Biogenesis and functions of acp3U have not been extensively investigated. Using a reverse-genetic approach supported by comparative genomics, we find here that the Escherichia coli yfiP gene, which we rename tapT (tRNA aminocarboxypropyltransferase), is responsible for acp3U formation in tRNA. Recombinant TapT synthesizes acp3U at position 47 of tRNAs in the presence of S-adenosylmethionine. Biochemical experiments reveal that acp3U47 confers thermal stability on tRNA. Curiously, the ΔtapT strain exhibits genome instability under continuous heat stress. We also find that the human homologs of tapT, DTWD1 and DTWD2, are responsible for acp3U formation at positions 20 and 20a of tRNAs, respectively. Double knockout cells of DTWD1 and DTWD2 exhibit growth retardation, indicating that acp3U is physiologically important in mammals.Transfer (t)RNAs contain a wide variety of post-transcriptional modifications, which play critical roles in tRNA stability and functions. 3-(3-amino-3-carboxypropyl)uridine (acp E. coli and human tRNAs contain 3-(3-amino-3-carboxypropyl)uridine (acp3U) modification. Here the authors identify E. coli TapT and human DTWD1/2 as tRNA aminocarboxypropyltransferases responsible for acp3U formation. Inhibition of acp3U modification results in genome instability in heat-stressed E. coli and growth defects in human cells. Transfer (t)RNAs are especially heavily modified; indeed, more than 80% of RNA modifications found so far were discovered in tRNA molecules from various organisms. By stabilizing tRNA tertiary structure and fine-tuning decoding capability, these modifications ensure that tRNAs function properly4.The emerging field of epitranscriptomics has revealed the chemical diversity and functional importance of RNA modifications. To date, about 150 species of RNA modifications have been identified in RNA molecules from all domains of life2. The second class of modifications is clustered in the tRNA core structure formed by the D-loop, TΨC-loop (T-loop), and variable-loop (V-loop). These tRNA modifications are structural modulators that contribute to correct folding and stabilization of tRNA3. Some of these modifications are also required for tRNA flexibility5. The 2′-O-methyl modifications found in the D- and T-loops, in particular, 2′-O-methylguanosine (Gm) at position 18, confer conformational rigidity on the tRNA core region by fixing C3′-endo ribose puckering6. Gm18 stabilizes the D-loop/T-loop interaction through base pairing with pseudouridine (Ψ) at position 55 in the T-loop8. Ψ55 stabilizes T-loop structure with additional hydrogen bond to the phosphate-ribose backbone9. 5-methyluridine  at position 54 in the T-loop confers thermal stability to tRNA10. In thermophilic organisms, additional modifications, including 5-methyl-2-thiouridine (m5s2U or s2T) and archaeosine (G+), are present in the tRNA core region13. These modifications stabilize tRNAs, enabling cell growth at high temperature15. The modifications in the core region play crucial roles in determining not only the physicochemical properties of tRNAs but also their cellular stability. Because properly modified mature tRNAs are required for accurate and efficient translation, living organisms have evolved a control quality system that degrades hypomodified tRNAs17.A wide variety of tRNA modifications are found in the anticodon loop, especially at positions 34 and 37. These modifications stabilize and modulate codon–anticodon interactions on the ribosome, thereby ensuring accurate and efficient decoding during translation3U) is a widely conserved modification found in tRNA core region in bacteria and eukaryotes19  are present at positions 20 and 47, respectively, in cytoplasmic tRNALys(TTT)21uridine 21Fig. . In seve3U derivative, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (m1acp3Ψ), is present at the P-site of small subunit of the eukaryotic and archaeal ribosome: positions 1191 and 1248 in 18S rRNA from S. cerevisiae and human, respectively24. In human, m1acp3Ψ1248 is synthesized via three steps. The biogenesis is initiated by pseudouridylation mediated by H/ACA snoRNP bearing SNORA1325, followed by methylation catalyzed by EMG126. Finally, TSR3 transfers the acp group of S-adenosylmethionine (SAM) to form m1acp3Ψ28. TSR3 contains a domain similar to SPOUT-class RNA methyltransferase28. The crystal structure of the archaeal homolog of TSR3 in complex with SAM revealed the molecular basis of acp3U formation28, although the enzymatic activity of TSR3 has never been described. Reduction in the level of the acp modification causes accumulation of 18S rRNA precursors and a reduction in the level of 40S ribosomal subunit, indicating that this modification is involved in 40S subunit assembly29.An acp3U modification in tRNAs from bacteria and eukaryotes, the biogenesis and physiological roles of this modification remain to be determined. The solution structure of the acp3U nucleoside does not affect its sugar conformation30. Structural analysis of D-acp3U-A trinucleotide suggested that acp3U20a binds to Mg2+, and this interaction may stabilize the local conformation of tRNA31. Chemical acetylation of the amino group of acp3U47 in E. coli tRNAPhe does not affect its binding affinity for phenylalanyl-tRNA synthetase or the ribosome32. In the tertiary structure of tRNAs35, the acp group of acp3U47 is oriented toward the solvent side of the tRNA structure and does not directly interact with the other residues. In 1974, enzymatic formation of acp3U in tRNA was carried out using E. coli lysate36. Specifically, Nishimura’s group successfully reconstituted acp3U47 formation in tRNAPhe in the presence of SAM, and demonstrated that the acp group of SAM is transferred to tRNA to form acp3U47  to systematically analyze total nucleosides of RNAs obtained from a series of strains harboring knockouts in uncharacterized genes. If a target RNA modification is absent in a certain knockout strain, we can identify the gene dedicated to biogenesis of the modification in a reverse-genetic manner. We have discovered dozens of genes responsible for RNA modifications in tRNAs41, rRNAs43, and mRNAs44. In this study, we apply ribonucleome analysis assisted by comparative genomics to identify an E. coli gene yfiP (renamed as tapT) responsible for acp3U47 formation. We successfully reconstitute formation of acp3U47 by recombinant TapT protein in the presence of SAM. Measurements of melting temperature reveal that acp3U47 stabilizes tRNA by 3 °C. We observe genome instability of the ΔtapT strain under continuous heat stress, indicating that this modification plays a physiological role in bacteria. We also identify eukaryotic homologs of tapT, including the human homologs DTWD1 and DTWD2, and show that they are responsible for the formation of acp3U at positions 20 and 20a of tRNAs, respectively. In human cells, double knockout of DTWD1 and DTWD2 causes slow growth, indicating that acp3U is also physiologically important in mammals.To achieve a deeper understanding of acp3U47 formation in E. coli, we used comparative genomics to narrow down the candidate genes 21, and we confirmed the presence of acp3U in the same tRNA from Leishmania major45. Using these data, we performed a comparative genomics analysis using the Microbial Genome Database46. Among 4144 E. coli ORFs, we selected 65 genes commonly present in E. coli, as well as in the two protists (T. brucei and L. major), but absent in the five bacteria mentioned above , and classified into the TDD superfamily, which includes the TSR3 (COG2042), DTWD1 (KOG3795), and DTWD2 families49.To identify the gene responsible for acpnes Fig. . Becauseove Fig. . Based oion Fig. . yfiP beyfiP is responsible for acp3U47 formation in tRNAs, we performed LC/MS nucleoside analysis of total RNAs from E. coli WT and ΔyfiP strains .To examine whether ins Fig. . As expe3U47 using the recombinant TapT protein. As a substrate, we isolated E. coli tRNAMet lacking acp3U from the ΔtapT strain. After the reaction, the tRNA was digested by RNase T1 and analyzed by LC/MS. We clearly detected an RNA fragment containing acp3U in the presence of both TapT and SAM  values of the tRNA with and without acp3U47 were 70.0 and 66.7 °C, respectively   Fig. . We conf51. Lack of N6-isopentenyladenosine (i6A) in tRNAs in the ΔmiaA strain induces transversion-type DNA mutation50. If similar translation stress is induced by loss of acp3U in the ΔtapT strain, the genomic mutation linked to the small colony phenotype might be enhanced by the TSM pathway51. To measure the genomic mutation rate of the ΔtapT strain, we performed a mutator assay52. Specifically, we cultured the cells continuously at 42 °C and then examined their resistance to nalidixic acid  showed a lower mutation rate. As reported, the ΔmiaA strain exhibited a mild mutator phenotype. However, in contrast to our speculation, the mutation rate of the ΔtapT strain was in the same range as that of the control strain, suggesting that the small colony phenotype of the ΔtapT strain under heat stress cannot be explained by a mutator phenotype.tRNA modification-deficient strains exhibit a moderate mutator phenotype mediated by the translational stress-induced mutagenesis (TSM) pathway3U is present at position 20 of tRNATyr54 and position 20a of tRNAAsn55. We reanalyzed high throughput sequencing of human tRNAs56 and observed an apparent U-to-C conversion at positions 20 and 20a of these tRNAs, indicating that acp3U causes misincorporation during cDNA synthesis. Based on the misincorporation signatures at positions 20 and 20a of several human tRNAs, we predicted the presence of acp3U20 in two tRNAs for Cys(GCA) and Tyr, and acp3U20a in three tRNAs for Asn(GTT), Ile(AAT), and Ile(TAT). We then isolated these cytoplasmic tRNAs from HEK293T cells and analyzed the tRNA modifications by capillary LC–nanoelectrospray ionization (ESI)–MS (RNA-MS)37. We confirmed the presence of acp3U at positions 20 and 20a in these five tRNA species   Fig. .Fig. 4HutapT belongs to the DTWD2 family and TDD superfamily49. Two paralogs of DTW-containing proteins in the TDD superfamily, DTWD1 and DTWD2, are encoded in the human genome, and we hypothesized that these proteins are responsible for acp3U at positions 20 and 20a. To examine this speculation, we knocked out each of these proteins in HEK293T cells using the CRISPR-Cas9 system. Two isoforms of human DTWD2 with different first exons are produced by alternative splicing: DTWD2L (long) and DTWD2S (short).  and Tyr were drastically reduced, with the modified bases converted to D20 and U20, although a small percentage of acp3U20 remained , Ile(AAT), and Ile(TAT) in the DTWD2 KO cells  from DTWD2L KO cells, and analyzed the status of acp3U20a modification 20a formation, and DTWD2S is redundantly responsible for the remaining modification.Regarding two isoforms of ion Fig. . Both acDTWD1 or DTWD2  formation.Intriguingly, D20 or D20a appeared upon knockout of WD2 Fig. . D20 and3U modifications in human tRNAs. In comparison with WT HEK293T cells, little growth reduction was observed in DTWD1 and DTWD2 single-KO cells , and diphthamide formed in EF-2. In other tRNA modification, the acp transfer reaction is involved in the synthesis of wybutosine (yW), a hypermodified nucleoside present in eukaryotic tRNAPhe16. The SAM-dependent class I methyltransferase TYW2 catalyzes the acp transfer reaction and synthesizes yW-86, a precursor of yW60. However, TDD superfamily proteins do not have sequence homology with these acp-transferases49. According to the crystal structure of archaeal Tsr3, which is a member of TDD superfamily protein28, it has a SPOUT-class RNA methyltransferase fold. The closest structural homolog of the TDD superfamily is a tRNA methyltransferase Trm10. Further structural studies of TapT and DTWD1/2 are necessary to reveal the molecular basis of tRNA substrate recognition and acp3U formation.In this study, we discovered that the 3U47 conferred thermal stability to tRNA, increasing the Tm value by 3 °C. In the tertiary structures of tRNAs35, the acp group of the modification does not interact with any other residues but resides in close vicinity to the backbone of the T-stem. We speculated that the acp group might stabilize the local conformation of tRNA by interacting with the T-stem via magnesium ion or water31. In addition, the acp group at the N3 atom position hinders Watson–Crick base pairing and may destabilize some metastable structures so as to ensure correct folding of tRNA, similarly to the function of m1A961.Measurement of melting temperature revealed that a single acpE. coli ΔtapT strain exhibited a small colony phenotype associated with motility