Starting from isolated full-length zebrafish (Danio rerio) cDNA for slc7a1a, we performed comparative and phylogenetic sequence analysis, investigated the conservation of the gene during vertebrate evolution, and defined tissue expression during zebrafish development. Whole mount in situ hybridization first detected slc7a1a transcripts in somites, eyes, and brain at 14 h post-fertilization (hpf) with additional expression in the distal nephron at 24 hpf and in branchial arches at 3 days post-fertilization (dpf), with significant increase by 5 dpf. Taken together, the expression analysis of the zebrafish Cat-1 system gene slc7a1a suggests a functional role(s) during the early development of the central nervous system, muscle, gills, and kidney.Cationic amino acid transporter 1 (Cat-1 The online version of this article (10.1007/s10695-020-00873-x) contains supplementary material, which is available to authorized users. In mammals, the Cat protein group consists of Cat-1, Cat-2a, Cat-2b, and Cat-3, all exhibiting a nearly identical substrate specificity for cationic l-amino acids  regions, with intracellular N- and C-termini  located at the intracellular face of the third TM, which is recognized to be necessary for the transport activity of mouse Slc7a1  that are glycosylated in mouse Slc7a1  and caveolin in pulmonary artery endothelial cells  and arginase enzyme activities, which eventually modulate proliferation and differentiation of epidermal skin cells  and focused on its spatial expression during the early stages of development. Our findings revealed conservation of functionally important amino acids in the zebrafish slc7a1a sequence along with the conservation of synteny, which suggests a common biochemical scheme in basic amino acid transport processes across teleost fish membranes, and from teleost fish to mammals. Also, in situ hybridization revealed embryonic and larval stage specific slc7a1a expression in eyes, somites, distal nephrons, and branchial arches. Taken together, these results suggest that slc7a1a has embryonic stage and organ specific expression and is possibly important for normal embryonic development and function.In this context and with special regard to the elucidation of the role of basic amino acid transport processes in teleost systems and compartments, where at least to our knowledge no information is available to date, we isolated the full-length 2, 0.33 mM MgSO4).Zebrafish were maintained and bred at HIB, University of Bergen, as described elsewhere . Predicted transcripts used for slc7a1a isolation refer to Ensembl Transcript ID: ENSDART00000008248 (Ensembl Gene ID: ENSDARG00000016439). The cloned zebrafish slc7a1a was sequenced and the translated sequence used for sequence comparisons.The nucleotide and protein sequences described in this study were obtained from Ensembl . The neighbor-joining (NJ) method-based phylogenetic tree was built using MEGA X (http://www.megasoftware.net).The SLC7A1/Slc7a1-type amino acid sequences used for sequence comparison are in Table http://www.cbs.dtu.dk/services/TMHMM/), which is part of the Simple Modular Architecture Research Tool (SMART) (http://smart.embl-heidelberg.de/). Potential N-glycosylation and protein kinase C recognition sequences were identified using the PROSITE 19.7 computational tools (http://www.expasy.org/prosite/).Putative transmembrane domains were predicted using TMHMM 2.0  was evaluated by gene database consulting at the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/gene).Conservation of the zebrafish slc7a1a cDNA was cloned from total RNA retro-transcription followed by PCR amplification  was used to predict the structures of the zebrafish Slc7a1a proteins (isoforms X1 and X2) from their sequences ; probe length was verified by agarose gel and then dissolved in 150 μl hybridization buffer and stored at − 20 °C until use.Plasmid DNA was linearized with appropriate restriction endonucleases for 5 h at 37 °C, purified using QIAquick Nucleotide Removal Kit , and the degree of linearization was examined on a 1% agarose gel. In vitro transcription to produce digoxigenin (DIG)-labeled RNA probe was carried out combining linearized plasmid, 1 μg DIG labeling mix , 2 μl transcription buffer, 2 μl RNase inhibitor , 1 μl T7/Sp6 RNA polymerase (Roche), and 2 μl RNase-free ddHZebrafish embryos, collected at 24 hpf, 3 dpf, and 5 dpf, were dechorionated, anesthetized with tricaine, washed with E3, and fixed overnight in 4% paraformaldehyde (PFA) at 4 °C. Twelve-fourteen hours post-fertilization embryos were fixed in 4% PFA before dechorionation. Pigmentation was prevented using 0.003% PTU in E3 medium. Fixed embryos were placed in 100% methanol at − 20 °C until use.slc7a1 RNA probes were used, the former as negative control.Whole mount in situ hybridization was carried out as described previously . Figures were generated using Adobe CS2 Photoshop™ and Illustrator™.Zebrafish were maintained and experiments conducted in compliance with the Norwegian Animal Welfare Act guidelines. No ethical permission was needed. According to the EU Directive 2010/63/EU on the protection of animals used for scientific purposes, implemented in Norwegian legislation as of December 12, 2014, early life stages of zebrafish are not protected as animals until the stage of being capable of independent feeding, i.e., 5 days post fertilization (dpf).slc7a1-type genes are present in the zebrafish genome (GRCz11 Assembly)  and slc7a1b . Both genes produce “predicted” alternative splicing mRNA forms. In the case of slc7a1a, there are four predicted transcript variants in GenBank, indicated as X1 (Acc. N. XM_678531.7), X2 (Acc. N. XM_021479384.1), X3 (Acc. N. XM_005155277.4), and X4 (Acc. N. XR_002459600.1), and three predicted transcript variants in Ensembl, indicated as -201 (Transcript ID ENSDART00000008248.9), -202 (Transcript ID ENSDART00000146370.3), and -203 (Transcript ID ENSDART00000183510.1). These converge into two predicted alternative amino acid sequences in GenBank, indicated as isoforms X1  and X2 , and into three predicted alternative amino acid sequences in Ensembl, indicated as -201 (Protein ID ENSDARP00000027285.8), -202 (Protein ID ENSDARP00000121637.1), and -203 (Protein ID ENSDARP00000152628.1)  (Protein Data Bank Acc. No. 5OQT). However, only 12 transmembrane domains occurred in these models, with transmembrane domains 11 and 12 of GkApcT corresponding to transmembrane domains 13 and 14 of the zebrafish Slc7a1a proteins , two .1) Fig. . The cloids Fig. . Such hyPredicted Slc7a1-type amino acid sequences from zebrafish, Atlantic herring, medaka, fugu rubripes, tropical clawed frog, chicken, cattle, mouse, rhesus monkey, and human were aligned using Clustal Omega, and phylogenetic tree analysis was performed Fig. . The phy107 of mouse Slc7a1, which has been shown to be necessary for the transport activity  lays with muts2a within a syntenic region common to teleost fish, amphibians, birds and mammals  and carnitine production; also, it helps the body absorb calcium, and it is required for the formation of collagen, which is crucial for bones and connective tissues including skin, tendon, and cartilage  family of proteins, which consists of more than 400 organic and inorganic carriers  syntenic conservation of the zebrafish, and teleost fish, slc7a1/Slc7a1/SLC7A1 series of higher vertebrates.All together, these conserved features strongly support the authenticity of the predicted zebrafish Slc7a1a and put it in the orthologous line of the 107 residue within the third TM domain, our sequence analysis identified a special feature represented by a set of conserved negatively and positively charged amino acids  observable within a larger region encompassing the fourth intracellular loop in the zebrafish Slc7a1a isoform X1. This is substituted in isoform X2 by a similar stretch of amino acids but with differently charged amino acids in the parallel positions due to a splicing event involving exon 6a and exon 6b  to human SLC7A2 and mouse Slc7a2  is not conserved in the non-mammalian species. Downstream to this motif, there are several conserved basic residues along with two cysteines. In mouse, this conserved N-terminal motif along with the conserved basic residues of Slc7a1 has been shown to give a regulatory signal for transport into or retention inside of diverse cell membrane compartments  leading to defective proximal tubules and consequent pericardial edema  cells   Perry . VariousMarshall . Consideslc7a1a to be expressed in the distal tubule segment of the nephron from 24 hpf and onwards, while it was expressed in the gills from 3 dpf. There is compelling evidence in animals (fish included) that, unlike storage of excess lipids and carbohydrate, the excess of amino acids and proteins is usually metabolized to ammonia  and the Rh and UT (ammonia and urea transporter) in the amino acid metabolism. However, detailed co-localization and functional studies are essential to establish this link.It was originally considered that ammonia and urea move passively through tissues along partial pressure or concentration gradients. It is now proved that it requires Rhesus (Rh) proteins for ammonia transport


Mycobacterium abscessus. Fragment library screening, followed by structure-guided chemical elaboration of hits, led to the rapid development of drug-like molecules with potent in vitro TrmD inhibitory activity. Several of these compounds exhibit activity against planktonic M. abscessus and M. tuberculosis as well as against intracellular M. abscessus and M. leprae, indicating their potential as the basis for a novel class of broad-spectrum mycobacterial drugs.Translational frameshift errors are often deleterious to the synthesis of functional proteins and could therefore be promoted therapeutically to kill bacteria. TrmD (tRNA-(N(1)G37) methyltransferase) is an essential tRNA modification enzyme in bacteria that prevents +1 errors in the reading frame during protein translation and represents an attractive potential target for the development of new antibiotics. Here, we describe the application of a structure-guided fragment-based drug discovery approach to the design of a new class of inhibitors against TrmD in  Mycobacterium tuberculosis, responsible for over 1.5 million deaths per year from tuberculosis and Mycobacterium leprae, the causative agent of leprosy, both associated with increasing rates of acquired drug resistance , has recently emerged as a major threat to individuals with Cystic Fibrosis (CF) and other inflammatory lung conditions  in prokaryotic tRNAs  is present in tRNAs containing a G36G37 sequence in the anti-codon region from all three domains of life, where G37 is the base adjacent to the anticodon at the 3′ end  and 37 (3′ and adjacent to the anticodon) of tRNA ,7. TrmD,s Figure . This moe 3′ end ,9. MutatS-adenosyl-l-methionine (SAM)-dependent methyltransferases known as the SpoU-TrmD (SPOUT) RNA methyltransferase superfamily or Class IV methyltransferases. Proteins belonging to this family are structurally unique due to the absence of a consensus methyltransferase