However, the molecular basis of its substrate recognition is still not fully understood. Using Escherichia coli LipB as a model enzyme, we show here that the octanoyl-transferase mainly recognizes the 4′-phosphopantetheine-tethered acyl-chain of its donor substrate and weakly binds the apo-acyl carrier protein. We demonstrate LipB can accept octanoate from its own ACP and noncognate ACPs, as well as C8-CoA. Furthermore, our 1H saturation transfer difference and 31P NMR studies demonstrate the binding of adenosine, as well as the phosphopantetheine arm of CoA to LipB, akin to binding to LplA. Finally, we show a conserved 71RGG73 loop, analogous to the lipoate-binding loop of LplA, is required for full LipB activity. Collectively, our studies highlight commonalities between LipB and LplA in their mechanism of substrate recognition. This knowledge could be of significance in the treatment of mitochondrial fatty acid synthesis related disorders.Lipoic acid is a sulfur-containing cofactor indispensable for the function of several metabolic enzymes. In microorganisms, lipoic acid can be salvaged from the surroundings by lipoate protein ligase A (LplA), an ATP-dependent enzyme. Alternatively, it can be synthesized by the sequential actions of lipoate protein ligase B (LipB) and lipoyl synthase (LipA). LipB takes up the octanoyl chain from C LipB functions as a cysteine/lysine dyad acyltransferase, cysteine 176 and Lys 142 of M. tuberculosis LipB (Cys169 and Lys 135 of E. coli LipB) functioning as acid/base catalysts (8-ACP (acyl carrier protein) to Cys176 of LipB  by insertion of two sulfur atoms.Most of the knowledge with regard to lipoic acid metabolism comes from studies conducted on s aureus . In the s aureus , 13, 14.s aureus , 14, 15.s aureus . In lipoatalysts , 19. A c of LipB . Subsequ8-ACP to the lipoyl subunit of GCS, (b) lipoic acid synthase (LIAS), a sulfur insertion enzyme that adds two sulfur atoms to the C8- chain to form lipoic acid, and (c) LipT1, an amidotransferase, that relays octanoyl-chain/lipoic acid from the H subunit of GCS to the lipoyl carrier protein subunit of other dehydrogenases, viz. PDH, BCKDHc, αKGDH. Malfunction of any of the gene products causes lipoic acid synthesis defects in neonatal patients, underscoring the need to understand the pathway at the molecular level. Disorders range from defective mitochondrial energy metabolism, toxic levels of certain amino acids, neurological problems, and even death, depending on which lipoic acid synthesis gene malfunctions  lipoyl transferase 2 (LipT2) that transfers octanoyl chain from Cunctions , 21, 22.unctions , malonylunctions . Defectsunctions , 26. Impunctions , 12, 13.E. coli, Thermoplasma acidophilum, Enterococcus faecalis, Streptococcus pneumoniae, and Mycoplasma hypopneumoniae. However, the biosynthetic enzyme LipB is relatively less explored. Structures are available from only two sources, M. tuberculosis and T. thermophilus for this enzyme. The mechanistic understanding of LipB interaction with its substrates is also sparse. Two recent studies on LipB have highlighted its ability to allosterically distinguish between different acyl-ACP's, viz. C6-, C8-, C10-, and C12-ACP based on differences in their surface conformation  as a donor substrate and lipoyl domain of dehydrogenases, for example, GCS H protein (GcvH), PDC, or 2-oxoglutarate dehydrogenase complex (OGDH) as octanoate acceptors  has been used as a structural model to interpret our findings.LipB (UNP60720) catalyzes a bisubstrate reaction, using Ccceptors . LimitedM. tuberculosis LipB comprises five central β-strands ↑β1,↑β2,↓β3,↑β7,↓β6, enclosed by three long helices, α2 and α3 on one face and α1 on the other side of β sheet, illustrated in Figure 1A. Some of the catalytically important residues of M. tuberculosis LipB are shown as sticks and labeled cyan. The corresponding E. coli LipB residues are also labeled black. A short stretch of beta strands comprising ↓β2',↓β1',↑β3', called the 'capping sheet', is also present in LipB, of unknown function. Figure 1B shows the molecular surface of MtLipB, colored based on coulombic charge, displaying the position of active site cavity. E. coli LipB amino acid sequence has been compared with other octanoyl transferases in E. coli LipB (UNP 60720) shares 32% sequence identity with M. tuberculosis LipB , 28% with human mitochondrial LipT2 (UNP A6NK58), 29% with Mus musculus LipT2, and 26% with Leishmania major enzyme (UNP Q4A1B1).8-ACP interaction, a catalytically inactive E. coli LipBK135A/C169A double mutant was prepared, and its interaction with 15N labeled E. coli C8-ACP followed in a 1H15N transverse relaxation optimized spectroscopy (TROSY)–heteronuclear single quantum coherence (HSQC) NMR experiment. C8-ACP was stable at room temperature for approximately 7 days, as shown in To gain insights into the LipB:CE. coli ACP displays a four-helix bundle fold, consisting of helix I (Thr 2-Gly 16), helix II (Asp 35-Asp 51), helix III (Asp 56-Thr 64), and helix IV (Thr 64-His 75). Titration with LipB resulted in perturbation of several C8-ACP backbone amides viz. Gln 14, Leu 15, Asp 35, Ser 36, Asp 38, Val 