defect under continuous heat stress. Because ectopic expression of tapT did not restore the phenotype, we speculated that genomic mutations in swarming-related genes might be involved. Curiously, translation stress in E. coli strains bearing mutations in tRNA or the tRNA-modifying enzyme causes mutator phenotypes63. These facts prompted us to speculate that loss of acp3U in the ΔtapT strain contributes to the small colony phenotype mediated by the TSM pathway51. Unexpectedly, however, ΔtapT strain did not exhibit any mutator phenotypes under continuous heat stress conditions, indicating that neither error-prone replication nor aberrant DNA repairing is involved in this phenotype. Dysregulation of transposable elements are another possible source of genome instability64, but this should be investigated in future studies.Strikingly, the 3U formation in the D-loop; and bacterial DTWD2 (TapT), which is responsible for acp3U formation in the V-loop.To investigate the phylogenetic distribution of the TDD family, we generated a phylogenetic tree of organisms possessing or lacking each homolog  and one DTWD2 ortholog (Tb927.3.4690) are encoded in the T. brucei genome49, indicating that these proteins are responsible for forming acp3U20 and acp3D47, respectively.Vibrio cholerae tRNAs identified acp3U and its derivative at positions 20b, 46, and 4765. Curiously, the Vibrio cholerae genome encodes three DTWD2 paralogs . According to the phylogenetic analysis  subfamily, this homolog might be responsible for acp3U46 formation. VC1980 forms a unique subfamily with other bacterial orthologs near the eukaryotic DTWD2 subfamily. Considering that eukaryotic DTWD2 is responsible for acp3U20a formation, we speculated that the VC1980 subfamily is responsible for acp3U20b formation. Pseudomonas species contain two DTWD2 paralogs  subfamily, and this paralog is predicted to localize in chloroplasts, implying that it is responsible for acp3U47 formation in plastid tRNAs. AT5G54880, along with other plant orthologs, forms a subfamily near the VC1980-like subfamily, implying that this paralog is required for acp3U20b formation. Experimental verification is necessary to confirm these speculations.In plants, acpDTWD1 and DTWD2 caused severe growth defects in culture cells, indicating the physiological importance of acp3U modification. Although the functional roles of this modification remain elusive, it has been suggested that acp3U20a can coordinate Mg2+, thereby stabilizing the local conformation of tRNA31. The stabilization of tRNAs by acp3U20/20a may lead to efficient translation and rapid growth. According to the Genotype-Tissue Expression project  or ΔyagA (JW0260) with kanamycin resistance markers (Keio collection)78 and their parental E. coli strain BW25113  were cultivated in LB medium with vigorous shaking. A. ferrooxidans  was cultured in 0.5 g/L MgSO4·7H2O, 0.4 g/L (NH4)2SO4, 0.2 g/L K2HPO4, 0.1 g/L KCl, 10 mg/L FeSO4·7H2O, and 0.025% yeast extract (adjusted to pH 2.0 with 2 M H2SO4) at 45 °C79. Synechocystis sp. PCC 6803  was cultured with BG11 medium80 under LED light at 34 °C. T. thermophilus HB27  was cultured in rich medium containing 0.8% polypeptone, 0.4% yeast extract, and 0.3% NaCl (adjusted to pH 7.5 with 1 M HCl) at 70 °C rich medium as previously described81. B. subtilis str.168  was cultured in LB medium at 37 °C82. M. mobile 163K  was grown in Aluotto medium (pH 7.8), consisting of 2.1% heart infusion broth (Difco), 0.56% yeast extract, 10% horse serum (inactivated at 56 °C), and 0.005% ampicillin at 25 °C83.ΔyfiP gene for complementation, the open reading frame of yfiP with promoter region was PCR-amplified from the E. coli genome using a set of primers listed in Supplementary Table To construct a plasmid encoding 2. To compare cell proliferation, WT HEK293T cells and a series of knockout cells were seeded into 96-well plates (4.0 × 103 cells per well). Each day, a 1/10th volume of 1 mM resazurin solution in PBS was added to each well, and the cells were incubated for 3 h. The fluorescence of reduced resazurin was measured using a fluorescence microplate reader  with excitation/emission at 560/590 nm.HEK293T  and Hela  cells were cultured in high-glucose DMEM  supplemented with 5% FBS (Gibco) and 1% penicillin–streptomycin , at 37 °C in a humidified atmosphere containing 5% CODTWD1 and DTWD2 single- and double-KO cells were generated using the CRISPR-Cas9 system, as described84. Two sgRNAs targeting exon 1 of the DTWD1 gene, and two sgRNAs targeting exon 2 or 3 of the DTWD2 gene, were designed. Sense and antisense oligonucleotides for the sgRNAs (Supplementary Table 85. HEK293T cells (1.0 × 105 cells per plate) were transfected with the vector bearing the sgRNA sequence, along with pEGFP-N1 (Clontech) and modified pLL3.7 vectors harboring a puromycin resistance gene. One day after transfection, the cells were seeded at low density and selected with 1 μg/mL puromycin. The KO cell lines were selected by confirming the frameshift mutations in the target region. DTWD1/DTWD2 double-KO cells were generated from the DTWD1 single-KO cells.E. coli were extracted as described86. Total RNAs of A. ferrooxidans, Synechocystis sp. PCC 6803, T. thermophilus HB27, B. subtilis str.168, and M. mobile 163K were extracted using the acidic phenol method as described83. Total RNAs were extracted from HEK293T cells using TriPure Isolation Reagent (Roche Life Science).Total RNAs of 87 using the 5′-terminal ethylcarbamate amino-modified DNA probes (Sigma Aldrich Japan) listed in Supplementary Table Individual tRNAs were isolated by reciprocal circulating chromatography as describedE. coli, total RNAs (1.6 μg) were digested with 0.8 U nuclease P1  at 37 °C for 60 min. The digests were adjusted to 50 mM ammonium bicarbonate (pH 8.2), followed by addition of 0.2 U phosphodiesterase I (Worthington) and incubated at 37 °C for 60 min, then 0.2 U of bacterial alkaline phosphatase  was added, and incubated at 37 °C for 60 min, as described86. Nucleosides were dissolved in 90% acetonitrile (10% water), and subjected to a hydrophilic interaction LC  coupled with ESI-MS on a Q Exactive hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific), equipped with an ESI source and an Ultimate 3000 liquid chromatography system (Thermo Fisher Scientific)88. The mobile phase consisted of 5 mM ammonium acetate (pH 5.3) (solvent A) and acetonitrile (solvent B), and the nucleosides were chromatographed with a flow rate of 100 μL/min in a multistep linear gradient; 90–40% B from 0 to 30 min, 40% B for 10 min, and then initialized to 90% B. Proton adducts of nucleosides were scanned in a positive polarity mode over an m/z range of 103–700. Xcalibur 3.0.63 (Thermo Fisher Scientific) was used for the system operation.For nucleoside analysis of 79 or a Q Exactive hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) equipped with an ESI source and an Ultimate 3000 liquid chromatography system (Thermo Fisher Scientific)88. The mobile phase consisted of 5 mM ammonium acetate (pH 5.3) (solvent A) and acetonitrile (solvent B). Xcalibur 2.0 SUR1 (Thermo Fisher Scientific) was used for operation of the LCQ Advantage system,.Nucleoside analyses for comparative genomics were performed by reverse-phase chromatography/ESI-MS. Total RNAs (16–160 µg) were digested in a reaction mixture containing with 20 mM ammonium acetate (pH 5.3), 0.04–0.08 U BAP, and 0.05–0.1 U nuclease P1, and subjected to InertSil ODS-3  or SunShell C18  column, then analyzed by an LCQ Advantage ion-trap mass spectrometer (Thermo Fisher Scientific) with an ESI source and an HP1100 liquid chromatography system (Agilent Technologies)1 in 20 mM ammonium acetate (pH 5.3) at 37 °C for 60 min. The digests were mixed with 1/10 vol. of 0.1 M triethylamine acetate (pH 7.0) and subjected to LC/nano ESI-MS on an LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific) equipped with a splitless nanoflow high-performance LC (nano-HPLC) system  using a nano-LC trap column  and a capillary column  as described37. Digested fragments were separated for 40 min at a flow rate of 300 nL/min by capillary LC using a linear gradient from 2 to 100% solvent B (v/v) in a solvent system consisting of 0.4 M 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) (pH 7.0) (solvent A) and 0.4 M HFIP (pH 7.0) in 50% methanol (solvent B). The eluent was ionized by ESI source in a negative polarity and scanned over an m/z range of 600–2000. Xcalibur 2.0.7 (Thermo Fisher Scientific) was used for the system operation.For RNA fragment analysis, the isolated tRNAs (2 pmol) were digested with 10 U of RNase Thttps://mods.rna.albany.edu/masspec/Mongo-Oligo) was used for assignment of the product ions in CID spectra.The LC/MS data were analyzed using Xcalibur Qual browser (Thermo Fisher Scientific). In nucleoside analysis, the peak of modified nucleoside was normalized by the peak area of the unmodified nucleoside. In RNA fragment analysis, the peak of each fragment was normalized by the sum of modified and unmodified fragments. Mongo Oligo Mass Calculator v2.08 (yfiP was PCR-amplified from the E. coli genome using the primers listed in Supplementary Table E. coli BL21(DE3) strain was transformed with the plasmid and cultured at 37 °C. When the absorbance at 600 nm (A600) reached 0.5, isopropyl-β-D-thiogalactopyranoside was added to a final concentration of 0.1 mM for inducing expression, and the cells were cultured at 18 °C for an additional 24 h. The cells were harvested and lysed by sonication in lysis buffer . The soluble recombinant protein was purified using a HisTrap HP column  and anion exchange chromatography  using the AKTA Purifier 10 system . The purified protein was dialyzed against a buffer consisting of 20 mM HEPES-KOH (pH 7.5), 200 mM KCl, 6 mM β-Me, and 10% glycerol, and then stored at −80 °C.To construct an expression plasmid for TapT recombinant protein, the open reading frame of Met from the ΔtapT strain by reciprocal circulating chromatography87. The tRNAMet transcript was prepared by in vitro transcription using T7 RNA polymerase, essentially as described89. The synthetic DNAs used for template construction by PCR are listed in Supplementary Table 2, 5 mM DTT, 2.5 mM spermidine, 0.01% Triton X-100, 30 nM DNA template, 2 mM GTP, 2 mM CTP, 2 mM UTP, 2 mM ATP, and 10 mM GMP. Subsequently, the reaction mixture was subjected to phenol–chloroform extraction and desalted by passing through a PD-10 columns . The transcribed tRNA was purified by running on 10% denaturing polyacrylamide gel electrophoresis (PAGE).For the substrates, we isolated tRNA3U modification was performed at 37 °C for 2 h in a reaction mixture containing 20 mM HEPES-KOH (pH 7.5), 10 mM MgCl2, 100 mM NaCl, 1 mM DTT, 1 mM SAM, 8.5 μM tRNAMet, and 0.75 μM recombinant TapT. The tRNA product was digested with RNase T1 (Thermo Fisher Scientific) and subjected to LC/MS analysis as described above.In vitro reconstitution of acp2, and incubated at 75 °C for 5 min, followed by cooling at room temperature for annealing. A UV–Vis spectrophotometer  equipped with an 8 multi-quartz micro cell array  was used to monitor the hyperchromicity of tRNA. The temperature gradient was as follows: 25 °C for 30 s, and then ramped at 5 °C/min to 40 °C and held for 5 min, and ramped at 0.5 °C/min to 90 °C.The tRNA (2.5 μg) was dissolved in degassed buffer containing 20 mM HEPES-KOH (pH 7.5), 150 mM NaCl, and 6 mM MgClE. coli strains were cultivated in 5 mL LB medium with vigorous shaking at 37 °C or 42 °C for 16–24 h until reaching the late stationary phase. Then, cells were diluted by a factor of 106 or 107 so that the concentration was suitable for counting the colony numbers. One hundred microliters of the diluted cells were spread on LB agar plate and cultured at 37 °C overnight. In parallel, 2.5 μL harvested cells was inoculated into 5 mL fresh LB medium and cultured at the same temperature. The process described above was repeated for 5–7 days. The frequency of small colonies appearing on the plate was calculated by counting the number of normal and small colonies on each plate.90. Precultured E. coli cells were diluted to 0.01 A600, and then 2 μL diluted cells was inoculated at the center of the soft agar plate . The plates were cultured at 30 °C for 16 h. The swarming diameter of each strain was averaged from five different sets of measurements.The motility assay was performed basically as described92. In addition to the ΔtapT strain, ΔmutS, ΔmiaA, ΔyagA strains were used as the strong mutator, mild mutator, and negative controls, respectively. E. coli cells were cultivated at 42 °C for 5 days and then plated on LB plates at every passage as described in the morphological