fold. TrmD and other proteins of the SPOUT family consist of a deep trefoil knot architecture at the catalytic region, which provides an L-shaped pocket for binding of SAM. In contrast, G37 methylation in eukaryotes is carried out by the enzyme Trm5 belonging to the Class I methyltransferase family  molecules. These fragments can then be chemically optimized into lead candidates, thereby more effectively exploring the chemical space available for binding to the target protein  is a promising approach for the development of new drugs, whereby the complexity of sampling chemical space is reduced by screening promiscuous small molecules (‘fragments’) for low affinity  protein . In thisEscherichia coli DH5α, used for cloning, was grown in LB Lennox  medium at 37°C. Mycobacterium abscessus ssp. massiliense CIP108297 was grown in Middlebrook 7H9-ADC broth  supplemented with 0.05% Tween 80 or 7H11-ADC agar  at 37°C. Kanamycin (Kan), streptomycin (Str) and hygromycin (Hyg) were added to final concentrations of 200, 200 and 2000 μg/ml, respectively. Homologous recombination at the trmD locus of M. abscessus ssp. massiliense CIP108297 was performed using a mycobacterial recombinase-based system in which the recombineering genes from mycobacteriophage Che9c (xylE-kan (a derivative of the pNitET-sacB-kan plasmid (sacB gene was replaced by the xylE colored marker) under control of an isovaleronitrile-inducible promoter. Isovaleronitrile-induced M. abscessus ssp. massiliense CIP108297 cells harboring pNitET-xylE-kan were electro-transformed with ∼300 ng of linear allelic exchange substrate consisting of the streptomycin-resistance cassette from pHP45Ω flanked by 1000 bp of DNA sequence immediately flanking the start and stop codons of trmD, and double-crossover mutants were isolated on Str-containing agar. Allelic replacement leading to the complete deletion of the trmD locus was checked by PCR using a pair of primers annealing outside the linear allelic exchange substrate.ge Che9c  are expr plasmid  generatetrmD gene from M. abscessus ssp. massiliense CIP108297 in the HindIII site of pMV306H. All primer sequences are shown in Plasmid pMV306H was constructed by replacing the kanamycin-resistance cassette of pMV306hsp   by a hygEscherichia coli BL21 (DE3) strain containing AVA0421 plasmid with an N-His-3C Protease site-TrmD full-length insert, kindly provided by the Seattle Structural Genomics Consortium, (A600 nm) reached 0.6. The expression of recombinant construct was induced by the addition of isopropyl β-d-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM and further allowed to grow at 18°C for 16 h.sortium,  was grow2, 1 mM TCEP, 20 mM imidazole). 0.1% Triton (Sigma), 10 μg/ml DNaseI, 5 mM MgCl2 and three protease inhibitor cocktail tablets (New England Biolabs) were added to the cell suspension. The cells were lysed in an Emulsiflex (Glen Creston) and clarified the lysate by centrifugation at 4°C for 40 min at 25 568 g.Cells were harvested by centrifugation at 4°C for 20 min at 5000 g and the pellet was re-suspended in buffer A  and subjected to dialysis against 2 l of buffer C  overnight at 4°C.The clarified lysate was filtered using a 0.45 μm syringe filter and passed through a pre-equilibrated (with buffer A), 10 ml pre-packed nickel- sepharose column . The column was washed with 5 column volumes of buffer A and the bound protein was eluted as 4 × 10 ml elutes using buffer B . The protein was analyzed on a 15% SDS-PAGE gel. Protein, after overnight dialysis and cleavage of N-His tag, was passed through a pre-equilibrated (buffer A) 5 ml HiTrap IMAC FF Nickel column .The flow through from the above column was concentrated to 3 ml using a 10 kDa centrifugal concentrator (Sartorius Stedim) and loaded onto a pre-equilibrated  120 ml Superdex200 16/600 column . 2 ml fractions were collected and analyzed on a 15% SDS-PAGE gel. Fractions corresponding to pure TrmD protein were pooled and concentrated to 25 mg/ml, flash frozen in liquid nitrogen and stored at –80°C. Identity of the purified protein was further confirmed by MALDI mass fingerprinting.M. abscessus TrmD apo crystals were grown in 48-well sitting drop plates (Swiss CDI) in the following condition: 0.08 mM Sodium cacodylate pH 5.8 to 6.8, 1–2 M ammonium sulphate. 24 mg/ml of the protein in storage buffer  at drop ratio 1 μl:1 μl (protein:reservoir respectively) were set up and equilibrated against 70 μl reservoir.Crystals for this experiment were grown at 19°C in 48-well sitting drop plates (Swiss CDI) in the following condition: 0.08 mM Sodium cacodylate pH 6.5 to 7.0, 1–2 M ammonium sulphate, 20 mg/ml of the protein in storage buffer  at drop ratio 1 μl:1 μl were set up and equilibrated against 250 μl reservoir. Further, the crystals were picked and allowed to soak in a 4 μl drop containing reservoir solution and 10 mM fragments/compound (in DMSO) which was then equilibrated against 700 μl of the corresponding reservoir solution overnight at 19°C in 24-well hanging drop vapor diffusion set up.2–5 mM final concentration of compound in DMSO/water was added to 20 mg/ml of TrmD protein, mixed and incubated for 2 h on ice. Crystals were grown in the following condition: 0.08 mM Sodium cacodylate pH 6.5 to 7.0, 1–2 M Ammonium sulphate or in sparse matrix screens: Wizard 1&2 (Molecular Dimensions), Wizard 3&4 (Molecular Dimensions), JCSG +Suite (Molecular Dimensions). The crystallization drops were set up at a protein to reservoir drop ratio of 0.3 μl:0.3 μl, in 96-well (MRC2) sitting drop plate, using mosquito crystallization robot (TTP labtech) and the drops were equilibrated against 70 μl of reservoir at 19°C.P212121 and consisted of two protomers in the asymmetric unit.The TrmD apo/ligand-bound crystals were cryo-cooled in mother liquor containing 27.5% ethylene glycol. X-ray data sets were collected on I04, I02, I03, I04-1 or I24 beamlines at the Diamond Light Source in the UK, using the rotation method at wavelength of 0.979 Å, Omega start: 0°, Omega Oscillation: 0.1–0.2°, Total oscillation: 210–240°, total images: 2100–2400, Exposure time: 0.05–0.08 s. The diffraction images were processed using AutoPROC , utiliziM. abscessus TrmD Apo structure was solved by molecular replacement using PHASER  as search model and TrmD ligand bound structures were solved by molecular replacement with the atomic coordinates of the solved M. abscessus TrmD Apo structure (PDB entry: 6NVR) as search model. Structure refinement was carried out using REFMAC , 5 mM compound, 5% DMSO and 5× Sypro orange dye. Appropriate positive  and negative  controls were also included. The measurements were performed in a Biorad-CFX connect thermal cycler using the following program: 25°C for 10 min followed by a linear increment of 0.5°C every 30 s to reach a final temperature of 95°C. The results were analyzed using Microsoft excel.M. abscessus TrmD: 50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol; M. tuberculosis TrmD: 25 mM HEPES pH 7.5, 500 mM NaCl). Sample cell and syringe solutions were prepared using the same storage buffer, with a final DMSO concentration of 2–10% according to ligand solubility in the buffer. TrmD concentrations of either 33 or 100 μM were used, with ligand to protein concentration ratios ranging from 10 to 20:1. Control titrations without protein were also performed and subtracted from ligand to protein titrations. Titrations were fitted with Origin software , using a one-site binding model with N fixed to 1 only for weakly binding ligands. Titrations were typically performed once (n = 1), with multiple isotherms obtained (n > 1) for key compounds of interest. Kd values are reported to two significant figures. Error provided by Origin software due to model fit is reported when n = 1, whereas standard deviation is reported when n > 1.ITC experiments to quantify binding of ligands to TrmD were done as described in  using MaAW1-7 were synthesised according to the procedures described in the supporting information. A more in-depth discussion around the medicinal chemistry strategy for fragment merging and lead compound development is described in a corresponding publication by Whitehouse et al. (AW1-7 are listed in this publication as follows: AW1 (Compound 23), AW2 (Compound 24f), AW3 (Compound 26f), AW4 (Compound 28), AW5 (Compound 29a), AW6 (Compound 31a) and AW7 (Compound 29d).The compounds e et al. . The comProUGG in the presence of 0–500 μM compounds in serial dilutions using assay buffer containing 50 mM Tris–HCl pH 7.5, 10 mM MgCl2, 24 mM NH4Cl, 5% DMSO and 1 mM DTT in nuclease free water. tRNA sequences were identified from the M. abscessus genome sequence using tRNAscan-SE algorithm, . The reactions were carried out for 1 h at room temperature followed by addition of 20 mM EDTA to stop the reactions. Each of the 20 μl samples were diluted ten-fold with the UPLC mobile phase solvent A (0.1% formic acid in water), centrifuged for 10 min at 13 000 g, to remove any precipitates, and the supernatant was aliquoted into 96-well plates. 40 μl samples were then injected into Acquity UPLC (Waters) T3 1.8 μM column and eluted using a gradient elution consisting of Mobile Phase A: 0.1% formic acid in water and mobile phase B: 0.1% formic acid in 100% methanol for 4 min. The absorbance was monitored using a photodiode array (PDA) detector (Waters) at wavelength range of ƛ: 220–500 nm. All reactions were carried out in triplicate. The blank corrected data were analyzed using Microsoft excel and non-linear regression analysis for IC50 determination were done using GraphPad prism version 7.00, GraphPad Software, La Jolla, CA, USA.Assays for quantifying TrmD methylation reactions were carried out in 20 μl reactions consisting of 6.25 μM SAM, 0.1 μM TrmD and 6.25 μM tRNAgorithm, ,39. The Mycobacterium abscessus ssp. abscessus (ATCC 19977) transformed with pmv310 plasmid expressing Lux ABDCE operon, grown in Middlebrook 7H9 broth supplemented with ADC . All the other NTM strains are clinical isolates. Minimum inhibitory concentrations (MIC) were determined for mycobacteria according to the Clinical and Laboratory Standards Institute (CLSI) method M07-A9. Briefly, mycobacteria were grown to optical density (A600 nm) of 0.2–0.3 in liquid culture and 1 × 105 bacteria were added to each well of 96-well plates containing serial dilutions of compound , in triplicate wells per condition, and incubated at 37°C until growth was seen in the control wells. MIC measurements using M. tuberculosis H37Rv were performed as reported in , 0.81 g/l NaCl, 0.3 g/l casitone, and 0.05% Tyloxapol. H37Rv was grown and diluted to a similar inoculum size as mentioned above prior to exposure to serial dilutions of compounds (starting at 100 μM), and the plates were incubated at 37°C for 2 weeks. The MIC value was determined as the last well which showed no bacterial growth.et al. (M. abscessus ATCC19977. The 20 nucleotides guides targeting yidC(MAB_4953c) and trmD(MAB_3226c) were annealed and cloned in between sphI and aclI of the pGRNAz. As a control the pGRNAz was left empty. The CRISPr-I containing strains were cultivated in Middlebrook 