to snake envenomation in Thailand [Calloselasma rhodostoma Kuhl (CR)  causes the greatest incidence of envenomation in the country [Venomous snakebite is an important public health problem in remote areas of Thailand since the economic activities in these areas are mainly agricultural. Thailand  and Call country . Among t2 (PLA2), hyaluronidase and L-amino acid oxidase (LAAO) are found in most snake venoms and are deeply involved in snake envenomation by inducing local effects such as severe inflammatory reactions, hemorrhage and necrosis of local tissues [2s (EC 3.1.1.4) are enzymes that specifically hydrolyzes the sn-2 ester bond of 3-sn membrane glycerophospholipids, generating lysophospholipids and free fatty acids, which themselves cause considerable erythrocyte membrane lytic and damage [2s exhibit wide varieties of pharmacological effects such as neurotoxicity, cardiotoxicity, myotoxicity, as well as necrotic, anticoagulant, hypotensive, hemolytic, hemorrhagic and edematogenic [2s from snake venoms are classified into groups I or II, based on their sequences and modes of disulphide pairings. Group I PLA2s are found in the venom of Elapidae snakes, whereas group II PLA2s are found in the venom of Viperidae snakes. PLA2s from different snake venoms share a high degree of homology in the amino acid sequence and enzymatic active sites. They hydrolyze phospholipids using a His-Asp doublet plus a conserved water molecule as a nucleophile, and a Ca2+ ion as a cofactor [2O2) [2O2[et al. [2O2 formation. Recently, Pawelek et al. [Snake venoms are complex mixtures comprised mainly of proteins and peptides possessing a variety of biological activities. Venom proteins have many diverse enzymatic activities . Enzymes tissues . PLA2s  . This en2O2) [2O2. In addi2[et al.  suggestek et al.  demonstrMangifera indica L. cv. ‘Fahlun’, Anacardiaceae) has a relatively high phenolic content and is composed of 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (PGG) (61.28%), methyl gallate (MG) (0.68%) and gallic acid (GA) (0.44%) [2, hyaluronidase and LAAO of CR and NK venoms by in vitro tests. The inhibitory effects of MSKE on the activities of these enzymes were also observed by following in vivo tests on the anti-hemorrhagic and anti-dermonecrotic activities of CR and NK venoms, respectively. Molecular docking studies using the Gold v3.2 program were carried out to investigate the binding mode of the extract’s principles to the enzymes. Plant extracts constitute a rich source of bioactive compounds with a variety of pharmacological activities. Tannins from plants have been shown to interact with enzymes from snake venoms and act as an antidote ,14,15,16 (0.44%) . The ext (0.44%) ,18. The 2 assay, the results revealed that the enzyme from both venoms exhibited indirect hemolytic activity. This was probably due to the activity of PLA2s in the hydrolysis of lecithins to lysolecithins and lysis of the cell membranes of red blood cells [2s of CR and NK venoms. It can be seen that MSKE and its phenolic principles clearly showed a dose-dependent inhibitory activity against PLA2s of both venoms. The order of potency as judged by the half-effective dose (ED50) was PGG (25.6 ± 1.3 µg) > MG (42.9 ± 2.3 µg) > MSKE (44.4 ± 0.4 µg) > GA (66.0 ± 2.5 µg) for CR venom, and PGG (30.2 ± 2.4 µg) > MSKE (57.9 ± 0.5 µg) > MG (108.7 ± 2.0 µg) > GA (123.5 ± 2.5 µg) for NK venom (2s of both venoms by MSKE may result in the prevention of red blood cell rupture.CR (17.1 µg) and NK (3.4 µg) venoms produced clear zone diameters of 21.1 ± 0.3 mm and 20.6 ± 0.3 mm, respectively, on agarose-erythrocyte egg yolk gel plate and these clear zones were selected as the minimum indirect hemolytic dose for each venom. From the indirect PLAod cells . Figure NK venom . The ext50) was PGG (3.4 ± 2.4 µg) > MG (5.9 ± 2.4 µg) > MSKE (6.0 ± 1.9 µg) > GA (16.0 ± 2.5 µg) for CR venom, and PGG (2.6 ± 1.7 µg) > MSKE (5.0 ± 2.6 µg) > MG (8.6 ± 2.1 µg) > GA (14.8 ± 1.4 µg) for NK venom  inhibited the activity of hyaluronidase in CR and NK venoms in a dose-dependent manner . The ordNK venom . The inh50) was PGG (0.9 ± 2.4 µg) > MSKE (1.5 ± 3.2 µg) > GA (1.7 ± 3.4 µg) > MG (2.3 ± 2.4 µg) for CR venom, and PGG (2.2 ± 2.0 µg) > MSKE (4.0 ± 1.7 µg) > MG (4.4 ± 2.0 µg) > GA (4.5 ± 2.8 µg) for NK venom  inhibited the LAAO activity