analysis section. At every passage, adequate amounts of the harvested cells were spread on LB agar plates containing 80 μg/mL nalidixic acid and cultured at 37°C overnight. Mutation rate was calculated from the ratio of colony numbers on the LB plates with and without nalidixic acid.The mutator assay was carried out basically as describedDTWD1/DTWD2 double-KO cells were dissolved by 10% denaturing PAGE, stained with SYBR safe (Invitrogen), and blotted onto a nylon membrane  in 0.5 × TBE using a Transblot Turbo apparatus (Bio-Rad). Hybridization was performed essentially according to the manufacturer’s instructions  at 42 °C with 2 pmol of 5′-32P-labeled oligonucleotides  from WT HEK293T and 5 cells per dish) were transfected with the pcDNA-3.1 vectors (Invitrogen) bearing the open reading frame of DTWD1, DTWD2L, or DTWD2S, each of which expresses the C-terminally flag-tagged protein. The cells were fixed with 3.7% formaldehyde, permeabilized with 1% Triton X-100, and blocked with 20% Ezblockchemi (ATTO). Immunostaining was performed with anti-DYKDDDDK IgG antibody (1:1000)  for the primary antibody and anti-mouse IgG Alexa fluor 488 (1:1000)  for the secondary antibody. Then, cells were stained with DAPI . The pictures were taken using DMI 6000 (Leica).Hela cells (4.0 × 10Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Peptides corresponding to N- and C-terminal heptad repeat regions  of gp41 can inhibit HIV-1 infection in a dominant negative manner by interfering with refolding of the viral HR1 and HR2 to form a six-helix bundle (6HB) that induces fusion between viral and host cell membranes. Previously, we found that HIV-1 acquired the mutations of Glu560 (E560) in HR1 of envelope (Env) to escape peptide inhibitors. The present study aimed to elucidate the critical role of position 560 in the virus entry and potential resistance mechanisms.The Glu560Lys/Asp/Gly (E560K/D/G) mutations in HR1 of gp41 that are selected under the pressure of N- and C-peptide inhibitors modified its molecular interactions with HR2 to change 6HB stability and peptide inhibitor binding. E560K mutation increased 6HB thermostability and resulted in resistance to N peptide inhibitors, but E560G or E560D as compensatory mutations destabilized the 6HB to reduce inhibitor binding and resulted in increased resistance to C peptide inhibitor, T20. Significantly, the neutralizing activities of all mutants to soluble CD4 and broadly neutralizing antibodies targeting membrane proximal external region, 2F5 and 4E10 were improved, indicating the mutations of E560 could regulate Env conformations through cross interactions with gp120 or gp41. The molecular modeling analysis of E560K/D/G mutants suggested that position 560 might interact with the residues within two potentially flexible topological layer 1 and layer 2 in the gp120 inner domain to apparently affect the CD4 utilization. The E560K/D/G mutations changed its interactions with Gln650 (Q650) in HR2 to contribute to the resistance of peptide inhibitors.These findings identify the contributions of mutations of E560K/D/G in the highly conserved gp41 and highlight Env’s high degree of plasticity for virus entry and inhibitor design. The HIV-1 envelope glycoprotein (Env) forms a trimer of noncovalently associated heterodimer of gp120 and gp41. The binding of the surface subunit gp120 to the CD4 receptor on host cells triggers conformational changes of Env that allow gp120 binding to the chemokine receptor CCR5 or CXCR4. After that, the hydrophobic fusion peptide at N-terminal region of transmembrane subunit gp41 inserts into cell membrane and the pre-hairpin intermediate forms . This prThe fusion process provides possibilities to block virus entry and prevent HIV-1 infection. The first fusion inhibitor licensed for clinical use, enfuvirtide  that mimics HR2 sequences of gp41 can package with the coiled coil core of HR1 in an antiparallel manner to form a heterogenous 6HB (inhibitor bundle) that interferes with the formation of the homogenous 6HB (endogenous bundle) –14. HowePeptides that mimic HR1 segment also inhibit virus fusion, but they are not yet in clinical use . The lowThe reported resistance mutations to T20 were consistent in vitro and in vivo. The highest frequency of mutations for T20 was within the region of amino acids 547–556 of Env , 34. ExcTo investigate the property and resistance mechanisms of these substitutions at position 560 in HR1, we constructed HIV-1 LAI pseudoviruses bearing Env with mutations E560K/D/G. The mutation E560K conferred resistance to N36 and IZN36 through increasing endogenous 6HB stability, but E560G or E560D as compensatory mutations destabilized the 6HB to reduce inhibitor binding and resulted in increased resistance to T20. The sensitivity of all mutants to soluble CD4 (sCD4) and the broadly neutralizing Abs (bNAbs) targeting membrane proximal external region (MPER), 2F5 and 4E10, was enhanced. The molecular modeling analysis also revealed the possible conformational changes of the E560K/D/G mutations indicating that the residue at the position 560 could regulate Env conformations through interactions with gp120 and HR2 of gp41. Our findings further clarified the molecular mechanism for the resistance mutations at position 560 to fusion peptide inhibitors and provided new insights for HIV-1 entry and inhibitor design.+CXCR4+ cells that express high levels of CD4 and CXCR4 receptors  and three mutations E560K/D/G were reported under treatment of T20 in clinical data , the senNext, we investigated whether these mutations altered the sensitivity to sCD4. All three mutants increased sensitivity by 1.63- to 4.62-folds to sCD4 compared to wild type, suggesting that the specific residues at the position 560 could influence the interaction between Env and CD4 receptors  in their thermal denaturation curves (ranging from 42 °C to 52 °C), consistent with the formation of 6HB structures  by 6 °C, but mutation E560D or E560G decreased the Tm (∆Tm) by 3 °C or 4 °C, respectively, compared to wild-type N36.Mutations in the HR1 would not only directly interfere with inhibitor binding to gp41 but also probably affect the affinity of endogenous 6HBs. We detected the effects of these mutations at the position 560 of 6HB structure using circular dichroism and native PAGE. The α-helicity and thermal stability of 6HB formed by the interaction of C34 with N36 or its mutants at equimolar concentration were analyzed , the major residue at position 560 is glutamic acid (E), and other residues such as aspartic acid (D), lysine (K) and glycine (G) are also observed in HIV-1 primary isolates. These data indicate that although the residue at the position 560 in the Env of HIV-1 strains is highly conserved, various mutants exist in nature.In our previous work the mutations E560D and E560G were found in the resistance LAI strains under T20 selection in vitro by increasing its concentration gradually (our unpublished data). And E560K is observed in the screened HIV-1 JRcsf  and LAI +CXCR4+ and RC4 cells than wild type. Meanwhile, the sensitivity of these three mutants to sCD4 increased  at the position 560 is the only negative charged residue in the N36 region and occupy position b in the 4,3-hydrophobic heptad repeat which oriented into outside of 6HB. The substitution of a positively charged residue Lysine (E560K), a negative charged residue Aspartic acid (E560D), or an uncharged residue Glycine (E560G) changed the size of side chain or charges and may disrupt the interactions between HR1 with HR2 or gp120. These changes may involve in the resistance mechanisms to fusion peptide inhibitors. The one universal mechanism of improving the stability of the endogenous 6HB to favor the virus might be a more efficient resistance mechanism, especially for an inhibitor that can bind to two different sites of Env.50 values to N36 and IZN36 and was sensitive to T20, and E560G mutant was sensitive to all tested fusion peptide inhibitors  of 6HB containing G547D mutation compared to that containing wild type based on CD analysis (our unpublished data). And as the analyzing above, E560D and E560G mutations reduced the stability of 6HB through rearrangements of hydrogen bonds between them and Q650 network  were kindly provided by Dan Littman (New York University). HEK 293T cells were purchased from America ATCC cell bank. RC4 cells that are derived from HeLa cells and express low levels of CD4 and CCR5 were a gift from David Kabat  [U87 cells expressing CD4 and CXCR4  . The expPeptides LAI N36 , LAI N36 560K (SGIVQQQNNLLRAIKAQQHLLQLTVWGIKQLQARIL), LAI N36 560D (SGIVQQQNNLLRAIDAQQHLLQLTVWGIKQLQARIL), LAI N36 560G (SGIVQQQNNLLRAIGAQQHLLQLTVWGIKQLQARIL), LAI IZN36 (IKKEIEAIKKEQEAIKKKIEAIEKEISGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL), LAI T20  and LAI C34  were synthesized by Shanghai JiEr biochemistry company. SDS-PAGE and analytic high performance liquid chromatography (HPLC) showed that the purity of all peptides were over 95%. The molecular weight of all peptides was confirmed by matrix-assisted laser desorption ionization-time-of-flight mass spectroscopy (MALDI-TOF–MS). The bNAbs 2F5 and 4E10 were provided by NIH . Soluble CD4 (sCD4) was purchased from Progenics Pharmaceuticals .env gene within pCMV/R-Env(LAI). Each plasmid was confirmed by sequencing the full-length env gene.Env-expressing plasmids pCMV/R-Env with E560K, E560D or E560G were created by overlapped PCR using pCMV/R-Env(LAI) wild type (Genebank No. K02013.1)  as a tem6 293T cells per 10 cm-diameter dish. Supernatants were collected and filtered at 48 h after transfection and quantified by a p24 enzyme-linked immunosorbent assay (ELISA) . Pseudoviruses containing 5 ng of p24 infected target cells U87CD4+CXCR4+ and RC4 cells (2 × 104 cells/well in 96-well plates), and their infectivity was normalized to wild type. The infectivity of serially diluted pseudoviruses was also detected and 50% tissue culture infectivity dose (TCID50) was calculated. The equivalent TCID50 inocula of pseudoviruses was added to each well of target cells (2 × 104 cells/well in 96-well plates) in the presence of peptide, sCD4 or bNAbs in a total volume of 100 µl supplemented with 2.5 µg/ml of DEAE (Sigma). At 48 h postinfection, the luciferase activity was measured using luciferase substrate  on a Modulus™II microplate multimode reader .HIV-1 pseudoviruses were generated by the cotransfection with 0.5 µg of the Env-expressing plasmid, 4 µg of Env-deficient HIV-1 genome plasmid (pCMVΔ8.2) and 4 µg of reporter plasmid (pHR’-Luc) in 5 × 10m) value was determined using Chirascan software. The Tm averages of at least two measurements for each complex were calculated.HR1 and HR2 peptides were mixed (10 µM each) in 50 mM sodium phosphate (pH 7.0) containing 150 mM NaCl and incubated at 37 °C for 30 min . These peptides or peptide mixtures were acquired on a Chirascan spectropolarimeter  at 20 °C using a 1.0 nm bandwidth, 0.1 nm resolution, 0.1 cm pathlength, 4.0 s response time, and a 5 nm/min scanning speed. The spectrums were corrected by subtraction of a blank corresponding to the solvent , 56. TheThe 6HB which is composed of HR1 and HR2 peptides was detected by native PAGE as described previously . The pep50 value) of peptide inhibitors for each pseudovirus was calculated by nonlinear regression analysis using GraphPad Prism software . The average IC50 for each mutant or inhibitor was compared to that for the wild-type pseudovirus using student t test with non-parametric assumption and p values equal to or less than 0.05 were considered significant.The 50% inhibitory concentration  Infectivity of pseudoviruses in U87CD4+CXCR4+ and RC4 cells. The relative luciferase infectivity is indicating that the ratio is IC50 for the indicated pseudovirus/LAIwt pseudovirus. The averages and SD values (error bars) of at least three independent experiments are shown. *p < 0.05 compared with LAIwt. (b) Western blot of pseudovirions bearing the envelope glycoproteins with mutations of E560. Top panel shows a blot probed with a monoclonal antibody  recognizing gp160 and gp41. Bottom panel shows the same blot probed with a monoclonal antibody VAK4 [Figure S2. Modeling the mutations of L544S, G547D and E560/D/G in the six-helix bundle (6HB). Mutation L544S is modeled in the 6HB conformation (PDB entry 1ENV) in a ribbon model in cross-section view (a) and longitudinal view (b). The 6HB of wild type (c), the mutants with mutations L544S, G547D and E560G (d) or with mutations L544S, G547D and E560D (e) are modeled. Interatomic distances are marked by the dashed lines.ody VAK4  recognizAdditional file 2: Table S1. Primer sequences for LAI mutant env creation.