7H9 broth supplemented with 1× ADC, 0.05% tween80 and 0.5% glycerol, hygromycin 1 mg/ml and zeocin 300 μg/ml. The cultures were inoculated at 10^6CFU/ml from an exponentially grown pre-cultures. The AW7 compound and doxycycline were added or not at a concentration of 25μM and 1.5625, 6.25, 25 or 100 ng/ml respectively. The OD600 was measured and the CFU counted at 72 h.The dCas9 encoding plasmid (pTetInt-dcas9-Km) and the second vector containing the sgRNA cassette (pGRNAz) were derived from the tetracycline inducible CRISPr Interference system of Choudhary et al.  and opti+ positive selection using the MACS Miltenyi Biotec Human CD14 microbead protocol (Miltenyi Biotec). CD14+ cells were differentiated into macrophages using recombinant human granulocyte-macrophage colony-stimulating factor (200 ng/ml GM-CSF) and recombinant human interferon gamma (50 ng/ml IFNγ) (Peprotech) in standard tissue culture DMEM media containing fetal calf serum, penicillin and streptomycin. Following removal of antibiotics, macrophages were infected at a multiplicity of infection of 10:1 with M. abscessus 19977 for 2 h, washed in sterile phosphate buffered saline, and then incubated in DMEM media with FCS and 25 μM of compound for 24 and 48 h. At the given time points, supernatant was saved for cell cytotoxicity studies, and M. abscessus survival within the macrophages calculated by macrophage lysis in sterile water, and colony forming unit calculation on Columbia Blood Agar plates (VWR BDH).Blood samples were donated by healthy volunteers who had undertaken informed consent in accordance with local Research Ethics Committee approval. Peripheral blood mononuclear cells were isolated from citrated peripheral blood samples by density gradient separation using Lympholyte (Cedarlane Labs), and subsequent CD14Lactate dehydrogenase (LDH) was measured as a biomarker for cellular cytotoxicity using the Pierce LDH Cytotoxicity Assay Kit. Cell supernatant was measured at 2, 24 and 48 h post-infection according to the kit protocol.Mycobacterium leprae (isolate Thai-53) was maintained in serial passage in the foot pads of athymic nude mice . Mice were inoculated in the plantar surface of both hind feet with 5 × 107 fresh, viable nude mice derived M. leprae. When the mouse foot pads became moderately enlarged (at ∼5–6 months), they were harvested for intracellular M. leprae as described previously  and were incubated for 7 days at 33°C. Media only and rifampin  at 2.4 μM were used as negative and positive controls. Following incubation aliquots of AW7 treated and control M. leprae were processed for radiorespirometry (RR) as described previously (Freshly harvested nude mouse foot pad derived eviously .2. The cells were infected with freshly harvested nude mice foot pad derived live M. leprae at a multiplicity of infection (MOI) of 20:1 overnight at 33°C and then washed to remove extracellular bacteria. AW7 was added at different concentrations (100–6.25 μM) and the cells were incubated for 7 days at 33°C. Media only and rifampicin at 2.4 μM were used as negative and positive controls. AW7 treated and control cells were lysed with sodium dodecyl sulfate  and the intracellular M. leprae processed for radiorespirometry  supplemented with 10% (v/v) fetal calf serum (Life Technologies), 25 mM/l HEPES , 2 mM/l glutamine , 50 μg/ml ampicillin  and 10 ng/ml of recombinant murine macrophages colony stimulating factor  for 6–7 days at 37°C and 5% COirometry .M. leprae was measured by evaluating the oxidation of 14C-palmitic acid to 14CO2 by radiorespirometry as described previously  were recorded and percentage inhibition of metabolism determined as compared to no drug control. Statistical significance between treatment groups and no drug control were determined by Student's t-test and P < 0.05 is considered as significant.Metabolism of a suspension of eviously . Levels trmD is essential in M. tuberculosis , as well as in complex with SAM and S-adenosyl-l-homocysteine (SAH) at 1.67 and 1.48 Å resolution respectively (PDB codes: 6NW6 and 6NW7). Data collection and crystallographic statistics are given in P212121 and consist of a homodimer in the asymmetric unit. Each non-crystallographic 2-fold symmetry-related protomer of TrmD interacts in an antiparallel manner and consists of two domains: a larger N-terminal domain spanning residues 1–161 and a smaller C-terminal helical domain (177–242) connected by a flexible inter-domain linker. The two domains of the individual protomers do not contact each other and the inter-domain region is largely disordered, with residues 162–177 not clearly visible in the apo structure  of the ribose ring forms a hydrogen bond with the backbone amide of Gly109. The methionine and homocysteine moieties further extend into the active site groove formed between the cover and wall loops, making further hydrogen-bonding interactions with water molecules in this region Figure .A structural superposition of the apo and SAM bound forms of TrmD reveals the wall loop undergoing a switch in conformation leading to a movement of about 5 Å, when measured at the Cα of Tyr111, to the outer edge of subunit A. This conformational flip of the wall loop from apo form to SAM-bound form and the subsequent change in positions of residues 110–113 help to accommodate the methionine moiety of the methyl donor Figure  and B.M. abscessus TrmD catalytic site, we initiated a structure-guided FBDD effort targeting M. abscessus TrmD by screening an in-house library of 960 small molecule fragments. The preliminary screening was performed using differential scanning fluorimetry (DSF), resulting in 53 hits within a thermal shift cut-off value of 3 standard deviations from the negative control (the TrmD protein and DMSO in the absence of ligand). These hits were then selected for validation by X-ray crystallography. Apo crystals of M. abscessus TrmD were soaked with each of the 53 fragments in independent experiments. The resulting crystal structure determinations allowed characterization of the binding modes of 27 fragments  and 24 , were chosen for further chemical development by a fragment-merging strategy. The choice of fragments for subsequent chemical optimization was based on a number of criteria, including binding affinity, ligand efficiency, synthetic tractability and their ability to make key binding interactions at the TrmD SAM binding site. Fragment 23 occupies the adenosine binding region of the TrmD AdoMet site, with its pyrazole ring making hydrogen bond contacts to the backbone amides of Tyr136 and Leu138 and the amino group making further hydrogen bonds with the backbone carbonyl oxygen of Gly134 and the side chain of Ser132, respectively. The 4-methoxyphenyl ring of the fragment extends into the ribose binding site, engaging hydrophobic and π-interactions with the residues of the cover loop , formed by merging the two fragments, adopts a similar conformation to that of the original fragments in the TrmD SAM site, as shown in Figures The indole ring of Fragment 2 Figure . CompounTo aid the structure-guided lead discovery, we examined the ligand binding propensities of TrmD using hotspot mapping, a computational approach developed by Randoux, Blundell, and colleagues . HotspotAW1 satisfy many of the predicted protein-hotspot interactions, the map also suggested further potential interactions that could stabilise elaborated fragments. As shown in Figure AW1 occupies Hotspot 1 at the base of the TrmD active site, where it satisfies the hydrogen-bond donor requirements by interacting with the backbone amide oxygen atoms of Gly134 and Tyr136. The merged compound AW1 also orients its pyrazole nitrogen atom in the acceptor map in this region where it forms a hydrogen bond with the backbone NH of Leu138  was performed, initially utilizing the indole nitrogen as a vector for growth. While the detailed medicinal chemistry strategy of all the compounds and intermediates are described separately   Table . The met0 Figure .AW2 was further improved by the addition of a nitrile group on the 4-position of its pyrazole ring, extending into the narrow space between residues 83PTP85 of the cover loop and 131VSI133 of the bottom loop respectively  was a significant improvement on AW2, with the addition of two heavy atoms affording a 25-fold decrease in Kd (12–0.50 μM), increasing the ligand efficiency to the level of the original merged compound AW1 (0.36), and a 100-fold decrease in IC50 (33 to 0.31 μM)  Table . The X-r3 Figure .AW3 through the attachment of a pyrrolidinyl ring via another methylene linker, with AW4  affording an additional 5-fold improvement in affinity  was tolerated with a >3-fold improvement in affinity (92–27 nM) and an increase in ligand efficiency (0.32–0.34)   showed a slight worsening of both affinity and IC50  Table . The X-rn Figure . The remM) Table , had a dp Figure . Explora50 Table , possibl4 Figure .M. abscessus and M. tuberculosis  and AW7 (Kd 0.33 μM)  compounds in later stages of development showed promising activity against 0.33 μM)  were in ia Table . Given tia Table , the varia Table ,55.AW6 and AW7 using lactate dehydrogenase (LDH) release assay. It was observed that at or below a concentration of 150 μM, neither compound caused cellular toxicity on human macrophages  or an empty vector control  was further tested against M. leprae maintained intracellularly in murine bone marrow macrophage. As a proxy for mycobacterial viability, relative inhibition of β-oxidation rates were measured using a radiorespirometry assay to track the metabolism of 14C-palmitic acid to 14CO2 after 7 days of incubation  and elongation (actinomycin)  or nomycin) , to whicFragments are powerful chemical tools that have huge capacity to explore the chemical space of proteins  with sevM. abscessus TrmD compounds developed in this study, not only showed potent affinity toward M. tuberculosis TrmD but also were active against several other mycobacteria, including M. leprae. While we succeeded in developing potent TrmD inhibitors, the disconnect observed between compound affinity in vitro and bacterial MIC for some of these molecules, will require further iterative efforts to optimize permeability, retention and/or intra-bacterial metabolic stability of the compounds. It will be also interesting to probe the effect of the TrmD inhibitors developed in this study on membrane protein and efflux pump biosynthesis in mycobacteria, as previously observed in gram-negative bacteria (Mycobacteria are a notoriously difficult group of microorganisms to develop new drugs against, due to their impermeable cell wall, efflux pumps, target modification enzymes, and extensive capacity to metabolise compounds . TrmD isbacteria . Such anCoordinates and structure factors related to this work been deposited in the PDB with accession numbers 6NVR, 6NW6, 6NW7, 6QO2, 6QO3,6QO4, 6QO6, 6QOA, 6QOC, 6QOD, 6QOE, 6QOF, 6QOG, 6QOH, 6QOI, 6QOJ, 6QOK, 6QOL, 6QOM, 6QON, 6QOO, 6QOP, 6QOQ, 6QOR, 6QOS, 6QOT, 6QOU, 6QOV, 6QOW, 6QOX, 6QQS, 6QQX, 6QQY, 6QR6, 6QR5 and 6QR8.gkaa539_Supplemental_FileClick here for additional data file.