of both venoms in a dose-dependent manner . The ordNK venom . In thiss of H2O2. The inh2, hyaluronidase and LAAO activities of both venoms. These results indicated that the anti-enzymatic potency of MSKE may be attributed to its major principle (PGG) and other unidentified constituents, since PGG exerted its effect at the lowest ED50 value and had the highest percentage content (61.28%) within the MSKE compared with GA (0.44%) and MG (0.68%). It could be noted that although GA and MG inhibited these enzymatic activities of both venoms, their percentage content within the MSKE were very low. Therefore, GA and MG may only possess a negligible effect compared with PGG. It has been reported that other plant extracts also contain compounds that capable of neutralizing PLA2, hyaluronidase and LAAO activities of snake venoms [It is seen from e venoms ,21,22. TP < 0.05) diminished the diameters of hemorrhagic lesions induced by three MHDs of CR venom in a dose-dependent manner, and the hemorrhagic activity could be completely inhibited at the extract amount of 67 mg/kg mouse (P < 0.05). Solvent and the extract alone did not induce necrotic lesion in rats.The minimum hemorrhagic dose (MHD) of CR venom was determined to be 20.4 µg/mouse, while the venom at three MHDs-induced hemorrhagic lesions with a diameter of 13.4 ± 0.4 mm. The MSKE significantly  in the PLAK venoms . GoldSco changed ,23. Furtchelator  and it io-aminobenzoate [et al. [o-aminobenzoate; i.e. Leu51, Arg90, Asn208, His223, Asp224, Arg283, Gly313, Tyr372, Ile430 and Gly464. It can be seen from The possible molecular orientation of the MSKE constituents  in the LAAO binding pocket of CR venom is shown in benzoate . Pawelek [et al.  reportedin vivo inhibition of viper venom-induced hemorrhagic and cobra venom-induced dermonecrotic actions by MSKE is proposed. The inhibition was due firstly to a non-selective mechanism as a result of complexation between the venom proteins and tannins in the extract at the injection site [2 and LAAO) of CR and NK venoms were blocked by the phenolic principles of MSKE as demonstrated in the molecular modeling study  since PGG exerted its effect at the lowest value of ED50 with the highest GoldScores and had the highest percentage content within the MSKE compared with GA and MG.This study shows a beneficial relationship between the Male Swiss albino mice (25–30 g) and male Sprague-Dawley rats (250–300 g) were used for the assays of anti-hemorrhagic and anti-dermonecrotic activities, respectively. Experiments were carried out according to the guidelines set by Mahidol University Animal Care and Use Committee (PY-ACUC). Lyophilized CR and NK venoms were obtained from the Queen Saovabha Memorial Institute, Thai Red Cross Society, Bangkok, Thailand. o-Dianisidine (≥ 97%), horseradish peroxidase (987 U/mg solid), hyaluronic acid (≥ 95%) and L-leucine (≥ 98%) were purchased from Sigma Chemical . All reagents used in the in vitro and in vivo experiments were obtained from commercial sources and were of analytical grade.Gallic acid  and methyl gallate  were purchased from Fluka . Pentagalloylglucopyranose  was obtained from Endotherm GmbH (Germany). Mangifera indica L. cv. ‘Fahlun’) were purchased from a local market. A voucher specimen (R.B. 20007) was deposited at the Museum of Natural Medicine, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand. The ethanolic extract from fresh mango seed kernels was obtained by chopping and homogenizing the seed kernels in a blender, using hot ethanol (80 °C) as an extracting solvent at a ratio of seed kernels/solvent = 1:2 (w/v) for 10 min at room temperature (30 °C) prior to centrifugation at 2000 rpm for 15 min in a Hattich Roto magna®. The extraction was performed three times. The ethanolic extracts were filtered, combined and concentrated in a rotary evaporator (Büchi Rotavapor R-200) at 40 °C. The extract was defatted with hexane, evaporated under reduced pressure, and then freeze-dried to afford a crude mango seed kernel extract (MSKE) with a yield of 9.36% (w/w). Fully grown unripened Thai mango fruits (3/MeOH/EtOAc/ethyl methyl ketone (6:1.6:2:2) with five drops of formic acid as the mobile phase for GA and MG and CHCl3/EtOH/formic acid (3:5:1) for PGG. The developed TLC plates were scanned at 286 nm and the amount of each compound  in