Pseudomonas aeruginosa (PA) is an opportunistic bacterium that causes severe and chronic infection of immune-depressed patients. It has the ability to form a biofilm that gives a selective advantage to the bacteria with respect to antibiotherapy and host defenses. Herein, we have focused on the tetrameric soluble lectin which is involved in bacterium adherence to host cells, biofilm formation, and cytotoxicity. It binds to l-fucose, d-mannose and glycan exposing terminal fucose or mannose. Using a competitive assay on microarray, 156 oligosaccharides and polysaccharides issued from fermentation or from the biomass were screened toward their affinity to LecB. Next, the five best ligands  were derivatized with a propargyl aglycon allowing the synthesis of 25 trivalent, 25 tetravalent and 5 monovalent constructions thanks to copper catalyzed azide alkyne cycloaddition. The 55 clusters were immobilized by DNA Directed immobilization leading to the fabrication of a glycocluster microarray. Their binding to LecB was studied. Multivalency improved the binding to LecB. The binding structure relationship of the clusters is mainly influenced by the carbohydrate residues. Molecular simulations indicated that the simultaneous contact of both binding sites of monomer A and D seems to be energetically possible.The Gram negative bacterium  The emergence of antibiotic-resistant bacterial strains calls for new anti-bacterial strategies. One of them consists of inhibiting virulent factors in particular lectins involved in bacterial adhesion and/or biofilm formation using monovalent glycomimetics  or multiPseudomonas aeruginosa (PA) is a Gram-negative opportunistic bacterium involved in severe infections of the respiratory and/or urinary tracts, skin and eyes +; found 461.1877.Azide solid support 157 ,40, N3-p 161–163  were pre164b   affording 4.04 g (78%%) of phosphoramidite derivative 165b. 1H-NMR : δ 1.16 ; 2.43 ; 2.63 ; 3.52 ; 3.72 ; 3.88 ; 4.14 ; 7.32 ; 7.77 . 13C-NMR : δ.22.26 and 22.34 (CH2CN), 23.6 (CH3Ar), 26.50, 26.57, 26.60, 26.67 (4× CH3CH), 44.95 and 45.12 (CHMe2), 60.37 and 60.61 (POCH2CH2CN), 64.46 and 64.69 , 70.65, 71.25, 72.50, 72.55, 72.64, 72.72 (C7-C12 OCH2), 73.15 and 73.24, , 119.8 (CN), 130.0 and 131.8 (ArH), 135.0 (Ar-S), 146.8 (Ar-C). 31P-NMR : δ 148.59. HRMS (ESI/Q-TOF): Calcd. for C24H42N2O8PS 549.2400 [M + H]+; found 549.2393.The same protocol was applied to tosylated tetraethyleneglycol v/v/v; Cap B: 10% N-methylimidazole in THF; Oxidizer solution: 0.1 M iodide in THF/pyridine/H2O, 78:20:2, v/v/v. Detritylation solution: 3% trichloroacetic acid (TCA) in CH2Cl2 and dry CH3CN for DNA synthesis were purchased from Biosolve . Cy3-amidite was purchased from GE Healthcare. All oligoglycooligonucleotides were purified and analyzed by C18 reversed-phase HPLC  on an Ultimate 3000 system  with a Reodyne  injector and a UV DAD 3000 detector. Oligonucleotides were dosed by UV-Vis spectrophotometry at 550 nm on a 300 Bio UV-Vis spectrophotometer .The oligonucleotide synthesis on solid support was performed on a 381A or 394 DNA synthesizer from Applied Biosystems . Reactions under microwave activation were performed on an Monowave 300 system . Solutions of Cap A, Cap B and iodide were purchased from Link Technologies as well as the commercial solid supports. Cap A: Acetic anhydride/pyridine/THF, 1:1:8, 158–163 (10 µmol in 100 μL H2O), freshly prepared degassed aqueous solutions of CuSO4  and sodium ascorbate , degassed water (400 μL) and MeOH (350 μL) were added to 5 µmol of azide solid support 157. The resulting mixture in a sealed tube G10 was heated to 60 °C for 60 min, stirring at 400 r/min, using a microwave synthesizer. The temperature was monitored with an internal infrared probe. The solution was removed, and CPG beads were washed with H2O (3 × 2 mL), MeOH (3 × 2 mL) and CH2Cl2 (3 × 2 mL), and dried affording the solid-supported polyol scaffolds. An aqueous solution of propargyl derivatives 165a or 165b using coupling, oxidation and capping steps. For the coupling step, benzylmercaptotetrazole (BMT) was used as an activator (0.3 M in anhydrous CH3CN) and phosphoramidites 165a or 165b (0.15 M in anhydrous CH3CN) were introduced four times with a 180 s coupling time. Oxidation was performed with oxidizer solution for 15 s. Capping was applied using CapA + CapB for 120 s.Introduction of tosyl linkers: Solid-supported polyol scaffolds (1 µmol) were treated, on a DNA synthesizer, with phosphoramidites 3CN) were introduced with a 20 s coupling time and Cy3 amidite (0.06 M in anhydrous CH3CN) with a 180 s coupling time. The capping step was performed with CapA + CapB for 15 s. Each oxidation was performed with oxidizer solution for 15 s. Detritylation was performed with 3% TCA in CH2Cl2 for 65 s.Then, the elongation of DNA sequences was carried out using a standard synthesis cycle at the 1 µmol scale by phosphoramidite chemistry. For the coupling step, BMT and commercially available nucleoside phosphoramidites (0.1 M in anhydrous CH3)  for 2 h at 65 °C. The beads were washed with acetonitrile (5 × 2 mL) and dried by flushing with argon.The solid-supported oligonucleotides bearing the tri—or tetra-tosyl scaffold (1 µmol) were treated with a solution of tetramethylguanidinuim azide  oligosaccharide 166–170 (40 nmol in 40 µL H2O), ~0.1 mg of copper nanopowder were added and the mixture was sonicated for 2 s and then stirred at 65 °C with microwave assistance for 90 min. Method A: Mono-conjugation. In a G4 microwave tube from Anton-Paar, Oligonucleotide-hexylazide (20 nmol in 20 µL H2O) oligosaccharide 166–169 (100 nmol in 100 µL H2O), ~0.1 mg of copper nanopowder were added and the mixture was sonicated for 2 s and then stirred at 65 °C under microwave assistance for 90 min. Method B: Tri-conjugation. In a microwave tube G4 from Anton-Paar, Oligonucleotide-pentafuranosyl triazide (20 nmol in 20 µL H2O), oligosaccharide 166–169 (100 nmol in 50 µL H2O), THPTA  and ~0.1 mg of copper nanopowder were added and the mixture was sonicated for 2 s and then stirred at 65 °C under microwave assistance for 60 min. Method C: Tri-conjugation. In a G4 microwave tube from Anton-Paar, Oligonucleotide-pentafuranosyl triazide (20 nmol in 20 µL H2O), oligosaccharide 166–169 (140 nmol in 140 µL H2O) and ~0.1 mg of copper nanopowder were added and the mixture was sonicated for 2 s and then stirred at 65 °C under microwave assistance for 90 min. Method D: Tetra-conjugation. In a G4 microwave tube from Anton-Paar, Oligonucleotide-hexapyranosyl tetraazide (20 nmol in 20 µL H2O), oligosaccharide 166–169 (100 nmol in 50 µL H2O), THPTA  and ~0.1 mg of copper nanopowder were added and the mixture was sonicated for 2 s and then stirred at 65 °C under microwave assistance for 60 min. Method E: Tri-conjugation. In a G4 microwave tube from Anton-Paar, Oligonucleotide- hexapyranosyl tetraazide , oligosaccharide 170 (100 nmol in 100 µL H2O), 0.4 M TEAAc buffer pH 7.5 (20 µL) and ~0.1 mg of copper nanopowder were added and the mixture was sonicated for 2 s and then stirred at 65 °C under microwave assistance for 120 min. Method F: sLex Tetra-conjugation. In a G4 microwave tube from Anton-Paar, Oligonucleotide-hexapyranosyl tetraazide (20 nmol in 20 µL H2O), oligosaccharide 170 (140 nmol in 140 µL H2O), 0.4 M TEAAc buffer pH 7.5 (20 µL) and ~0.1 mg of copper nanopowder were added and the mixture was sonicated for 2 s and then stirred at 65 °C under microwave assistance for 120 min. Method G: sLe18 reverse-phase HPLC. The products were co-evaporated with water, and then lyophilized.The crude solutions were centrifuged and the supernatant was directly purified by Cv/v) containing 10% of ammonium citrate and a few beads of DOWEX 50W-X8 ammonium sulfonic acid resin were added. Then, 1 µL of the mixture was placed on a plate and dried at room temperature and pressure.MALDI-TOF mass spectra were recorded on a Voyager DE mass spectrometer  equipped with a nitrogen laser. MALDI conditions were: accelerating voltage 24,000 V; guide wire 0.05% of the accelerating voltage; grid voltage 94% of the accelerating voltage; delay extraction time 500 ns. 1 µL of sample was mixed with 5 µL of a saturated solution of HPA in acetonitrile/water . When they are purchased from other providers, their origin is then specified.v/v/v). Microarrays were made from Nexterion glass D slides (Schott). They were microstructured with 40 square wells . The slide microstructuring was performed using xurography and oxide-buffered etch (BOE): (Ammonium fluoride/hydrofluoric acid mixture)/(37% hydrochloric acid)/; 1:2:2 ) for 30 min in an ultrasound bath and rinsed with water (3 × 5 min). A structured vinyl adhesive mask  was stuck on each slide and slides were incubated in the BOE mixture for 2 h at room temperature under agitation. Then, the slides were rinsed with water (3 × 5 min) and the masks were removed from the slides.The slides were washed with a piranha mixture (30% H−1 mbar). After introduction of the tert-butyl-11-(dimethylamino)silylundecanoate (TDSUM), it was vaporized under reduced pressure and allowed to react for 12 h at pressure ranging between 4 × 10−1 and 10−1 mbar and the temperature was set at 145 °C. The slides were washed with tetrahydrofuran (THF) and then water . The tert butyl ester functionalized slides were then converted into the corresponding acid by incubation for 7 h in glacial formic acid at room temperature. After washing with dichloromethane and water , acid functions were activated for amine coupling using N-hydroxysuccinimide  and N,N’-diisopropylcarbodiimide (0.1 M) in dry THF overnight at room temperature. The slides were finally rinsed with THF and dichloromethane .The microstructured slides were functionalized by silanization using a gas phase protocol previously described . BrieflyTwo amino-modified oligonucleotides (sequence c1 and c2)  were used. Each oligonucleotide was prepared at 25 µM in phosphate-buffered saline (PBS) 10× (pH 8.5) and spotted using the sciFLEXARRAYER s3 piezo-electric system . Thirty two spots (16 spots per amino-modified oligonucleotide) of 0.3 nL were spotted at the bottom of each well of the microarray. The substitution reaction between the amino-modified oligonucleotides and NHS-activated microarray surface was performed for 3 h under water-saturated atmosphere and overnight in normal atmosphere to allow for the gentle drying of the spots. To remove the excess of oligonucleotides, the slides were washed for 30 min at 70 °C in sodium dodecyl sulfate (SDS) 0.1% and rinsed with water.To avoid nonspecific adsorption, the slides were incubated for 2 h with bovine serum albumin (BSA) at 4% in PBS 1× (pH 7.4). Then, the slides were washed in 0.05% PBS 1×-Tween20 (3 × 3 min), in PBS 1× (3 × 3 min) and rinsed with water. BSA blocking was repeated following the same procedure, after the oligoglycocluster immobilization.F1 [Cy3-oligonucleotide oligoglycoclusters F1   was screened in a competitive assay. The binding of the 156 carbohydrates was screened toward the lectin LecB . Lectin was labeled as previously described [® 647 Microscale Protein Labelling