We report thermal, X-ray diffraction (XRD) and cytotoxicity studies of complexes of fluconazole (FCZ) with Cu (II), Fe(II), Cd(II), Co(II), Ni(II), and Mn(II). From XRD measurements, FCZ and its metal complexes were identified as polycrystalline. Marked differences in the X-ray patterns of drug and its metal complexes revealed that the complexes are indeed different compounds and not just the mixture of the starting materials. Unlike pristine FCZ, which did not exhibit cytotoxicity, three complexes derived from Fe(II), Cu(II) and Co (II) proved to be effective in the cytotoxicity assay. The Cu(II)-FCZ exhibited significant activity against SNB-19, HCT-15, COLO-205, and KB-3-1 cell lines, while Fe(II)-FCZ and Co(II)-FCZ were found cytotoxic only to KB-3-1 cell line. For the pure FCZ, thermogravimetry revealed massive weight loss in the temperature range of 215 to 297 °C, due to the volatilization of FCZ. All the complexes followed multi-stage degradation profiles, eventually resulting in the formation of metal oxides. For pure FCZ, differential scanning calorimetry revealed melting point at 137 °C, followed by two further endothermic transitions at 294 °C and 498.44 °C representing the volatilization and subsequent degradation of FCZ, respectively. The absence of endothermic FCZ melting peak at around 137 °C indicates that the complexes represent different compounds. All complexes exhibit endothermic transitions at around 240-300 °C, representing melting and removal of ligand moiety, followed by another endothermic transition at around 498-499 °C, representing the ligand decomposition. Over the last few decades, growing resistance of microorganisms against existing drugs have been a significant concern for researchers as it poses a considerable burden on the health departments of nations around the world -3. SubstCandida lusitaniae. For patients suffering from candidial osteomyelitis who are unable to start or complete the required course of amphotericin B, FCZ is a reasonable alternative and has been successful in achieving desired treatment results  remains the most widely explored owing to many advantages it offers , 14. Due results , 30.Scedosporium, Fusarium, and Mucorales has also been reported  sample was kindly provided by NabiQasim Pharma (PVT) Ltd, Karachi, Pakistan. Hydrated chlorides of copper, iron, manganese, cadmium, cobalt, and nickel  were all used as received. Methanol  was used as received. For all experiments, freshly distilled water was used.Synthesis of the ComplexesA series of metal complexes of FCZ with Co, Mn, Fe, Ni, Cu, and Cd were synthesized. Details of the synthesis procedure and characterizations of the resulting complexes have already been described in our previous paper . A generCharacterizationX-ray diffraction studies were performed to explore the crystallinity of fluconazole and its metal complexes using a Bruker AXS D8 (Germany) X-Ray diffractometer operated at 40 kV and 60 mA and equipped with a Cu-Kα1 radiation (1.54 Å). The data were recorded at a scanning speed of 5◦/min in the range of 10° < 2θ < 60° using a step size of 0.02°/point. All samples were analyzed as dry powder.-1. The parameters, such as onset temperature and peak temperature, were recorded from heating scans. Thermogravimetric analysis (TGA) was performed on a TA Instruments STD Q600 system using approximately 10 to 12 mg samples placed in crucibles. The samples were heated from room temperature to 700 °C at a heating rate of 10 °C min-1 in nitrogen atmosphere.Thermal properties of complexes were studied using differential scanning calorimetry (DSC) performed on a TA Instruments STD Q600 system under nitrogen atmosphere (50 kPa pressure). For all measurements, around 10 to 12 mg of powder samples, enclosed in DSC pans, were heated from room temperature to 700 °C at a scan rate of 10 °C min2.Four cell lines including human astrocytoma SNB-19, human Dukes’ type C colorectal adenocarcinoma HCT-15, human Dukes’ type D colorectal adenocarcinoma COLO-205, and human epidermoid carcinoma KB-3-1 were obtained from the American Type Culture Collection . The cell lines were grown in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin in a humidified incubator at 37 °C with 5% CO50 values (50% inhibitory concentration) were calculated.MTT  assay was used to evaluate the cytotoxicity of the test compounds. The cells were inoculated in 96-well plates at the density of 5,000 cells per well and allowed to adhere and grow for 24 h. The test compounds in different concentration were incubated with the cells for 72 h. After drug incubation, 4 mg/mL MTT solution was added to the cells by 20 μL per well followed by a four-hour incubation. The cell viability was measured by reduction of the yellow dye MTT to a blue formazan product that were dissolved in DMSO . The absX-Ray Diffractionet al., although there are few more spikes, which are likely due to the presence of more than one polymorphic form -FCZ, Cu(II)-FCZ, Fe(II)-FCZ, Mn(II)-FCZ, and Ni(II)-FCZ are depicted in Thermal Stability-1. The resulting thermograms are shown in Thermal stabilities of FCZ and its metal complexes were explored using thermogravimetric analysis (TGA) which was performed on powder samples under inert atmosphere employing a heating rate of 10 °C minet al. is attributed to the volatilization of molecular FCZ The DSC patterns recorded for FCZ and its complexes are shown in Anticancer Activity50 values of the FCZ and its metal complexes on the human cancer cells used were summarized in 50 values (mean ± standard deviation) of 60.10 ±7.85 μM, and 60.90 ± 4.58 µM, respectively. Also, only Cu(II)-FCZ had mild cytotoxicity on SNB-19 cells, but with a relatively lower IC50 value of 27.80 ± 4.16 µM. KB-3-1 cells exhibited higher sensitivity to the drugs than the other three cell lines. Among the seven compounds, Fe(II)-FCZ, Cu(II)-FCZ, and Co(II)-FCZ had IC50 values lower than 100 µM on KB-3-1 cell line, which were 81.33 ± 11.35 µM, 13.04 ± 5.72 µM, and 62.03 ± 19.84 µM, respectively -salen-like complex displayed remarkable anticancer activity against human chronic myelogenouserythroleukemia cell line and breast adenocarcinoma cell line, and further mechanistic studies supported that the resulting cancer cell apoptosis was probably led by certain superoxide dismutase (SOD) mimic activity and the subsequent local imbalance in superoxide/hydrogen peroxide levels. HoweverClinical and commercial importance of fluconazole (FCZ) has become an inspiration for researchers to explore new strategies and formulations to improve its growing ineffectiveness as antifungal. Complexation of drugs with metal ions is a well-established approach in medicinal chemistry to improve the efficacy of various drugs. The present contribution was intended to explore six complexes of FCZ with Cu (II), Fe(II), Cd(II), Co(II), Ni(II), and Mn(II) for their thermal, XRD, and cytotoxicity properties.Our results revealed that pure FCZ and its metal complexes were of polycrystalline nature. Contrary to the pure FCZ, three complexes demonstrated cytotoxicity against four human cancer cell lines used in the cytotoxicity assay. The Cu (II)-FCZ complex had cytotoxic activity against all four cancer cells, while Fe (II) and Co (II) complexes of FCZ showed some cytotoxic activity against KB-3-1 cancer cell, which implied the important role of metal complexes in anticancer activity. In case of pure FCZ, thermogravimetry revealed massive weight loss in the temperature range of 215 to 297 °C, due to the FCZ volatilization. The complexes; however followed multi stage degradation profiles, eventually resulting in the formation of metal oxides. From differential scanning calorimetry, the melting transition for pure FCZ was identified at 137 °C. Remarkably, this transition was not observed for all the six complexes indicating that these complexes represent definite compounds. Additional endothermic transitions for pure FCZ and its metal complexes were also observed.Although not all metal complexes showed effectiveness, the obtained results indicate activity of Cu (II)-FCZ, Fe (II)-FCZ, and Co (II)-FCZ for the chosen strains. It can be said that the FCZ metal complexes embody a starting point towards the development and optimization of more effective anticancer and antifungal drugs and will likely lead to new direction for studies of novel antifungal formulations. More studies on evaluation of these metal complexes and further preparation of different derivatives are needed.


Although RSV and Q increased pgc1α expression, they did not significantly change either glucose oxidation or β-oxidation. Q increased AMPK, insulin receptor substrate 1 (IRS-1), and AS160 phosphorylation in basal conditions and glycogen synthase kinase 3 (GSK3β) in insulin-stimulated conditions. RSV tended to increase the phosphorylation rates of AMPK and GSK3β. Both of the polyphenols increased insulin-stimulated glycogen synthesis and reduced lactate production in human myotubes. Thus, physiological doses of RSV or Q may exhibit anti-diabetic actions in human myotubes.Phenolic compounds have emerged in recent years as an option to face insulin resistance and diabetes. The central aim of this study was: (1) to demonstrate that physiological doses of resveratrol (RSV) or quercetin (Q) can influence glucose metabolism in human myotubes, (2) to establish whether AMP-activated protein kinase (AMPK) and protein kinase B –PKB- (Akt) pathways are involved in this effect. In addition, the effects of these polyphenols on mitochondrial biogenesis and fatty acid oxidation were analysed. Myotubes from healthy donors were cultured for 24 h with either 0.1 μM of RSV or with 10 μM of Q. Glucose metabolism, such as glycogen synthesis, glucose oxidation, and lactate production, were measured with D[U- Insulin resistance and diabetes currently represent pandemic diseases at a global level. In 2016, diabetes caused an estimated 1.6 million deaths, and high blood glucose levels were responsible for 2.2 million deaths glucose (1μCi/mL) with or without 100 mM of insulin. Following incubation, the cells were solubilised in KOH 30% and glycogen synthesis was determined, as previously described glucose (1 μCi/mL) in the presence or absence of 100 mM of insulin. Following incubation, glucose oxidation was determined by counting the 14CO2 released into the culture medium. All of the assays were performed in duplicate, and the data were normalized to cell protein content, as previously described palmitate  and non-labelled (cold) palmitate, without glucose. Palmitate was coupled to a fatty acid (FA)–free BSA in a molar ratio of 5:1. After incubation, 14CO2 and 14C-ASM were measured, as previously described [Myotubes were pre-incubated for three hours with [1–escribed . All of After cell treatment, aliquots of the incubation media were removed and analysed for lactate quantification using a commercial kit and following the manufacturer´s instructions. The results were expressed as mmol/L.pgc-1α, tfam, . Hk2, pkm and ldha were measured by TaqMan® Gene Expression Assays  in MyiQ™ Single-Color Real-Time PCR Detection System . The Rplp0 mRNA levels were similarly measured and served as the reference gene. The amplification reaction was performed in duplicate on 0.67 µL of cDNA and the amplification parameters were, as follows: 50 °C for two minutes, 95 °C for 10 min, 40 cycles of 95 °C for 15 s, and 60 °C for one minute. All fo the sample mRNA levels were normalized to Rplp0 values and the data were expressed as relative fold changes of threshold cycle (Ct) value relative to controls using the 2−ΔΔCt method [The RNA samples from treated cells were extracted while using RNeasy mini kit  following the manufacturer´s instructions. The integrity of the RNA extracted from all of the samples was verified and quantified using a NanoDrop ND-1000 spectrophotometer . 1 μg of total RNA of each sample was reverse-transcribed to first-strand complementary DNA (cDNA) using the MultiScribe reverse transcriptase method  on a GeneAmpPCRSystem 9700 . Relative t method .® TGX™ Precast Gels , electroblotted onto PVDF membranes , and immunodetected with ChemiDoc MP imaging system  while using the following primary antibodies: GLUT4 , Ser9 pGSK-3β, Thr172pAMPK, Ser473 pAkt , Tyr-989 pIRS-1 , and Thr642 pAS160 . Histone H3  served as an internal control, with the exception of pAkt, where the internal control was GAPDH . The bound antibodies were visualized by an ECL system  and quantified using Chemi-Doc MP imaging system .The myotubes were incubated for 20 min in DMEM-low glucose-Glutamax in the presence or absence of 100 nM insulin. Afterwards, the cells were harvested in a RIPA buffer  complemented with 10 µL/mL protease inhibitor, 10 µL/mL phosphatase I inhibitor and 10 µL/mL phosphatase II inhibitor. Afterwards, the protein concentration was determined by BCA reagent . 20 µg of total protein were run on 4–15% Mini-PROTEANt test. Statistical significance was represented, as follows: * p < 0.05, ** p < 0.01; *** p < 0.001The results are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed using SPSS v. 26.0 . The analysed variables were normally distributed, according to Shapiro–Wilk´s test. Because our interest lay in determining the effectiveness of each phenolic compound and not in comparing the effects among them, comparisons between the cells treated with each compound and the control cells were made using Student’s It can be concluded that, under our experimental conditions, neither RSV nor Q modify glucose uptake in primary myotubes, at physiological doses. Conversely, both are able to enhance glycogen synthesis and reduce lactate content, two effects that could represent a beneficial effect for glucose homeostasis and exercise endurance.