MSKE was calculated from the calibration curves [GA, MG and PGG were used as chemical markers and the calibration curves for each compound were obtained by densitometric scanning of different quantities of the chemical marker bands on developed chromatographic plates. An aliquot of the crude extract (MSKE)  was applied along with serial amounts of the chemical marker stock solution. The thin-layer chromatographic (TLC) plates were developed in a pre-saturated twin trough glass tank using CHCln curves .2 activity in CR and NK venoms was measured using an indirect hemolytic assay on agarose-erythrocyte-egg yolk gel plates to define the minimum indirect hemolytic dose (MIHD) [2 activity by MSKE and its isolated phenolic principles was determined against one MIHD of CR and NK venoms. Test solutions and the venoms (0.05 mL each) were pre-incubated for 1 h at 37 °C. After centrifugation at 10,000 rpm for 10 min, the supernatant (20 µL) was tested for PLA2 activity. The anti-PLA2 potential of the extract and its phenolic principles was presented as percent inhibition of the enzymatic activity versus dose, in which 100% inhibition should produce no clear zone. ED50, the effective dose that PLA2 activity of the venoms was reduced by 50%, was calculated. PLAe (MIHD) , the doset al. [50, the effective dose that hyaluronidase activity of the venoms was reduced by 50%, was calculated.Hyaluronidase activity was determined according to the methods of Ferrante  and Yinget al.  with slio-dianisidine. After incubation at 25 °C for 3 min, 0.1 mL CR or NK venom in PBS (1 mg/mL) was added, and an increase in absorbance at 436 nm (Shimadzu UV-VIS spectrophotometer UV-160A) was measured against blank. Enzymatic activity was calculated from the linear portion of the time–absorbance plot, where one unit of enzymatic activity was defined as the amount of enzyme that caused an increase of 0.001 absorbance units per minute. The percent inhibition of LAAO activity by MSKE and its isolated phenolic principles was evaluated by pre-incubating CR or NK venom  with test sample in sterile water for injection (SWI) (0.1 mL) for 1 h at 37 °C. After pre-incubation, the mixture was centrifuged at 10,000 rpm for 5 min, and supernatant (0.1 mL) was evaluated for LAAO activity. Venoms, test sample, and solvents alone were used as controls. The anti-LAAO potential of the extract and its phenolic principles was presented as percent inhibition of the enzymatic activity versus dose. ED50, the effective dose that LAAO activity of the venoms was reduced by 50%, was calculated.LAAO activity of CR and NK venoms was determined by using an enzyme-coupled assay . The asset al. [et al. [et al. [Hemorrhagic activity of CR venom was determined according to the method of Kondo et al. . The min [et al.  and Guti [et al. . Twenty et al. [The methods of Kondo et al.  and Theaet al.  were useO-galloyl-β-d-glucopyranose (PGG)  [2  from CR and NK venoms were built by homology modeling technique using Geno3D web server [2 structures from CR venom, the sequence of CRV-S1E6a [2 from venom of Agkistridon piscivorus piscivorus (PDB code 1vap) which has the 65% sequence identity. Homology modeling of amino acid sequences of PLA2 (CM-III) from NK venom [2 from Naja naja naja venom (PDB code 1a3d), which had a high sequence identity of 90%. Hydrogen atoms were added to all structures by using the SYBYL 7.2 program.Structures of gallic acid (GA), methyl gallate (MG) and 1,2,3,4,6-penta-se (PGG)  were conde 1f8r) . Proteinb server . Geno3D b server . For homRV-S1E6a  was baseNK venom  was base2 from CR and NK venoms. The radius of the binding site was set to 10 Å. For LAAO structure, the binding site was defined by using the center of mass of citrate which is in the active site. For both PLA2 structures, the possible binding sites were predicted by using PASS (Putative Active Sites with Spheres) program [2 structure [The molecular docking method was done using Gold version 3.2 program  to inves program . PASS idtructure , was useP < 0.05) between means were assessed by one-way ANOVA, followed by Tukey’s honesty significant difference test or Dunnett’s T3 test for multiple comparisons.Results were expressed as mean ± S.E.M. for two determinations. Statistical analysis was carried out using SPSS 13.0 for Windows. Significant differences (