Kit (Invitrogen).A library of 156 sugars extracted from biomass or produced by bacterial fermentation see  issued fescribed  using thl-fucose (as a control) were tested. The inhibitory effect of each oligosaccharide was determined at four concentrations 0.1, 1.0, 5.0 and 15 µM and at 0.5, 2.5, 5.0 and 7.5 mg/L for polymers: Aliginate, galacturonan, galactomannan, glucomannan, mannan, ulvan, xylan and the glucan. To this end, the labeled LecB lectin , BSA , CaCl2  in PBS 1× (pH 7.4) were mixed with the solutions of oligosaccharides . For each concentration, 1.5 µL of the mixture was poured into a microwell, where F1 was previously immobilized. The slides were incubated for 3 h at 37 °C in a water-saturated atmosphere. In each microwell, a competition between the two types of glycoclusters (F1) and oligosaccharide occurred to bind LecB. The microarrays were washed with 0.02% PBS-Tween20 for 5 min at room temperature. The fluorescence signal emitted by the complex glycocluster/Alexa 647-lectin was detected and an average of the mean fluorescence signal of 16 spots per glycocluster was calculated. The percentage of inhibition was calculated as follows: The alexa-647 fluorescent signal for a given sugar was normalized by the Alexa 647 fluorescent signal measured at 0.1 µM of fucose. Under these conditions, no inhibition of the interaction between LecB and the two fucosylated clusters by fucose is observed (IC50 10 and 20 µM for fucose).On each slide, nine oligosaccharides and α-x pentasaccharide, Lex tetrasaccharide, Leb tetrasaccharide, 3FL and Lea tetrasaccharide), two oligoglycoclusters were immobilized per well by DDI with 32 repetition spots. In order to assess the relative surface densities of the oligoglycoclusters, the slides were scanned at 532 nm (excitation of Cy3) using the Axon microarray scanner, GenePix 4100A software package. The fluorescence signal of each oligoglycocluster was determined as the average of the mean fluorescence signal of 32 spots. The fluorescent signal of the oligoglycoclusters deviated by less than 15%.The interaction of LecB with the oligoglycoclusters was measured with Alexa 647 –labeled LecB. For the screening of the library of oligonucleotide oligoglycoclusters bearing several oligosaccharides  were added in each well. Incubation was performed at 37 °C for 3 h in a water-saturated atmosphere. The slides were washed with 0.02% PBS-Tween20 for 5 min and rinsed with water. The slides were scanned at 635 nm  using the Axon microarray scanner, GenePix 4100A software package . The Alexa 647-lectin fluorescence signal for each oligoglycocluster was determined as the average of the mean fluorescence signal of 32 spots. Error bars were calculated from these 32 spots.1.5 µL of Alexa 647-LecB . Docking experiments were performed with the GOLD software . The six heavy atoms of the ligand fucose ring found in the structure file were used as a scaffold in the active site. The rest of the ligand was considered as flexible during the docking process. Side chains of the following residues in the vicinity of the active site of the same monomer, S22, S23, T45, E95, D96, T98, D99, N100, D101 and D104 were also defined as flexible during the docking procedure. For each ligand, 10 poses that are energetically reasonable were kept while searching for the correct binding mode of the ligand. The decision to keep a trial pose is based on a computed ligand-receptor interaction energy (score) of that pose. The ChemPLP fitness scoring function is used to rank the poses. The ChemPLP scoring function is the default in Gold version 5.2 used here. Additionally, an empirical potential energy of interaction ΔE for the ranked complexes is evaluated using the simple expression (2): For that purpose, the Spectroscopic Empirical Potential Energy function SPASIBA and the corresponding parameters are used ,50. MolePseudomonas aeruginosa is an opportunistic bacterium. It is a causative agent of lung infections, the major cause of morbidity and mortality among cystic fibrosis patients and it remains a leading pathogen in nosocomial infections. It causes severe and chronic infections due to its ability to form a biofilm that gives a selective advantage to the bacteria with respect to antibiotherapy and host defenses. During the formation of the biofilm, several virulent factors are involved. Among these virulence factor, lectins play a key role. Therefore, molecules inhibiting these lectins are envisioned as new anti-bacterial agents. The soluble homotetrameric fucophilic lectin LecB of PA is classified as a virulent factor and is involved in biofilm formation, host/bacteria and bacteria/bacteria interaction, cytotoxicity and inhibition of ciliated removal. The gram negative bacterium a, Lewisb, Lewisx tetrasaccharides, sialyl-Lewisx pentasaccharide and 3’-Fucosyllactose (3FL), exhibiting good affinity for LecB, were selected for the synthesis of further oligoglycoclusters. To this end, they were first propargylated and then introduced by CuAAC on furanoside or pyranoside scaffolds exhibiting di- or tetra-ethyleneglycolazide arms. A library of 55 glycoclusters  exhibiting one, three or four times the five selected oligocarbohydrates and a DNA tag was prepared. Thanks to the DNA tag, they were easily immobilized on a DNA chip allowing for their rapid screening against LecB using only few tens of picomoles of material. The highest bindings were found for Lea, closely followed by 3FL and then by Leb, Lex and with the lowest affinity for sLex. From these two screenings, LecB seems to preferentially bind Fucα1-4 > Fucα1-3 > Fucα1-2 oligosaccharides. Additional fucose on the side chain does not necessarily increase the binding. The clusters built from a galactose core were found to be the best. However, multivalence does increase the binding and simulation seems to be in favor of a divalent contact. However, the loops V69–P73 and D96–D99 seem to generate a geometrical constrain. However, the stoichiometry of the binding remains to be confirmed by Isothermal Titration Calorimetry.Herein, 156 glycans were screened for their binding toward LecB through screening on a glycoarray, where they competed with an immobilized fucocluster. This screening by competition is straightforward and allows for the rapid evaluation of the binding of simple to complex oligosaccharides using only a few mg of them. The glycans to be evaluated were directly used without any derivatization. Among these 156 glycans, 36 were able to interact with LecB with different bindings from low to high. Five oligosaccharides, Lewisa tetrasaccharide and 3FL display higher affinity to LecB than the mannose-centered tetrafucocluster PC_2, confirming the interest to search for new glycans to improve the affinity of glycoclusters to LecB.The data showed that oligoglycoclusters built from LeSeveral authors have demonstrated that LecB inhibitors are able to reduce biofilm formation, bacterial adhesion and consequently to provide protection against PA infections. ,31 There


This fungus was isolated from deep-sea sediment collected at a depth of 1235 m and characterized. Piezotolerance was successfully tested under high pressure-low temperature (100 bar pressure and 20ºC) microbial cultivation system. Production of secondary metabolites was enhanced at optimized culture conditions. The in-vivo antifungal activity of sesquiterpenes was studied using the Caenorhabditis elegans – Candida albicans model system. The sesquiterpenes affected the virulence of C. albicans and prolonged the life of the host C. elegans. These findings suggest that sesquiterpenes are attractive antifungal drug candidates. The 5-HT2C receptor agonist is a potential target for the development of drugs for a range of central nervous system disorders. The interaction of 5-HT2C agonist ascotricin with the receptor was studied through bioinformatic analysis. The in silico molecular docking and molecular dynamic simulation studies demonstrated that they fit into the serotonin 5-HT2C active site and the crucial amino acid residues involved in the interactions were identified. To our knowledge, this is first report of in vivo antifungal analysis of sesquiterpenes and in silico studies of serotonin 5-HT2C receptor-ascotricin complex.The potent antifungal agent sesquiterpenes and serotonin 5-HT MD simulation of interaction of ascotricin with 5-HT2C receptor The growth and purification of the isolate were performed on potato dextrose agar (PDA), which was comprised of 200 g potato, 20 g dextrose and 15 g agar in 1000 mL filter sterilized seawater. The culture was stocked at 4°C and subcultured once in every 3 weeks.2.2.For genomic DNA extraction the fungus was cultured on the PDB at 28ºC for 5 days. The mycelia mat was transferred to 1.5 mL tube and DNA was extracted using high salt method. The 18S rRNA was amplified using Euk 18S 555F – Euk 18S 1269R primers and the ITS region was amplified using ITS1 – ITS2 primers. Total reaction volume 20 µL composed of 10 µL PCR master mix (Takara), 1 µL forward and reverse primers (10 pmol), 2 µL genomic DNA (3 ng), 1 µL of filter-sterilized DMSO and 5 µL of nuclease-free water. PCR products were purified and sequenced using an automated sequencer.2.3.The DNA sequences were compared to the reference sequences within the NCBI database using the BLASTN algorithm. All the chosen relative sequences were aligned using BIOEDIT ver.7.2.5 Hall . Then th2.4.in situ deep-sea conditions. In stationary growth phase, the cells were harvested by centrifugation at 8000g for 15 min. The isolate was cultivated on PDB (½ concentration) prepared in sea water and incubated at 10 bar pressure in 20ºC for 1–2 days, after which pressure was increased gradually up to 100 bar for 7–10 days. The growth of the fungus was examined in phase contrast microscope  after exposure to elevated pressure conditions.To examine the piezotolerance, the isolate was incubated at 100 bar pressure at 20ºC using customized pressure vessels  simulating 2.5.