N,O-acetals derived from 2-amino-1,4-naphthoquinone  against methicillin-resistant Staphylococcus aureus (MRSA). The derivatives 7b and 7c, specially 7b, caused strong impact on biofilm accumulation. This inhibition was linked to decreased expression of the genes fnbA, spa, hla and psmα3. More importantly, this downregulation was paralleled by the modulation of global virulence regulators. The substitution of 2-ethoxymethyl (7b) in comparison with 2-propynyloxymethyl (7c) enhanced sarA-agr inhibition, decreased fnbA transcripts (positively regulated by sarA) and strongly impaired biofilm accumulation. Indeed, 7b triggered intensive autolysis and was able to eliminate vancomycin-persistent cells. Consequently, 7b is a promising molecule displaying not only antimicrobial effects, but also antibiofilm and antipersistence activities. Therefore, 7b is a good candidate for further studies involving the development of novel and more rational antimicrobials able to act in chronic and recalcitrant infections, associated with biofilm formation.Despite the existing antibiotics, antimicrobial resistance is a major challenge. Consequently, the development of new drugs remains in great demand. Quinones is part of a broad group of molecules that present antibacterial activity besides other biological properties. The main purpose of this study was to evaluate the antibiofilm activities of synthetic  Staphylococcus aureus can cause pathological conditions varying from skin/soft tissue infections to more severe diseases, such as pneumonia, bloodstream infections (BSI), osteomyelitis, and endocarditis. These bacteria are a major cause of both hospital-associated and community-acquired infections worldwide1. The S. aureus genomic plasticity has facilitated its accelerated evolution as the consequence of the lateral acquisition of new traits, including antimicrobial resistance genes2.Despite being part of the human microbiota, S. aureus (MRSA) was reported in 1961, in England3. After that, MRSA rapidly spread throughout the world, becoming one of the main hospital-associated pathogens1. The pathogenicity of S. aureus infections is multifactorial and directly related to the production of a plethora of virulence factors and their interactions with the host4. The development of biofilm by S. aureus is considered the most important mode of growth lifestyle for some infections, mainly those related to implantable medical devices such as catheters, and to cardiac and orthopedic prostheses5. Also, biofilms can have other negative impacts on bacterial infections because bacterial cells in the biofilm growth do not efficiently respond to either antimicrobials or the killing promoted by the immune system7.The first case of methicillin-resistant 8. The occurrence of subpopulations of bacteria that display antimicrobial persistence reinforces the importance of the discovery of new molecules that can kill persistent cells. However, the development of new antibacterial agents has decreased in recent decades10. In previous studies, using broth macrodilution for MIC determination, we have shown that some N,O-acetal 2-amino-1,4-naphthoquinone derivatives presented promising action against planktonic Gram-positive bacteria11. In the study presented here, we demonstrated that 2-ethoxymethyl 2-amino-1,4-naphthoquinone, besides presenting acceptable pharmacological and toxicological parameters through in silico and ex-vivo studies, also displays other desirable properties associated with antibiofilm and antipersistence effects. In addition, we investigated the mechanisms involved in the modulation of biofilm accumulation by this compound.Some hypotheses have been proposed to explain the antimicrobial refractory/persistence in biofilms: (1) impairment of the antimicrobial penetration in the extracellular matrix; (2) activation of efflux pumps; (3) activation of stringent response with generation of persister cells due to increased concentration of the penta- or tetra-phosphate alarmone, commonly referred as (p)ppGpp; (4) specific genetic background of the bacterium; and (5) stochastic phenomenon, where a small population acquires a specific gene expression profile that leads to antimicrobial persistenceN,O-acetal derivatives against MRSA: NY/Japan-USA100/ST5-SCCmecII (strain CR15-071), USA300/ST8-SCCmecIV (strain USA300-0114), USA400/ST1-SCCmecIV (strain USA400-0051), Brazilian epidemic clone-BEC/ST239-SCCmecIII (strain BMB9393), pediatric clone-USA800/ST5-SCCmecIV (strain USA800-0179), and OSPC-USA1100/ST30-SCCmecIV (strain 07-040). The S. aureus ATCC 25923 was used as a control for the experiment of drug susceptibility. The biofilm-producing strain BMB9393 (ST239-SCCmecIII) was chosen for biofilm assays because its superior capability to accumulate biofilm matrix compared with other MRSA strains12. For biofilm production, tryptic soy broth  was supplemented with 1% (w/v) glucose (TSB-Glu). Tryptic soy agar  was used for bacterial growth. The Mueller-Hinton broth  and Mueller-Hinton agar  was used for susceptibility tests. All strains were maintained in 10% (v/v) glycerol stocks and stored at − 80 °C.Strain representatives of six globally spread clones/lineages of MRSA were used in this study to test the antimicrobial activity of the N,O-acetals derivatives from 2-amino-1,4-naphthoquinone (6a), a methodology that employs microwave irradiation was used as described previously11. The 6a and its derivatives 7a , 7b  and 7c  were characterized using spectroscopic methods, such as 1H and 13C NMR11. Stock solutions of these derivatives were prepared in DMSO 100%. In the experiments, the final concentrations of DMSO were ≤ 2% (v/v), which showed in previous tests performed in this study to cause no effect in S. aureus planktonic or sessile growth.To obtain 6a and its derivatives 7a, 7b, and 7c was tested as previously described using paper disks impregnated with each compound13. The derivatives were also analyzed quantitatively using microdilution assays in 96-well inert polystyrene microtiter plates  to determine the minimal inhibitory concentration (MIC)14. Biological control, as well as growth conditions, were performed following CLSI recommendation16. Additionally, the MIC values of the MRSA strains BMB9393 (BEC) and CR15-071 (USA100), and the control strain ATCC25923, for the derivatives 7a, 7b and 7c were also determined using Mueller Hinton agar dilution following CLSI recommendation16. Controls were also performed using 2% (v/v) DMSO only.The antibacterial activity of l-glutamine (amino acid), and 100 U/mL penicillin, during 24-h incubation period at 37 °C and 5% carbon dioxide (CO2). After that, the supernatant was removed, and the cell monolayers were incubated for 24 h with different concentrations of 6a or its derivatives 7a, 7b, or 7c, ranging from 8 to 512 µg/mL. For comparative purposes, these experiments were also performed with the commercially available antibiotics, tetracycline and trimethoprim-sulfamethoxazole, using the same range of concentrations. All compounds were diluted in DMSO for a final concentration of 1% (v/v) in the cell monolayers. In the experimental controls, DMSO was added to the drug-untreated cell monolayers for a final concentration of 1% (v/v). After incubation, the supernatant was removed, and 100 µL fresh medium and 20 µL 3--2,5-diphenyltetrazolium bromide  were added into each well. Plates were incubated for 1 h, the supernatant again removed, and 200 µL dimethyl sulfoxide (DMSO) were added to each well to dissolve the formazan crystals for 10 min. Absorbance was measured at 570 nm using an ELISA reader . The cytotoxic concentration 50% (CC50) value was defined as the concentration of a derivative required for reducing cell viability by 50%. The selectivity index (SI) was calculated as follows: SI (µg/mL) = CC50/MIC17. Three independent experiments were performed in triplicate. Controls were performed using 2% (v/v) DMSO only.The Vero cell line was cultivated to form monolayers on 96-well flat-bottom culture plates with Dulbecco’s modified Eagle’s medium  supplemented with 5% (v/v) fetal bovine serum , 1 µg/mL fungizone, 2.5 Mm 7a, 7b, and 7c were also evaluated for their hemolytic activity, using final concentrations of 25 µg/mL, 50 µg/mL, 100 µg/mL and 300 µg/mL in 1% (v/v) DMSO, according to Sathler and collaborators18. Erythrocytes were collected from three human volunteers in a citrate tube and washed three times (3×) with PBS (pH 7.4). All derivatives were incubated with the PBS-erythrocyte suspension for 3 h at 37 °C. The hemolysis with the release of hemoglobin was quantified by the spectrophotometric reading of the supernatant at 545 nm. Hemolysis less than 10% represented hemocompatibility and non-toxicity against erythrocyte membranes19. For the positive control, the human erythrocytes were lysed with 1% (v/v) Triton X-100. The negative control was performed with 1% (v/v) DMSO only . This protocol was approved by the Human Research Ethics Committee of the Universidade Federal Fluminense and under the number #20870414.9.0000.5243. All methods were carried out in accordance with relevant guidelines and regulations. In addition, informed consent was obtained from all volunteers. Three independent experiments were performed in triplicate.2-Amino-1,4-naphthoquinone and its derivatives 6a and its derivatives 7a, 7b and 7c were evaluated using pkCSM—pharmacokinetic webserver (https://biosig.unimelb.edu.au/pkcsm/)20, Pro-Tox-II webserver (https://tox.charite.de /protox_II/)21 and SwissADME webserver22. These results were also compared with the profiles of an antimalarial (atovaquone) and six antimicrobials . The theoretical pharmacokinetic properties evaluated were absorption, distribution, metabolism, and excretion (ADME). The absorption parameters were tested through simulation modelling of human intestinal absorption using Caco-2 permeability, human intestinal absorption, P-glycoprotein substrate, and P-glycoprotein I/II inhibition. For drug distribution, it was analyzed the steady-state volume of distribution (Vss) and the blood–brain barrier penetration (BBBP) predictions. Drug metabolism was analyzed by the theoretical activity of cytochrome P450 (CYP) enzymes. Lastly, excretion parameters including theoretical total clearance and renal OCT2 substrate were evaluated. Toxicological analyses comprised toxicity target, hepatotoxicity, hERGI/II inhibitors, and toxicity endpoints including carcinogenic, mutagenic, immunotoxicity, and cytotoxicity parameters.The in silico pharmacokinetic properties and toxicity profiles (ADMET) of 23. The compound 6a (1MIC = 128 µg/mL) and its derivatives 7a (1MIC = 256 µg/mL), 7b (1MIC = 128 µg/mL), and 7c (1MIC = 64 µg/mL) were added, separately, into each well of a polystyrene microtiter plate (Nunc) at concentrations of 1/4MIC 1/8MIC, or 1/16MIC in trypticase soy broth with 1% glucose . The same volume of the bacterial culture in TSB-Glu  diluted 1/100 in the same broth was added to the wells. After incubation (37 °C/24 h), supernatants were gently removed, rinsed with distilled water, and treated with crystal violet solution as described23. The adhered biomass was suspended in ethanol PA, and the OD read at 570 nm using the SpectraMax Plus 384 . Eight independent tests with three replicates each were performed. Controls were inoculated with the same bacterial inoculum but using only TSB-Glu with 2% (v/v) DMSO, final concentration.Biofilms were formed on polystyrene surfaces using the BEC representative, strain BMB9393. It had previously been shown that this method has a good correlation with an in vivo biofilm model7b showed the stronger reduction in biofilm accumulation, this compound was elected for these and other experiments designed to unveil the mechanisms by which this molecule may affect biofilm development. For comparative purposes and validation of the experiments performed here, the derivative 7c, which exhibited a lesser but still important reduction on biofilm accumulation, was also included in these studies. For CLSM experiments, after 24 h of incubation, the supernatant containing planktonic cells was gently removed, and biofilm treated with 25 nM SYTO 9 DNA-intercalating stain , as previously described24. Before visualization, SYTO 9 solution was removed and the material visualized using a confocal laser scanning microscope . The microscope was configured with the argon laser (458 nm/477 nm/488 nm/514 nm). The images were randomly captured with the Neorlu-Plan 406/06 Korr. Controls were performed using 2% (v/v) DMSO only . Three independent experiments were performed in quadruplicate.Because derivative N,O acetal derivatives 7b (32 µg/mL) or 7c (16 µg/mL). Gene expression analyses were performed using real-time RT-qPCR with Power SYBR Green RNA-to-CTTM 1-Step Kit . The platform used was a One Step Real-Time PCR System (Applied Biosystems). The genes chosen were those well characterized as associated