−1 in minimal salt medium (MSM) with the following composition: 1gL−1 NaCl, 0.1 g L−1 KCl, 1gL−1 KH2PO4, 1.5gL−1 K2HPO4, 0.3 g L− 1 MgSO4 · 7H2O (per liter of 1:1 seawater and distilled water). To this, filter sterilized trace element solution  and carbon source 20 g L− 1 dextrose were added to the pre-sterilized MSM media to evaluate the nitrogen source influencing bioactive compounds production. The flasks were inoculated with 10% of 5-day old fungal broth culture. The flasks were then incubated in dark under shaking conditions (120 rpm) at 28 ± 2ºC for 7 days. The fungal biomass growth was monitored by measuring mycelia dry weight and extracellular protein. Metabolic activities were monitored by measuring the reducing sugars and secondary metabolites every day up to 7 days and all analyses were carried out in duplicates.Optimization of parameters such as temperature , pH  and salinity  were tested in potato dextrose broth (PDB). The effect of carbon source  at a concentration of 1% and the effect of nitrogen source  at a concentration of 20 mmol L2.6.Amylomyces rouxii MTCC 386 and Aspergillus fumigatus MTCC 2550 .Pure culture slant was inoculated into the PDB  prepared in sea water and incubated at 28ºC under shaking conditions for 7 days. The secondary metabolites were extracted from the culture broth with an equal volume of n-butanol. The organic layer was separated and concentrated in a rotary evaporator . The n-butanol extract obtained from culture broth was subjected to a Silica Gel GC 60–120 mesh column chromatography using chloroform/methanol 90:10–50:50 solvent systems to yield different fractions. All the fractions were tested against two type strains, namely, 2.7.1H spectra and 13C-NMR spectra for compound I (colorless powder) in DMSO-d6 was recorded at 40ºC in high-resolution liquid state NMR (Jeol ECA 500 MHz). ESI-MS analysis was performed using Agilent 6545 Q-TOF mass spectrometer. The FTIR analysis was carried out in the range of 4000 to 400 cm−1 with Schimadhzu IR affinity spectrometer. The solvent fraction (compound II) was dissolved in 0.5 mL methanol and injected into the GC-MS instrument , and the separated compounds were identified using library match.A milligram of bioactive subfraction was dissolved in MeOH and filtered through 0.22 µm syringe membrane filter. This filtrate was then subjected to HPLC analysis on an Agilent C18 column  at a flow rate of 1.0 mL/min. The elution gradient (methanol-water) was setup as 0 ~ 5 min, 5% as 0 ~ 5 min, 5%; 5 ~ 65 min, 5%~100%; 65 ~ 75 min, 100%B; 75 ~ 80 min, 100%~5%B. Bioactivity-guided fractionation of extracts led to the isolation of two compounds, colourless powder (compound I) and solvent fraction (compound II). 2.8.Cryptococcus albidus (MTCC 4746), Candida albicans (MTCC 227), Aspergillus fumigatus (MTCC 2550), Aspergillus flavus (MTCC 277), Penicillium chrysogenum (MTCC 2725), Amylomyces rouxii (MTCC 386), Aspergillus terreus (MTCC 3572), Aspergillus niger (MTCC 282), Trichophyton rubrum (MTCC 296) and Saccharomyces cerevisiae (MTCC 307) were from the Institute of Microbial Technology (IMTECH), India. The test compound was dissolved in DMSO (5%) at a final concentration of 10 mg/mL, and serial dilutions were prepared using the same solvent. The MIC was determined on 96 well microdilution plates according to the standard protocols ((NCCLS) National Committee for Clinical Laboratory Standards The solvent fraction (compound II) was evaluated for antifungal activity against test pathogens. The test fungi used in the present study including 2.9.C. elegans were grown in nematode growth medium (NGM) in petri dishes and fed with E. coli OP50 as food source. Three different (sesquiterpenes) metabolites concentration were tested 100 ng, 10 µg and 100 µg using a balanced salt solution (M-9) of composition 1.5 g KH2PO4, 3.0 Na2HPO4, 2.5 g NaCl, 0.5 mL 1M MgSO4 in 500 mL distilled water , following by incubation for approximately 20 h at 30ºC. Kanamycin was added to prevent growth of any residual E. coli OP50 strain carried on the nematode during the experiment. For control experiments using only E. coli OP50, kanamycin was omitted from the liquid medium. Worms were scored daily into one of three categories: alive, dead with hyphae piercing the cuticle, and dead without hyphae piercing the cuticle. Worms were considered dead if they did not move in response to mechanical stimulation with a pick. Dead worms were removed from the assay. Microscopies of nematodes were performed by using Zeiss Axio Imager microscope.A previously described protocol for infecting 2.11.in vitro agonist activity as described earlier with minor modification (Lambert and Lauder 2C receptor was carried out using the CDOCKER module for Discovery studio V4.0 (DS). The structure of the receptor 5-HT2C was obtained from protein data bank (PDB Id: 6BQG). The active site was predicted and docking was carried out subsequently. Best poses with lowest energy values associated with favourable region of binding were obtained. Molecular dynamics (MD) simulation studies were carried out using the MD simulation module in DS. The atoms were typed according to CHARMM force field and were solvated. The surface charges were neutralized by adding Na2+ and Cl2- counterions. Prior to simulation, the system was energy minimized using steepest descent and conjugate gradient methods. The system was equilibrated with a time step of 2 fs at a temperature of 300 K and 1 atm pressure. The production run was carried out for a time period of 100ps and the trajectories were saved for every 1ps. The RMSF, total potential energy of the system and other parameters were analysed using the various modules of DS. The crucial residues that were involved in the interaction between ascotricin A and 5-HT2C receptor were further studied based on the conformations generations through MD simulation.The compound I (ascotricin A) was evaluated for 3.3.1.Ascotricha sp. based on the combination of morphological characteristics including ascomata, ascomatal hairs, conidiophores and spores. The mycelium of the isolate produced yellow tint mat-like colonies on potato dextrose agar at 28ºC and changed to dark green on sporulation. The monoculture matured within 144 h with sporulation phases and produced secondary metabolites extracellularly. The molecular characteristics including 18S rRNA and ribosomal intergenic spacer region (ITS) confirmed that the deep-sea fungus belongs to the genus Ascotricha sp. and is most closely related to Ascotricha lusitanica showing 98% homology similarity. The sequences were imported to BIOEDIT ver.7.2.5 . Microscopical analysis elucidated the pressure induced alterations in the morphology. Much reduced mycelial hyphae were observed in fungus grown at 100 bar pressure. The size was reduced to 4–6 times in cells grown at elevated pressure compared to the growth at 1 bar pressure . Alth3.3.Ascotricha sp. showed a log phase from 48-120h and this phase were followed by a prominent decline in the mycelia biomass until 168 h. In submerged culture this fungus exerted accelerated growth metabolism and showed high mycelial production. The amount of secondary metabolites remained significantly higher than in the log phase. Therefore, it was a capable fungus with fascinating features.The growth 3.4.Ascotricha sp. was cultured in the flask at different pH (5.0–10.0) conditions. Our results elucidated the potential of strain to grow and produce the secondary metabolites at a broad spectra of pH ranging from 5.0 to 9.0. This fungus showed the highest growth of culture biomass (31.3 g/L) and production of secondary metabolites (1.14 g/L) when incubated in PDB for 120 h with an initial pH value of the medium at 9.0. Thus, the pH 9.0 was maintained in fermentation medium throughout the experimental studies. The effect of temperatures at 4, 10, 20, 30 and 40ºC on growth and metabolites synthesis by Ascotricha sp. was studied with pH 9.0. The growth of fungus increased while the temperature was increased from 10 to 30ºC, both biomass (30.9 g/L) and secondary metabolite production (1.12 g/L) was estimated to be highest at 30ºC. High growth and metabolites synthesis were observed at both 2% and 4% salinity concentration. The mycelial biomass (36.8 g/L) and secondary metabolites (1.29 g/L) production were observed to reach a peak value at 4% salinity followed by biomass (34.1 g/L) and secondary metabolites (1.01 g/L) production at 2% salinity concentration. The mycelia of Ascotricha sp. grew well at 0% salinity however it affected the biosynthesis of secondary metabolites.3.5.Ascotricha sp. was examined in the basal medium with various carbon sources, namely, dextrose, sucrose, maltose, starch and cellulose. Among the tested sources, sucrose gave significantly highest mycelium biomass (33.15 g/L) and bioactive metabolites production (0.92 g/L). Second high mycelium (26.6 g/L) was observed in dextrose but the secondary metabolites (0.66 g/L) were produced at moderate level. This was followed by maltose, starch and cellulose, which were considered as a poor source of carbon for the growth of this isolate. The cellulose was the least utilized carbon source and retarded growth was observed. Thus sucrose was provided as the sole carbon source for further studies. Nitrogen is an important factor influencing not only fungal growth and differentiation, but also on the secondary metabolites biosynthesis. Effects of different nitrogen sources (inorganic and organic) such as ammonium sulphate, ammonium dihydrogen phosphate, ammonium nitrate, yeast extract, beef extract, peptone, tryptophan, tyrosine and phenyl alanine on fungus growth and secondary metabolites production were analysed. Various nitrogen sources stimulated the growth of the Ascotricha sp. and some sources increased the production of secondary metabolites. Based on the inorganic nitrogen sources tested, the ammonium sulphate was the best nitrogen source and it supplementation significantly increased the mycelia biomass (34.4 gL−1) and secondary metabolites (0.86 gL−1) production. In addition, among the organic nitrogen sources tested, peptone influenced the biomass (31.5 gL−1) and secondary metabolites (0.92 gL−1) production. Interestingly, the addition of aromatic amino acid phenylalanine stimulated both the mycelial biomass (34.2 gL−1) and secondary metabolites (1.17 gL−1) biosynthesis on day 5 in the culture medium.The mycelia dry weight, pH values, total protein and extracellular secondary metabolites of the culture filtrates of 3.6.1H NMR, 13C NMR, and mass spectroscopic analysis. The FTIR spectra showed the presence of carboxylic acid group at 1734 cm−1 and alkyl groups at 2950 and 2922 cm−1 (Supplementary File 1). The 1H NMR spectra of compound I (colourless powder) elucidated the presence of aromatic protons and hydroxyl peaks (13C NMR spectrum confirmed the presence of methylene and aromatic peaks (m/z of compound I was found to be 557.19 [M +Na]+ (Supplementary File 2). The GC-MS analysis of the bioactive compound II yielded more than 4 molecules such as diphenylmethane, 1,6-Cyclodecadiene, sesquiterpenes and dihydro-cis-alpha-copaene-8-ol. The major compound was identified as sesquiterpenes which constituted about 66% (Bioactive subfractions yielded compound I (colourless powder) and compound II. The HPLC analysis of compound I confirmed the presence of single dominant peak and structural elucidation was carried by FTIR, yl peaks ,b). The ic peaks ,b). Thesic peaks . The ESIbout 66% .10.1080/3.7.Mucor rouxii, 6.0 µg/mL – Aspergillus fumigates and 5.0 µg/mL – Candida albicans. No activity was found against Aspergillus spinulosus, Aspergillus terreus and Peniclillium chrysogenum. Using C. elegans-C. albicans model system  nematode survival was diminished, even compared to the nematodes in the untreated control group. Probable this toxicity is present at even low concentrations, but the beneficial effect from the antifungal outweighs the toxic effect.The sesquiterpenes (compound II) inhibited the growth of fungi and MIC was found to be 4.0 µg/mL – l system ), the in defence ). This i defence ). The phcuticles ). These nfection ). Bioact10 µg/mL ). The se3.8.C. elegans survival at MIC compound concentration. In the initial phase of the curve within 12 h, there was death of nematodes in both positive control and test sample. Time-dependent toxicity was not observed as the nematodes entered a strong recovery phase and stabilized with time intervals. From this result, it was confirmed that secondary metabolites exerted short-term toxicity causing death of the C. elegans with minimal concentration (10 µg/mL) followed by stabilized phase of recovery.This screening allowed assessment of the compound toxicity level based on 3.9.2C agonist activity of compound I (ascotricin) and further attempts were made to elucidate the molecular interactions between the molecule and the receptor. Molecular Docking studies confirmed ascotricin A and 5-HT2C docked with a binding energy of −47.34 kcal/mol. The results indicated that the residues ASN331, SER219 and VAL215 formed favourable hydrogen bond with ascotricin A and the residues LEU209, TRP324, PHE327, VAL354 were involved in hydrophobic interaction. Conformations generated using MD simulations suggested that the binding energy of ascotricin A and 5-HT2C complex has increased further to −62.5827 kcal/mol indicating more compact binding in the active site. The plots indicating the changes in root-mean-square fluctuation (RMSF) and the total potential energy of the system are given in Initial screening confirmed the human serotonin 5-HT2C structure before and after MD simulation was 3.149 Å.Post-MD simulations, the residues LEU209, ASP211, SER334, CYS207, SER219 and ASN331were found to form hydrogen bonds with ascotricin A . The num4.in vitro and in vivo antifungal activity of the sesquiterpene derivatives extracted from the deep-sea piezotolerant ascomycetes fungus Ascotricha sp. Marine fungi biosynthesis variety of secondary metabolites with unique structures which have gained much importance for discovering new bioactive molecules, for eg., the antimicrobial agent cephalosporin was extracted and purified from a marine fungus, Cephalosporium acremonium  significantly affect the secondary metabolism , Aspergillus fumigatus (6.0 µg/mL) and Candida albicans (5.0 µg/mL). The option to treat invasive fungal infections caused by Aspergillus and Mucorales is limited. However, the morbidity and mortality for both pathogens remains high. Mucor rouxii elicits dimorphism morphogenetic phenomenon: mycelium or yeast cells based on physiological conditions and influence of growth factors . Secondly, the resistance towards pathogens were transferred to offsprings and healthy ones were generated. This functioning of metabolites in attenuating pathogenic fungus sheds new light on the antifungal drug discovery. The 5-HT2C receptor is a novel drug target to treat several neural disorders. Recently, the crystal structures of the 5-HT2C was solved. Many receptor subtypes have been screened and completely identified, consisting of three members of the 5-HT2 subclass characterized by close sequences homologies  also suggests a significant identity in terms of the binding pocket residues. This construes the fact the ascotricin-A binds to the similar crucial residues as that of ergotamine, thereby acts as a serotonin receptor agonist. The simulation analysis pinpoints amino acid difference at various positions in the 5-HT2C receptor that impacts translation of molecules for neural related disorders.To the best of our knowledge, this is the first report on the te NCCLS . The MICae Huang . This suAscotricha sp. from deep-sea. The important characteristic of this fungus was its novel piezotolerance, production of broad-spectrum anti-fungal metabolite sesquiterpenes and a serotonin 5-HT2C receptor agonist ascotricin. The in-vivo antifungal analysis confirmed that sesquiterpenes is a potent antifungal drug candidate. The in silico molecular docking and simulation studies confirmed ascotricin as a novel serotonin receptor agonist. These results confirm that deep-sea fungus Ascotricha sp. extract could be exploited as a potential source for antifungal compounds and 5-HT2C receptor-specific agonist which can be used for several medical applications.The present study is the first to report a piezotolerant


N-(2-(4-chlorophenyl)-4-oxoquinazolin-3(4H)-yl)acetamide derivatives was synthesized and its chemical structures were confirmed by physicochemical and spectral characteristics. The synthesized compounds were evaluated for their in vitro antimicrobial (tube dilution technique) and anticancer (MTT assay) activities along with molecular docking study by Schrodinger 2018-1, maestro v11.5. The antimicrobial results indicated that compounds 3, 8, 11 and 12 displayed the significant antimicrobial activity and comparable to the standards drugs (ciprofloxacin and fluconazole). The anticancer activity results indicated that compound 5 have good anticancer activity among the synthesized compounds but lower active than the standard drugs (5-fluorouracil and tomudex). Molecular docking study demonstrated that compounds 5 and 7 displayed the good docking score with better anticancer potency within the binding pocket and these compounds may be used as a lead for rational drug designing for the anticancer molecules.In the present study, a series of 2-(4-(2-chloroacetyl)piperazin-1-yl)- The ever-increasing microbial antibiotic resistance leads to ongoing testing of new biologically efficient compounds of either natural or synthetic origin for infectious diseases . Subsequ®)  Fig.  as an anMolecular docking analyses provide the most comprehensive illustration of the interaction between drug receptors and produced a modern rational approach to drug design . The RAWI) essential for antimicrobial activity. Rajveer et al.  [I) (0.01 mol) was reacted with hydrazine hydrate (0.02 mol) in the presence of ethanol and refluxed for 3 h (30 °C) to obtain II. The reaction mixture was cooled and the resultant precipitate was filtered off and recrystallized with ethanol [2-(4-Chlorophenyl)-4benzo[e] , 3 oxazi ethanol .H)-one (II) (0.01 mol) and chloroacetyl chloride (0.01 mol) with a few drops of glacial acetic acid in absolute ethanol (20 mL) was refluxed for 8 h (30 °C). The reaction mixture was then cooled in ice cold water and resultant precipitate of III was filtered, washed with water, dried and recrystallized with ethanol.A mixture of 3-amino-2-(4-chlorophenyl)quinazolin-4(3N-(2-(4-chlorophenyl)-4-oxoquinazolin-3(4H)-yl)acetamide (III) (0.01 mol) and piperazine (0.01 mol) with a few drops of glacial acetic acid in absolute ethanol (20 mL) was refluxed for 10 h (40 °C). The reaction mixture was cooled in ice cold water and resultant precipitate of IV was filtered, washed with water, dried and recrystallized with ethanol.A mixture of 2-chloro-N-(2-(4-Chlorophenyl)-4-oxoquinazolin-3(4H)-yl)-2-(piperazin-1-yl)acetamide (IV) (0.01 mol) was further treated with chloroacetyl chloride (0.01 mol) in the presence of few drops of glacial acetic acid in absolute ethanol (20 mL) and refluxed for 8 h (30 °C) yielded of V. The reaction mixture was then cooled in ice cold water and resultant precipitate was filtered, washed with water, dried and recrystallized with ethanol. 2-(4-(2-Chloroacetyl)piperazin-1-yl)-N-(2-(4-chlorophenyl)-4-oxoquinazolin-3(4H)-yl)acetamide (V) (0.01 mol) was reacted with different corresponding aniline (0.01 mol) with a few drops of glacial acetic acid in absolute ethanol was refluxed for 10 h (40 °C) to synthesize the title compounds (1–17). The reaction mixture was then cooled in ice cold water and the resultant precipitate was filtered, washed with water, dried and recrystallized with ethanol.S. aureus MTCC3160, B. subtilis MTCC441, Gram-negative bacterium: E. coli MTCC443 and fungal species: C. albicans MTCC227 and A. niger MTCC281 was determined by tube dilution method [The synthesized derivatives of quinazolinone were tested using ciprofloxacin  and fluconazole  as reference drugs for their in vitro antimicrobial potential. The antimicrobial activity towards Gram-positive bacteria: n method . To given method .2. Anticancer activity of synthesized compounds was assessed at distinct concentrations using MTT assay, as Mosmann (1983) explained but with minor alteration after 72 h of incubation. Using a spectrophotometer at 520 nm, assay sheets were read. Data produced were used to determine a dose–response curve from which the concentration of test compounds needed to kill 50% of the cell population (IC50) was determined [It has been determined the activity of synthesized compounds and control drugs against human colon (HCT116) and cancer cell lines of the mouse monocyte macrophage leukaemic (RAW 264.7). In RPMI 1640 (Sigma) the selected cancer cell strains were developed, supplemented by 10% heat-inactivated bovine fetal serum (FBS) (PAA Laboratories) and 1% penicillin/streptomycin (PAA Laboratories). Cultures were kept at 37 °C in a humidified incubator in an environment of 5% COtermined .Kinase inhibitors are extremely efficient in the therapy of cancer, specifically targeting specific mutations that mainly drive tumorigenesis. They are categorized according to their capacity to catalyze ATP terminal phosphate transfer to substrates that usually contain serine, threonine or tyrosine residues . Cyclinev11.5) acquired the docking score and targeted the binding site and formed the grid. The active site grid covered the major amino acids that interact with the receptor. Using their specific PDB ID: 5FGK for human colorectal carcinoma and PDB ID: 5JVY for mouse monocyte macrophage   in synthesized compounds is established by the existence of an IR absorption band around 1195 cm−1. The appearance of IR stretching 1591–1636 cm−1 in the spectral data of compounds (1–17) specified the existence of N=CH group. The appearance of IR stretching 1250–1253 cm−1 in the spectral data of synthesized compounds specified the existence of C–N group. The proton NMR displayed the multiplet signals between 6.03 and 8.05 δ ppm in the aromatic ring of the synthesized compounds. The compounds exhibited singlet at 8.91 δ ppm due to the existence of N–NH group. Compounds displayed singlet at 2.36–2.38 δ ppm due to the existence of –CH3 group. The compounds displayed singlet at 3.71δ ppm due to the existence of OCH3 of Ar-OCH3. The 13C–NMR spectra of aromatic ring exhibited the carbon atoms in the range of 170.9, 164.9, 164.6, 161.3, 151.9, 138.4, 135.8, 135.5, 133.5, 132.3, 129.5, 129.4, 128.0, 127.9, 127.6, 127.4, 126.9, 122.4, 121.7, 120.6, 118.3, 114.6, 58.7, 52.5, 52.4, 52.3, 46.8, 46.7 of the synthesized compounds. The elemental analysis was found within ± 0.4% of the theoretical results of compounds.A new series of quinazoline derivatives was synthesized using synthetic Scheme 3 (MICbs = 4.81 µM) emerged as most effective antibacterial agent toward B. subtilis. Compounds 11 and 12 (MICsa = 2.56 µM) displayed the promising activity toward S. aureus. Compound 8 (MICec = 5.54 µM) emerged as most active candidate against Gram-negative bacterium E. coli and comparable to standard drug ciprofloxacin. In the case of antifungal