with biofilm development, including fnbA, spa, mecA, hla, and psmα3—encoding fibronectin-binding proteins A (FnBPA), staphylococcal protein A (Spa), penicillin-binding protein 2a or 2′ (PBP 2a or 2′), α hemolysin, and phenol-soluble modulin α3, respectively. To confirm the data from real-time RT-qPCR for the biofilm-associated genes, and to further understand the mechanisms by which 7b caused a profound effect on MRSA biofilm, the same experiments were performed with the derivative 7c. Additionally, these data were also validated by testing important biofilm and global virulence regulators, involved in the modulation of the biofilm-associated genes, such as RNAIII (the main effector molecule of the Agr quorum sensing), sarA , and sigB , using new RNA preparations. The validated PCR primers used for each gene tested are listed in the Supplementary Table Total RNA was obtained using the RNeasy Kit  from sessile cells treated with 1/4MIC of the 7b or 7c was measured by quantifying atlA cDNA by real-time qRT-PCR, as described above. To validate the RT-qPCR data for derivative 7b, the same experiment was performed for derivative 7c. Additionally, to confirm these data, quantification of extracellular DNA (eDNA) was performed in the supernatant of bacterial growth treated and untreated with 1/4MIC of these two derivatives , using a previously described method25. Controls were performed using 2% (v/v) DMSO only. Three independent experiments were performed in triplicate.The autolytic activity in the presence and absence of the derivatives mecII lineage (NY/Japan-USA100 clone), since this MRSA has replaced the ST239-SCCmecIII (the Brazilian clone) in this country, and is now the most frequent MRSA lineage in Brazilian hospitals1. A high bacterial load (~ 109 CFU/ml) was inoculated on the surface of the TSA plate containing 8 µg/mL vancomycin alone, corresponding to 4MIC, or containing 4MIC vancomycin plus the derivatives 7b or 7c at concentrations of 1MIC (128 µg/mL), 1/2MIC (64 µg/mL), 1/4MIC (32 µg/mL), or 1/8MIC (16 µg/mL). The plates were incubated at 37 °C/24 h before being examined. For controlling the activity of the antimicrobials, plates containing vancomycin or each derivative alone at low bacterial load (~106 CFU) were also assayed. Bacterial growth was determined by removing persistent cells from the plates and determining UFC/ml. Controls were also performed using 2% (v/v) DMSO only. Three independent experiments were performed in triplicate.For this test, we choose the MRSA strain CR15-071 representative of the ST5-SCCt-test for all experiments, except for the biofilm data that were analyzed by Tukey’s test following One-Way ANOVA, considering a significance level of α = 0.01.All experiments were independently performed in triplicate with at least three technical replicates. The results shown are the mean ± standard deviation (SD). Statistical significance was determined by unpaired two-tailed Student’s N,O acetals derived from 2-amino-1,4-naphthoquinone (6a), the compounds 7a, 7b, and 7c, showed antimicrobial effects against the representatives of major MRSA international clones (Supplementary Table mecIII). This MRSA lineage shows resistance to several classes of antimicrobials besides β-lactams, including aminoglycosides, quinolones, and tetracycline2, but showed to be  susceptible to all derivatives tested here. The antimicrobial properties of these compounds were also evaluated using the minimal inhibitory concentration (MIC) method. The MIC of the derivatives using broth microdilution for the reference strain ATCC  25923 and MRSA clones ranged between 16 to 128 μg/mL and 64 to 256 μg/mL, respectively (Table The 7a), 2.7 ± 0.3% (7b), 1.5 ± 0.5% (7c), and 3.0 ± 0.9% (6a) in the highest concentration evaluated (300 µg/mL) and thus were unable to cause membrane lysis, with no release of hemoglobin  was less toxic compared with 6a, 7a, and 7c derivatives. SI values were calculated to evaluate the selectivity of the derivatives for the bacteria. The 7b derivative (SI = 1.72) showed some degree of selectivity for eukaryotic cells. Despite that, this value was better than that of sulfamethoxazole-trimethoprim (SI = 1.01) (Table The cytotoxic concentrations 50% (CC50) for Vero cells showed that N,O-acetal derivatives were submitted to in silico ADME/Tox  analysis  according SwissADME webserver  − log Vss > 0.45—suggest that the drug will be distributed in body tissue rather than plasma. On the other hand, lower values are considered when log Vss < −0.15. The intermediate values suggest that the drug presents appropriate plasma distribution profileer Table .6a, 7a and 7b did not inhibit CYP2C19, CYP2C9, CYP2D6, and CYP3A4, but showed inhibitory properties against CYP1A2. 7c derivative did not inhibited CYP2C9, CYP2D6, and CYP3A4, despite their inhibitory effect on CYP1A2 and CYP2C19. Among the referential drugs, only atovaquone showed inhibitory effects on CYP1A2, CYP2C19, and CYP2C9  values and the possibility of these compounds acting as substrates for organic cation transporter 2 (OCT2). The values of Log (CLtot) of all derivatives ranged from − 1.273 to 0.987, and thus none of them would apparently be a substrate of renal OCT2  varied from 2.503 log mg/kg b.w. per day to 2.776 mg/kg b.w. per day, and the maximum tolerated dose ranged from 0.516 log mg/kg b.w. per day to 0.778 log mg/kg b.w. per day  of 7c reduced by 75.6% ± 3.7%. This inhibition was more specific for biofilm development since such a strong impact in growth reduction was not observed for planktonic cells at these concentrations  and 7a (64 μg/ml), although showing antimicrobial effects, were much less effective at reducing biofilm development compared with 7b or 7c  of S. aureus genes are well known for their role in biofilm development. Therefore, due to the strong biofilm inhibition promoted by the derivative 7b, we investigated the role played by this compound in the expression of important biofilm-associated genes, to get some insights on the mechanisms by which this molecule strongly impairs biofilm production. Additionally, the derivative 7c, which also affects biofilm accumulation, but in lesser extent, was also included for comparison purpose. Among these genes, the mecA encoding PBP2A was not significantly affected by 1/4MIC of 7b (32 µg/mL) or 7c (16 µg/mL) . However, for the 7c derivative, the reduction for this gene was only 24.64% ± 3.5% (p < 0.01)  was considerably reduced by 90.0 ± 0.6% and 86.5 ± 1.0% after treatment with derivatives 7b and 7c, respectively (p < 0.0001). Also, an important reduction—by 95.4 ± 0.9% for 7b and 74.82 ± 4.9% for 7c (p < 0.0001)—was observed for the spa gene, which encodes the staphylococcal protein A. The expression of another important biofilm-associated gene, the psmα3 gene encoding the phenol soluble modulin alpha 3—was reduced by 97.75 ± 0.6% for 7b and 87.73 ± 4.3% for 7c (p < 0.0001)  and 7c (16 µg/mL) in the expression of rnaIII , sarA , and sigB  were tested . Derivative 7b also reduced importantly sarA gene expression at 65.5 ± 6.0% (p < 0.001). However, no important effect on sarA expression was observed for the derivative 7c. Moreover, derivative 7c increased sigB transcripts at 57.22 ± 5.2% (p < 0.01) while 7b did not affect the expression of the sigB transcription factor  on the autolytic activity was evaluated in the biofilm cell growth of the strain BMB9393. An important increase of 3.5-times (p < 0.0001) in the atlA transcripts was observed. This result was validated by the fact that 1/4MIC 7c (16 µg/mL) led to three-fold increase in the atlA expression (p < 0.0001)  for the derivative 7b (p < 0.0001), which triggered an increased expression of atlA in comparison with 7c  of the strain (CR15-071) representative of the clone USA100 were recovered from vancomycin plates (4MIC). However, when 1MIC of the derivatives 7b (128 µg/mL) or 7c (128 µg/mL)—both effectives to inhibit biofilm development—were added to the plates containing vancomycin (4MIC), a complete inhibition of persistent cells was achieved for derivative 7b (p < 0.0001), and an important reduction of 88.3% (p = 0.0002) in the total of persistent cells were caused by the association with the derivative 7c . After 18 h incubation, refractory/persistent cells  and CR15-071 (USA100). Actually, previous studies have already demonstrated disagreement in MIC data obtained with different recommended methods for some drug/strain combinations, including for S. aureus27. The MRSA lineages  tested for these derivatives are currently causing the majority of hospital and community infections worldwide, including bacteremia and biofilm-associated infections1. Notably, despite the infection control measures, bacteremia caused by MRSA remains at high frequency and accounts for a mortality rate of about 25%28.The 7a, 7b, and 7c did not display a hemolytic profile, according to Dobrovolskaia and collaborators19. The toxicity tests using monolayers of the Vero cell line revealed that the 7b derivative showed better SI value indicating that 7b displayed not only antibacterial effect but also higher selectivity against MRSA strain than toxicity to eukaryotic cells. Theoretical pharmacokinetic properties are important parameters to assess potential compounds during drug discovery process. All N,O-acetal derivatives showed good predictions for absorption values in the human intestine, similar to atovaquone, ciprofloxacin, and furazolidone. Indeed, the compounds 7b and 7c showed absorption values greater than 90%, close to that of ciprofloxacin, which is a good estimative for oral bioavailability29.The in vitro toxicity assays revealed that the derivatives N,O-acetal derivatives and atovaquone showed permeability to Caco-2 cells, which has been used as a model for human intestinal absorption of drugs30. The results also suggested that none of the N,O-acetal derivatives seem to affect P-glycoprotein, which is involved in drug exclusion31. All together, these data reveal an optimal in silico prediction of oral bioavailability for the N,O-acetal derivatives. Derivatives 6a, 7b and 7c showed intermediate Vss values, representing good predictions for adequate plasma distribution profiles. In addition, the analysis of BBB values, according to SwissADME calculation, suggested that N,O-acetal derivatives could cross blood brain barrier32.Interestingly, only 33. None of the derivatives tested was substrate for cytochrome P450 isozyme CYP2D6 and CYP3A4. Moreover, these derivatives were only presumed to be inhibitors of CYP1A2, except 7c derivative that was predicted to inhibit CYP2C19. Interestingly, CYP1A2 is one main xenobiotic metabolizing enzyme in humans, and a recent study associated this enzyme with the bioactivation of procarcinogens, including 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco specific and potent pulmonary carcinogen34.It has been estimated that CYPs could be able to metabolize around 75% of the commercially available drugs6a, all derivatives tested presented high Log(CLtot) values, but still acceptable. Actually, these values were lower than those observed for referential drugs such as ciprofloxacin, doxorubicin, furazolidone, and nitrofurantoin. None of the N,O-acetal derivatives tested was predicted to show hepatotoxicity. Also, they were not expected to inhibit hERG I and II channels. Drug-hERG channel interactions have been considered a therapeutic challenge as a major cause acquired-long QT syndrome. Actually, a relationship between chronic heart failure (CHF) and S. aureus bacteremia was suggested. CHF patients with S. aureus bacteremia showed a significantly higher mortality rate compared with patients with normal heart functions36. This observation reinforces the need of non-cardiogenic effect for anti-MRSA drug candidates and highlights this promising profile of our derivatives.Drug clearance was measured by determining Log(CLtot). Except for 7b derivative showed good toxicological parameters, except for the mutagenic profile. This feature could be related to the mechanism of action of these derivatives. Besides oxidative stress, another mechanism of action generally associated with naphthoquinones is the ability to bind to topoisomerase complex causing damage to the DNA replication process37. Despite that, in vitro studies using V79 Chinese hamster cells did not confirm a mutagenicity effect for 1,4-naphthoquinone38.Data from the ProTox-II platform revealed that the 7b derivative resulted in the best antibiofilm and antipersistence effects. Furthermore, contrarily to the substitution by 2-methoxymethyl or 2-propynyloxy, the 2-ethoxymethyl substituted amino-1,4-naphtoquinone did not inhibit four of five CYPs enzymes tested, was not a substrate of CYP3A4 and CYP2D6, and also was predicted to have the best intestinal absorption, comparable to ciprofloxacin.Besides the antimicrobial activity against MRSA and the improvement of pharmacological parameters, the