activity, compound 3 (MICca = 4.81 and MICan = 2.40 µM) was found to be the most potent antifungal agent against C. albicans and A. niger and had better antifungal activity than standard drug fluconazole . Results of antimicrobial activity indicated that the synthesized compounds possessed a higher antifungal activity than antibacterial activity. As far as antibacterial activity is concerned, the synthesized compounds were more active against Gram negative bacterium (E. coli) than Gram positive bacteria (S. aureus and B. subtilis).The synthesized compounds were tested by tube dilution technique against Gram positive and Gram negative bacterial and fungal strains for their in vitro antimicrobial activity and the MIC values of control drugs and synthetic compounds are shown in Table 5  was found to be the most active one and compared to the 5-fluorouracil and Tomudex (used as standard drugs). The anticancer screening (IC50 = µM) results are shown in Table The synthesized compounds were evaluated for their in vitro anticancer activity against human colorectal cancer (HCT116) and mouse monocyte macrophage RAW 264.7) cancer cell lines using MTT assay. Compound .7 cancer5 and 7 for the cancer cell lines of human colorectal carcinoma (HCT116) and mouse leukaemic monocyte macrophage (RAW 264.7). The compound 5 and standard drugs (5-fluorouracil and tomudex) were docked in the active site of the cycline-dependent kinase cdk8 (PDB ID: 5FGK) co-crystallized acuity 5XG ligand and in the active site of the S121P murine COX-2 mutant (PDB ID: 5JVY) co-crystallized acuity ligand COH were also docked. The binding mode of native ligand 5XG have docked score   (− 8.72) and COH ligand have docked score (− 8.93) showed good interaction with crucial amino acids residues with their respective proteins  have docked score (− 11.054) with better anticancer activity (0.82 μM) which also developed H-bond with crucial amino acid  with better anticancer potency (1.20 μM) , native ligands and standard drug (tomudex) which showed good to better docking score with their respective proteins. Docking results and interacting residues are shown in Tables The synthesized derivatives of quinazolinone showed excellent docking performance and the chosen proteins were discovered to communicate with significant amino acids. Molecular docking was performed to analyze the binding mode of active compounds ore − 8.9 showed gge RAW 26.7. The cThe docking findings therefore indicate that the synthesized compounds may be of excellent importance in effective chemotherapy. The selected database of protein i.e. (PDB ID: 5FGK) for human colorectal carcinoma and (PDB ID: 5JVY) mouse monocyte macrophage may be the target protein of derivatives of quinazolinone for their anticancer activity  and chloro with nitro (NO2) substitution on the phenylamino ring attached to the chlorophenyl quinazolinone piperazine-acetamide nucleus increased the antibacterial activity.In the case of antibacterial activity towards Results of anticancer activity revealed that the presence of electron withdrawing groups i.e. dichloro on the phenylamino ring attached to the chlorophenyl quinazolinone piperazine acetamide nucleus increased the anticancer activity against both cancer cell lines (RAW 264.7 and HCT116).The SAR of synthesized compounds can be summarized Fig.  as follo3 showed promising antimicrobial activity due to the presence of electron releasing group at meta–position of the substituted benzylidene nucleus and comparable to the control drugs. In case of anticancer activity indicated that compound 5 (meta/para-Cl) and compound 7 (meta-NO2) displayed moderate anticancer activity towards human colorectal carcinoma and mouse leukaemic monocyte macrophage cancer cell lines due to the presence of EWG on the substituted benzylidene nucleus. Molecular docking analysis demonstrated that compounds 5 and 7 showed the better docked score with better potency and comparable to the standard drug and native ligands of the proteins. The findings of the docking are compatible with the assays of anticancer. Docking information stay in excellent correlation with the outcomes of anticancer activity and these molecules may be used as a lead in the design of new anticancer agents.In the present study, the synthesized quinazolinone derivatives i.e. compound Additional file 1. Web link for PDB ID: 5FGK and 5JVY proteins.Additional file 2. Synthetic scheme with chemical structures.Additional file 3. Docking results of active compounds.


AbstractPimelodus LaCépède, 1803 comprises 35 formally recognized species distributed along the major neotropical river basins. Despite conservatism in diploid number with 2n=56, an intense variation of chromosomal morphology (karyotypic formula) has been documented in Pimelodus species. In the present study, we analyzed karyotypes of 20 specimens, identified as Pimelodusblochii Valenciennes, 1840 and collected from the lower courses of the Tapajós, Amazonas and Trombetas Rivers. The karyotypes were characterized by Giemsa conventional staining, C-banding, silver staining (Ag-NOR) and fluorescent in situ hybridization (FISH) with 5S and 18S rDNA probes. The karyotypes showed 2n=56 chromosomes in fish from the Tapajós River. In contrast, fish from the Amazonas and Trombetas Rivers had 2n=58. The nucleolus organizing regions were labeled on the short arm of an acrocentric chromosome as demonstrated by silver staining and FISH. Signals for 18S and 5S rDNA were co-localized on one chromosome pair. Our results demonstrate karyotypic divergence between Tapajós and Amazonas-Trombetas populations of P.blochii, interpreted as supporting the existence of a species complex in this taxon.The genus  Pimelodus LaCépède, 1803  comprises 35 valid species exclusively distributed in neotropical freshwater drainages. It is commonly recorded in the Amazonas, Orinoco, Araguaia-Tocantins, São Francisco, and Paraná-Paraguay River basins. In the Amazon basin, seven species of Pimelodus have been recorded: Pimelodusalbofasciatus Mees, 1974, Pimelodusblochii Valenciennes, 1840, Pimelodusaltissimus Eigenmann & Pearson, 1942, Pimelodusjivaro Eigenmann & Pearson, 1942, Pimelodusornatus Kner, 1858, Pimeloduspictus Steidachner, 1876, and Pimelodustetramerus Ribeiro & Lucena, 2006  in Calophysusmacropterus Lichtenstein, 1819, Pinirampuspirinampu Spix & Agassiz, 1829 and Luciopimelodusplati Valenciennes, 1835 species  and fluorescent in situ hybridization (FISH) technique with 5S and 18S rDNA probes.In the present paper, we investigate the karyotype of Twenty (20) specimens were collected from four localities in the Tapajós, Amazonas and Trombetas Rivers Table . The fisIntra-abdominal colchicine (0.0125%) injection was performed at 0.01 ml/g  to stop Conventional staining was performed with 5% Giemsa solution . The C-banding protocol from Sumner (1972) was followed with minor changes. The NORs were stained with silver nitrate following the FISH was used for mapping 18S and 5S rDNA loci . Double The PCR products were labeled by nick translation with biotin-14-dATP  and digoxigenin-11-dUTP . The slides were treated with RNase solution (5 µl RNase 10 mg/mL diluted in 975 µl 2×SSC) for a short period of time. The fixed chromosomes were denatured in 70% formamide (pH 7.0 2×SSC) and heated at 70 °C for 5 min. The hybridization solution mixture was prepared with 20 µl formamide + 8 µl of 50% dextran sulfate + 4 µl of each probe + 4 µl of 20×SSC. The slides were incubated in 2×SSC solution in a humidified and heated (37 °C) chamber overnight.Post-hybridization washes were performed with 15% formamide at 42 °C for 10 min, three washes in 0.1×SSC at 60 °C for 5 min, and 0.5% Tween20 at room temperature for 5 min. For signal detection, slides were placed in NFDM buffer  for 15 min, followed by two washes in 5% Tween20 for 5 min at room temperature.The hybridized probes were applied in a mixture containing 20 µl anti-digoxigenin-rhodamine (1:200)  + 4 µl FITC-Avidin (1:100)  + 26 µl of C buffer  for 60 min. The slides were coated with anti-fading reagent Vectashield H-1000  and chromosomes were counterstained with DAPI .At least 30 metaphases were counted to determine the diploid chromosome number. The best spread metaphase plates were photographed with a CCD camera (Moticam 10 MP) coupled to a Zeiss Axioskop40 microscope for conventional/banding images, and a Nikon Eclipse CI for FISH images. The contrast and brightness were adjusted with ADOBE PHOTOSHOP CS3. The chromosomes were arranged as metacentric (m), submetacentric (sm), subtelocentric (st) and acrocentric (t) following The diploid chromosome number was observed as 56 in the Tapajós River fish (Itaituba population). On the other hand, 2n=58 chromosomes were recorded from Amazonas (Santarém and Almeirim populations) and Trombetas Rivers (Oriximiná population) samples, with minor variation in the karyotypic formula, as 30m/sm+28a and 26m/sm+32a, respectively ; this syntenic pattern also occurred in the Santarém population but on just one homologous chromosome. The Tapajós River specimens’ karyotypes showed a distinct position for 18S and 5S rDNA Figs , 5.Pimelodusblochii from the Tapajós, Amazonas and Trombetas Rivers are compatible with a previous report (Pimelodusblochii from the Tapajós River with 2n=56 (36m/sm+20a) showed a resemblance to the population from the Araguaia River  (The karyotype macrostructure of s report  (Table 2s report . The speo State) .Pimelodusmaculatus and Pimelodus sp.  is the most common pattern observed in the ryotypes . This pabritskii . In the ris 1900 , Ancistrner 1854 , Hemibagker 1862 , Corydorède 1803 , Panaqolaml 2001  and Bunoner 1855 . This syner 1855 . Additioner 1855 .P.blochii from Guyana. The A variety has an ashy body pigmentation without dots or stripes, whereas the B variety exhibits four lateral body stripes with the fourth stripe possibly absent or fragmented into dots. Pimelodusalbofasciatus (Mees 1974) maintains a close resemblance to the B variety but is distinguished based on eye morphology and dorsal spine length , have the typical pigmentation for the A variety, whereas the specimens from the Santarém population, collected at the confluence of the Amazonas and Tapajós Rivers, had the B variety pigmentation. Although both populations conserved the diploid number 2n=58 and karyotypic formula (30m/sm+28a), they diverged in their 18S and 5S rDNA locations  distinguished through body pigmentation and characterized by a 2n=58 karyotype with minor variations. Additional studies of this group are needed in order to clarify the evolutionary dynamics of the 18S and 5S rRNA genes as well as to acquire morphological and molecular data to evaluate the taxonomy and phylogeny of Pimelodus species.Pimelodusblochii from the lower courses of Amazonas, Tapajós and Trombetas rivers presented differentiated karyotypes based on variation in diploid number and chromosome morphology. The specimens collected from the Tapajós River, with 2n=56, are clearly distinguished from the others and may constitute a new, undescribed Pimelodus species.The populations of