introduction of the 2-ethoxymethyl radical generating the S. aureus ability to cause infections and persist into the host5. In the present study, the N,O acetal derivatives from 2-amino-1,4-naphthoquinone, 7b and 7c, were able to reduce biofilm accumulation in 88% and 75%, respectively, at 1/4MIC without causing similar massive reduction in viability of planktonic cells. Oja and collaborators39 also found that other naphthoquinone derivatives [biosynthetic pyranonaphthoquinone (PNP) polyketides] impaired biofilms formed by methicillin-susceptible S. aureus (MSSA), ATCC 25923, and Newman strains. It is important to remark that vancomycin, considered one of the last resources to treat serious infections by MRSA, did not impair biofilm development in the study model chosen. Not only that, induction of biofilm by vancomycin had already been observed for some S. aureus strains40.It is well known that biofilm development enhances fnbA gene5. The expression of fnbA is upregulated by the sarA in an agrRNAIII-independent mode41. The derivative 7b, that showed a drastic reduction in biofilm accumulation in 1/4MIC, also presented a deeper decrease in the expression of the fnbA gene paralleled by an also important decline in sarA transcripts. Actually, it is well known that inhibition of sarA also leads to impairment of biofilm development in S. aureus42, which might explain the important reduction observed in the biofilm accumulation caused by the 7b derivative. In agreement with these data, the decrease of fnbA transcripts for MRSA treated with 7c was less pronounced when compared with 7b. Indeed, 7c also presented a lower ability to impair biofilm development compared with 7b. Moreover, the poorer effect of 7c in the decreasing sarA expression could be explained by the increase in the expression of sigB caused by 7c. SigB is a sarA-positive regulator that has also been implicated in the modulation of the maturation phase of S. aureus biofilms44.In one of the first stages of biofilm development, bacteria adhere to biotic or abiotic surfaces, in a mechanism mediated by bacterial surface adhesion molecules, such as the fibronectin-binding protein A (FnBPA) encoded by the spa gene that can also provide S. aureus with self-aggregation property, which is an important condition for biofilm development46. The MRSA treatment with both 7b and 7c derivatives caused an important reduction in the spa expression, which was more pronounced for 7b, and thus it might also account for the stronger biofilm impairment caused by these derivatives. The control of spa expression is complex and involves an intricate regulatory network47.Protein A is an antiphagocytic protein encoded by the 7b and 7c showed antibiofilm effects associated not only with a strong inhibition of fnbA and spa genes but also with an important attenuation in the expression of cytotoxicity-associated genes, such as hla encoding the staphylococcal α-hemolysin, hld encoding δ-hemolysin , and psmα3 encoding phenol-soluble modulin alpha 3 peptide48. It is very well known that PSMs are multifactorial molecules in staphylococcal pathogenesis. PSMs are broadly cytolytic, inducing the killing of different cell types. Furthermore, PSMs are important for the architecture of the S. aureus biofilms49. The α-hemolysin (Hla) is the prototype for small β-barrel pore-forming cytotoxins, which induces lysis of different host cells, including monocytes, neutrophils, pneumocytes, and endothelial cells. Besides its action promoting biofilm development50, it was observed that Δhla mutants lead to attenuated bloodstream infections in animal models51. It is notable that the inhibitory effects in toxin-associated genes were also more pronounced for 7b than 7c. Due to the importance of these genes for S. aureus pathogenesis, although we did not perform studies using animal models, it seems logical to suppose that the attenuation of cytotoxic genes, caused by these derivatives, is likely to result in antivirulence effects51.Notably, the derivatives agr-RNAIII can positively or negatively regulate biofilms depending on the background of the strains, and that agr-RNAIII is a positive biofilm regulator for strains with the ST239-SCCmecIII background, such as BMB939324. In fact, agr-RNAIII was impaired by both derivatives 7b and 7c, and again 7b caused a deeper effect by diminishing RNAIII transcripts. The impairment of RNAIII agrees with the reduction observed in hla and psmα3, since RNAIII is a positive regulator for these genes. AtlA is the main S. aureus muramidase that has an important role in cell wall biogenesis and the septation process during bacterial cell division52. It was found that small fractions of bacterial cell lysis enhance biofilm formation due to the ligation of eDNA to proteins and polysaccharides of the biofilm matrix53. However, it was demonstrated that the uncontrolled expression of atlA causes increased cell lysis, resulting in a defective biofilm formation54. Thus, the triggering of the autolytic system by 7b and 7c is likely to play a role in the impact of these naphthoquinone derivatives in MRSA biofilm development.It was previously found that 55. A hypothesis suggested to explain these failures was linked to a decreased penetration of vancomycin into the biofilm matrix57. Also, it was proposed that stress conditions in the hyper-populated environment, such as those encountered in biofilm growth or high bacterial load, might induce the generation of persistent cells58. However, the exact mechanisms through which bacteria become refractory/persistent to different antimicrobials in the biofilm environment are still to be completely clarified5.Therapeutic failures during the administration of vancomycin have been described, and in some cases, involved patients with MRSA infections7b derivative at 1MIC (128 µg/mL) was able to eradicate subpopulations of persistent cells that survived to concentrations of 4MIC (8 µg/mL) vancomycin. The ability of these derivatives to trigger cell autolysis might be involved in the killing of persistent cells. It was previously shown that the deletion of the operon msaABCR of S. aureus increased the processing of the major S. aureus autolysin by proteases, leading to the activation of the muramidase activity. In addition, it was found that msaABCR deletion enhanced the effectiveness of antibiotics against persistent cells, which was attributed to the increased cell lysis54. Thus, it is interesting to further investigate the role played by the autolytic system in the killing of vancomycin-persistent cells by these naphthoquinone derivatives and other active molecules.In this study, we found that S. aureus are outpacing the development of completely new antibiotics, and antibiofilm agents might provide an interesting alternative. In this context, the 2-(ethoxymethyl)-amino-1,4-naphthoquinone (7b) derivative, besides the antimicrobial action, also showed strong antibiofilm and antipersistence effects against multidrug-resistant MRSA strains. These effects were paralleled by the ability of 7b to concomitantly affects (directly or indirectly) important S. aureus virulence regulators (agr-RNAIII and sarA) and biofilm-associated genes . Finally, these properties associated with the biological and pharmacological aspects of this compound identified 7b derivative as an interesting model for the design of potentially promising and more effective drugs against MRSA (Fig. In conclusion, the accelerated evolution and dissemination of antimicrobial resistance in RSA Fig. .Figure 6Supplementary information.


We identified Met and Gly as candidate amino acids that fortify mitochondrial activity in sake yeast during fermentation. To confirm this biochemically, we measured reactive oxygen species (ROS) levels in sake yeast fermented with Met and Gly. Yeast cells supplemented with Met and Gly retained high ROS levels relative to the non-supplemented sake yeast. Moreover, Met-supplemented cells showed a metabolome distinct from that of non-supplemented cells. These results indicate that specific amino acids such as Met and Gly stabilize the mitochondrial activity of sake yeast during fermentation and thus manipulate brewing profiles of yeast.Addition of amino acids to fermentation media affects the growth and brewing profiles of yeast. In addition, retaining mitochondrial activity during fermentation is critical for the fermentation profiles of brewer’s yeasts. However, a concrete mechanism linking amino acids in fermentation media with mitochondrial activity during fermentation of brewer’s yeasts is yet unknown. Here, we report that amino acids in fermentation media, especially methionine (Met) and glycine (Gly), stabilize mitochondrial activity during fermentation of sake yeast. By utilizing  The nutrients in fermentation media affect the characteristics of brewer’s yeasts. For example, assimilable nitrogen increases the growth rate of yeast  and glycine (Gly) as candidates to activate mitochondrial activity. Consistent with the hypothesis, these amino acids strengthened the mitochondrial activity, as detected by reactive dye 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), which binds to the reactive oxygen species. Reactive oxygen species (ROS) are byproducts of the mitochondrial respiratory chain in aerobically growing cells (MATa/αhis3/his3 (atg32△::kanMX/atg32△::NAT1 (m/V) Bacto peptone, 1% (m/V) Bacto yeast extract , and 2% (m/V) glucose. For the fermentation tests, a minimal synthetic medium containing 0.67% (m/V) yeast nitrogen base without amino acids (Becton Dickinson), 790 mg/L complete supplement mixture (CSM) Dropout: Complete , and 15% (m/V) glucose with amino acids was used.Sake yeast RAK1536 m/V) glucose in a 100-mL flask. The flask was incubated at 30 °C overnight under aerobic and shaking conditions at 200 rpm for pre-enrichment culture. Then, yeast cells (106 cells/mL) were inoculated in 100 mL minimal synthetic medium containing 15% (m/V) glucose with or without amino acids in a 300-mL Erlenmeyer flask equipped with an air lock at the top, and cultured statically at 30 °C for 9 to 11 days. The culture mass was determined daily.Yeast cells were inoculated in 60 mL of minimal synthetic medium containing 2%  according to the manufacturer’s instructions, as described previously  at 7000×g for 1 min. Then 20-µL sample was mixed with 3 mL of colour reagent and incubated for 5 min at 37 °C. The absorbance was then measured at 505 nm. Standard solution was also prepared in the same way, the absorbance was measured and glucose mass fraction was calculated from the calibration curve.Glucose mass fraction was measured with a Glucose CII-kit  at 505 nm using a UV spectrophotometer  as described previously  at 3200×6 cells/mL) were inoculated in 10 mL minimal synthetic medium containing 15% (m/V) glucose with or without amino acids and incubated for 2–3 days at 30 °C with liquid paraffin overlaid on the culture. After fermentation, 107 cells/mL yeast cells were recovered and incubated with 13 µL DCFH-DA, 100 µL phosphate-buffered saline (PBS), pH=7.4, and 887 µL sterile water at 30 °C for 20 min in a shaker  at 200 rpm. After incubation, the cells were washed three times with sterile water, centrifuged  at 13 000×g for 1 min and 100 cells were sampled under fermentation stress, treated with DCFH-DA and the ROS content was analyzed by fluorescence microscopy .2,7-Dichlorodihydrofluorescein diacetate  stock solution of 1 mg was prepared in 100 µL ethanol and stored at -20 °C. Yeast cells  and centrifuged again under the same conditions. The pellet was then soaked in liquid nitrogen for 3.5 min, freeze-dried for 12 h, and stored at -80 °C. Next, 10 mg of the dried pellet were mixed with a mixture of chloroform, methanol  and water at ratio 2:5:2 for extraction, followed by the addition of 60 µL ribitol  to the solvent, and incubation for 3 min at 30 °C with shaking at 1500 rpm. Then, 800 µL supernatant were collected after centrifugation (4 °C and 16 000×g for 3 min) and 400 µL MilliQ water (Millipore Inc) were added, mixed and centrifuged again under the same conditions. We then evaporated 800 µL of the supernatant for 3 h and freeze-dried it for 12 h. Next, 100 µL methoxyamine  were mixed with the freeze-dried extract and incubated at 30 °C for 90 min with shaking at 1500 rpm, followed by the addition of 50 µL N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) and incubation at 37 °C and 1500 rpm for 30 min with shaking. Then, 70 µL of the solvent were transferred to a vial and the metabolites were analyzed using a gas chromatograph with flame ionization detector  with a CP Sil8CB column . The data were analysed with Lab Solutions v. 5.71 SP1  weak reactive oxygen species activity compared to the parental strain. The fluorescence intensity of sake mutant atg32△ was 30.2±0.4 and of its parental strain was 34.7±0.7, indicating that the atg32△ sake mutant yeast has defective mitochondrial activity. It was thus considered that atg32△ sake mutant yeast shows high ethanol fermentation ability . In terms of maximum fermentation rate, amino acids such as Met, Cys, Gly, Ala, Val, Pro, Ile, Tyr, Ser and Lys were effective (g/(L·day)). Moreover, in terms of final ethanol volume fraction, amino acids such as Met, Cys, Gly, Pro, Leu, Tyr, Trp, Ser, Gln, Lys, Arg, His and glutamic acid (Glu) showed similar characteristics. To examine this, we analyzed the growth profile (A600 nm·107 cells/mL) of wild-type and sake mutant atg32△ yeasts with or without the addition of amino acids. As for growth profile, Met, Cys, Gly, Val, Pro, Tyr, Ser, Gln, Arg and Asp showed similar effects in this study. The above results suggested that Met, Cys, Gly, Pro, Tyr and Ser showed common significant interaction effects for main fermentation characteristics among all amino acids , they have significant interaction . On the other hand, if the slope ratio is very low and positive , they do not have significant interaction.To identify the amino acid that activates mitochondrial activity during fermentation, we invented a system using ke yeast . As a reno acids . In Tablatg32△ sake yeast during fermentation. To test this hypothesis, we measured the mitochondrial activities of atg32△ sake mutant yeast supplemented with these amino acids by measuring the levels of reactive oxygen species (ROS) using DCFH-DA. Among these amino acids, only the addition of Met and Gly significantly (p<0.05) increased the fluorescence intensity of atg32△ sake mutant yeast  generated by the glycine cleavage system might have entered the mitochondria and activated their metabolism. The mechanism of the effect of Gly on the mitochondria of sake yeast needs further research.After Gly incorporation into yeast cells by the general amino acid permease Gap1 (The most abundant amino acids in sake are Asp, Arg, Pro, Ala, Gly, Glu and Leu (atg32Δ system and analyzing the fermentation profiles, we have revealed that methionine and glycine stabilize the mitochondrial activity of sake yeast. This new knowledge will be a new approach to stabilize mitochondria during fermentation.It has been accepted that mitochondria of brewer’s yeasts lose their activity as fermentation proceeds. By adopting the


Under optimal experimental conditions, this method could quantitatively detect cefquinome from 0.1 to 8 μg L−1 and with the limit of detection (LOD) of 0.16 μg L−1, much lower than the maximum residue limit (MRL) of 5 μg L−1 set by the European Union. In addition, the LOD of spiked milk samples with cefalexin, cefquinoxime, cefotafur, penicillin G and ampicillin were 14.88 μg L−1, 2.46 μg L−1, 17.16 μg L−1, 0.06 μg L−1, 0.21 μg L−1 and the limits of quantitation (LOQ) were 36.09 μg L−1, 5.40 μg L−1, 41.45 μg L−1, 0.13 μg L−1, 0.42 μg L−1, respectively. The sensor showed a favorable recovery of 84.89–102.44%. Moreover, the electrochemical receptor sensor was successfully applied to assay β-lactam antibiotics in milk, which showed good correlation with the results obtained from liquid chromatography-tandem mass spectrometry (LC-MS/MS).In antibiotics, β-lactam is one kind of major concern acknowledged as an unavoidable contaminant in milk. Thus, a facile and sensitive method is essential for rapid β-lactam antibiotics detection. In our work, a specific electrochemical receptor sensor based on the graphene/thionine (GO/TH) composite was established. The mechanism of the electrochemical receptor sensor was a direct competitive inhibition of the binding of horseradish peroxidase-labeled ampicillin (HRP-AMP) to the mutant BlaR-CTD protein by free β-lactam antibiotics. Then, horseradish peroxidase (HRP) catalyzed the hydrolysis of the substrate hydrogen peroxide (H EU Regulation 96/23/EC also establishes the maximum residue limits of β-lactam antibiotics in different animal tissue samples and the maximum residue limits (MRLs) of penicillin G and ampicillin in milk and muscle tissues are 4 and 50 μg Kg−1, respectively. These factors underscore the importance of the development of a rapid, simple, accurate and sensitive method for the detection of β-lactam antibiotics to ensure food safety 3−/[Fe(CN)6]4− couple and obtained a larger interface area, herein improving the loading amount of BlaR-CTD. However, a stepwise growth in semicircle diameters corresponding with the increased resistances happened after the sequential fabricating of BlaR-CTD and BSA (curves c and d). The reason for that was the protein layer acting as the inert electron, while mass-transfer blocking layer significantly deterred the diffusion of ferricyanide towards the electron surface. The above EIS results indicated that electrochemical receptor sensor had been successfully constructed [3−/4−, containing 1 M KCl at 50 mV/s scan rate. When the GO/TH deposited onto the GCE surface, the redox peak current went up dramatically (green curve), indicating that the GO/TH could accelerate electron transfer speed. In sequence, when receptor protein (BlaR-CTD) was dropped onto the activated electrode surface, apparently the peak current decreased, owing to the steric hindrance and an obstacle of insulated BlaR-CTD for the electron transfer, explaining that BlaR-CTD was successfully immobilized on the GO/TH/GCE surface (purple curve). After the receptor sensor was incubated with BSA, a further decrease in peak current was produced for the increasing of electron transfer resistance (red curve), implying that we successfully obtained the fabricated electrochemical biosensor for binding with BSA [In this research, both CV and EIS methods were adopted to test the interface features of the stepwise modified receptor sensor. structed ,29,30,31with BSA ,32.2O2 mixture at 50 mV/s scan rate under the optimal experiment conditions. The dilution ration of mutant protein I188K/S19C/G24C and HRP-AMP greatly affected the sensitivity and enzymatic reaction rate. In −1) was used in the experiment as the optimal mutant protein ratio. Similarly, as displayed in −1) was chosen as the best optimal ratio.In order to best analyze β-lactam antibiotics, we optimized several key experimental parameters. CV was used for the detection of protein and HRP-AMP, due to its high sensitivity and good resolution. After the incubation of the protein in different concentrations, the CV signals of them were recorded in phosphate buffered solution (PBS) and H2O2 mixture at 50 mV/s scan rate. The PH value of PBS plays a crucial role in electron transfer rate and influences the structure and function of the HRP protein. We prepared PBS with different PH values  to explore the effect of PH on the enzyme activity. As shown in −1, producing a correlation coefficient of 0.9946  (µg L−1) + 117.06, presented in −1 (linear range: l g 0.1 (μg L−1) – l g 8 (μg L−1), R2 = 0.9946).The limit of detection (LOD) of the sensor was calculated using a linear regression curve based on the method reported in the literature ,33. LOD As shown in −1), with the same batch of sensors. As shown in To evaluate the reproducibility and repeatability of this developed electrochemical receptor sensor, we carried out five reduplicative tests of cefquinome at six different concentrations  and standard deviation (SD) of 20 blank milk samples were sequentially calculated. According to the formula Z = C + 3/10 SD, the LOD and limits of quantitation (LOQ) values of cefalexin, cefaquinoxime, ceftiofur, penicillin G, and ampicillin in milk samples were obtained .To validate the applicability of the presented method for sensitively detecting those five kinds of β-lactam antibiotics , the spiked milk at concentrations of 1 × LOQ, 2 × LOQ and 4 × LOQ were assessed with the suggested method. As shown in Cefalexin and Ampicillin were purchased from the China Institute of Food and Drug Verification . Cefquinome, Ceftiofur and Penicillin G were provided by Dr. Ehrenstorfer . BlaR-CTD receptor protein had been developed in our laboratory. The NI-NTA affinity nickel column was purchased from GE Company, Waltham, MA, USA. BCA-100 Protein Quantitative Detection Kit (Enhanced), Tris-HCl  and Tris-HCl  were obtained from the Biyuntian Instituie of Biotechnology. EDTA, Kanamycin, NHS, EDC, HRP and BSA were purchased from Beijing Solebo Technology Co., Ltd. . IPTG was purchased from Anhui Biosharp Company . All other chemicals, such as sodium dihydrogen phosphate and potassium dihydrogen phosphate, were of analytical grade and were purchased from Sinopharm Chemical Reagent Co., Ltd. .All the electrochemical experiments including CV and EIS were performed with the Autolab PGSTAT128N . All measurements were carried out using a conventional three-electrode system that was composed of Ag/AgCl, which was saturated with KCl as reference electrode, a platinum wire as auxiliary electrode and a GCE with a diameter of 3 mm that was modified with the graphene/thionine working electrode at room temperature. The scanning electron microscope images and atomic force microscope images were photographed using a SU8010 scanning electron microscope  and Multimode 8 .E. coli BL21 (DE3) harboring mutant plasmid were cultured overnight in 1 L of LB broth containing 50 μg mL−1 kanamycin at 37 °C in shaker. The culture was diluted 100-fold into a fresh LB broth containing 50 μg mL−1 kanamycin and incubated with vigorous shaking at 37 °C, until the OD600 reached 0.6. Then, 1 mM IPTG was added and the culture was incubated at 18 °C for 12 h. Bacteria were collected by centrifugation at 12,000 rpm for 10 min, and were washed 3 times with ice cold PBS. Then, 50 mL binding buffer  was added into the precipitate, and ultrasonic fragmentation was carried out after resuspension of the precipitate. The suspension was centrifuged at 12,000 rpm for 40 min at 4 °C, and the soluble fraction was loaded onto a 2 mL Ni2+-charged chelating sepharose resin column. After washing by different concentrations of imidazole eluent, the protein was eluted at an imidazole concentration of 60, 100 and 200 mM. The purified mutant proteins were dialyzed against PBS, and were confirmed by SDS-PAGE with Coomassie Brilliant Blue staining and Western blot [ern blot .Ampicillin was coupled to HRP, as described by Zhang J et al., with some modifications . Briefly−1) was then added. The mixture was agitated at room temperature for 24 h, after which the composite was centrifuged. After being washed with ultra-pure water, the graphene thionine composite was stored in PBS solution at 4 °C. The GO/TH was characterized by UV-vis spectrometry.To prepare the graphene thionine composites, graphene was dispersed in PBS solution and 2 mL thionine solution (5 mg mL2O3 powder with different sizes (by using them with a size of 0.3 µm and 0.05 µm in sequence). GO/TH/GCE was prepared by drop-casting of GO/TH suspension onto the GCE. (ii) Then, we immediately dropped 10 μL of 1 µg·mL−1 BlaR-CTD protein onto the electrode surface and incubated it for 45 min at 37 °C. (iii) Following this, we rinsed the built electrode with PBS and next treated it with 10 μL of 1% BSA solution for 30 min at 37°C to block the inactive sites. (iv) Finally, we thoroughly rinsed the electrode with PBS again. The electrochemical receptor sensor was successfully prepared and stored at 4 °C [The electrochemical receptor sensor was prepared by the following steps. (i) The GCE was polished by α-Al at 4 °C ,34. The −1) with 10 µL of antibiotic standards solution, with different concentrations from 0.1 to 8 µg·L−1 in PBS. We added the miscible liquids onto the modified BlaR-CTD protein electrochemical receptor sensor surface and then incubated it at 37 °C for 45 min. In the incubation process, a competitive reaction happened between the immobilized HRP-AMP and free AMP toward a rationed BlaR-CTD protein on the electrode. Then, we washed the resulting electrode with PBS and finally immersed it into the freshly prepared phosphate buffered solution containing hydrogen peroxide. The potential cycling range of cyclic voltammetry (CV) was from −0.2–0.8 V; the scanning rate was 50 mV/s.In order to perform the competition assay, we firstly blended 10 µL of the diluted HRP-AMP –l g8 (µg L−1); the detection limit was 0.16 µg L−1. Moreover, the sensor showed excellent reproducibility, high sensitivity and accuracy, and might also be applicable to other kinds of β-lactam antibiotics. The system is effective and reliable, has a wide range of potential applications and may be ensured to guarantee food safety in the future.In conclusion, we developed a novel electrochemical receptor sensor based on GO/TH/GCE nanocomposites, with the high affinity mutation BlaR-CTD protein as a specific bio-recognition element used for the rapid detection of β-lactam antibiotics in milk. The signal amplification strategy on the GO/TH prominently improved the sensitivity of β-lactam antibiotics determination. Simultaneously, the HRP possessing excellent catalytic activity towards H