40, Glu 41, Val 43-Leu 46, Glu 49, Thr 52, Ile 54, Asp 56, Glu 60-Thr 64, Glu 66, and Ala 68-Tyr71 (C). These residues are present in helix II, III, and IV of ACP. The peaks for Asp 35, Glu 57, Val 65, and Ala 68 were broadened beyond 1:1 ACP:LipBK135A/C169A ratio, shown by a (∗) in the figure. Leu 37 and Glu 48 displayed chemical exchange line broadening right from the first titration point, shown as (•) in the figure. One SD has been used to demarcate significant chemical shift change. Perturbations have been mapped to the E. coli holo-ACP structure  represented as a worm in Figure 1D. Thickness of the worm is directly proportional to the magnitude of chemical shift change. Fig. S3A shows the multiple overlaid 1H15N TROSY-HSQC spectra for C8-ACP:LipBK135A/C169A titration. Each titration point is shown in a different color: free C8-ACP is colored red; C8-ACP:LipBK135A/C169A 1:0.25 M ratio blue; 1:0.5 magenta, 1:0.75 cyan, 1:1 green, 1:1.5 orange, and 1:2 purple. Same color coding has been used throughout for all the titrations reported in this study.68-Tyr71 C. These 8-ACP was determined by surface plasmon resonance (SPR) measurements, immobilizing LipBK135A/C169A on the surface of a sensor chip, and passing increasing concentrations of C8-ACP over the chip surface. Fig. S4A shows the SPR sensorgram and Fig. S4B binding curve for C8-ACP:LipBK135A/C169A interaction. The two proteins interact in micromolar range, with a dissociation equilibrium constant (KD) of 36.4 ± 0.19 μM.The binding affinity of LipBK135A/C169A for C8-ACP, but lacks the octanoate group. The contribution of C8-chain in the interaction of C8-ACP with LipB was also investigated. 15N-labeled holo-ACP (containing 15N-labeled 4′-phosphopantetheine arm) was titrated with unlabeled LipBK135A/C169A. Noticeable changes in the amide chemical shift were observed for several holo-ACP residues. As shown in Figure 1E, Gln 14, Glu 21, Val 29, Ser 36, Leu 37, Thr 39, Val 40, Glu 41, Leu 42, Val 43, Met 44, Ala 45, Leu 46, Glu 47, Asp 51, Thr 52, Glu 53, Ile 54, Glu 57, Ala 59, and Glu 60 displayed significant perturbations. The aforementioned residues are present in helix I, loop I, helix II and helix III of ACP, shown as a worm in Figure 1F. Fig. S3B displays multiple overlaid 1H15N TROSY-HSQC spectra for holo-ACP upon titration with LipBK135A/C169A. The direction of chemical shift change with increasing LipBK135A/C169A concentration is shown by a forward pointing arrow. Interestingly, one of the two NH resonances of 4′-phosphopantetheine moiety (15N-labeled phosphopantetheine expressed in holo-form in E. coli) observed at a chemical shift of 8.1 ppm in the 1H15N TROSY-HSQC spectra also displayed significant change during the titration .Holo-ACP contains a phosphopantetheine modification akin to CC shows the SPR sensorgram, and Fig. S4D shows binding curve for the interaction of LipBK135A/C169A with holo-ACP. The binding curve did not reach saturation, and therefore, the estimate of affinity has been reported as apparent binding constant. A KDapp of 100 ± 0.43 μM was observed for the LipB:holo-ACP interaction.Fig. S41H15N apo-ACP was titrated with unlabeled LipBK135A/C169A (G). Insignificant changes in the amide chemical shift were observed (<0.06 ppm) upon addition of up to 2 M equivalents of LipBK135A/C169A. In the superimposed spectra, most of the amide resonances of apo-ACP overlapped with the peaks in presence of LipBK135A/C169A . A worm figure of apo-ACP is also shown, displaying insignificant changes upon interaction with LipBK135A/C169A (H).In the cell, ACP is expressed as an apo-protein and posttranslationally modified to holo-ACP by the catalytic action of 4′-phosphopantetheinyl transferase. The importance of 4′-phosphopantetheine arm in the interaction of LipB with its substrate was also probed. 5A/C169A G. Insign5A/C169A H.8- and holo-ACP titrations. Fig. S4E shows the SPR sensorgram, and Fig. S4F shows binding curve for the interaction of LipBK135A/C169A with apo-ACP. A KDapp of 2.1 ± 0.18 mM was obtained for the binding interaction.SPR measurements were also performed to understand the strength of this interaction. Relatively higher concentration of apo-ACP was required, compared to C8-ACP substrates was also investigated. The enzyme assay comprised 5 μM E. coli LipB, an octanoate donor , and an octanoate acceptor GcvH (25 μM). Three separate assays were performed using different octanoate-donors: (a) E. coli C8-ACP (UNP 0A6A8), (b) C8-LmACP , and (c) C8-PfACP . The product formed after the assay (C8-GcvH) in each case was qualitatively analyzed on a 12% SDS-PAGE gel (A). Lane 1 is pure apo-GcvH, lane 2: C8-EcACP, lane 3: C8-LmACP, lane 4: C8-PfACP (used as a reference/control). Notably, all three ACPs have a molecular weight of ∼8 kDa, but their migration pattern on SDS-PAGE suggests a much higher value. As shown in Figure 2A1, the molecular weight determined from its migration on the 12% SDS-PAGE gel is 18 kDa, consistent with several previous studies  (B), 74% with C8-LmACP (L. major ACP) (C), and 85% when C8-PfACP  was used as an octanoate donor, illustrated in Figure 2D.Cross reactivity of LipB with noncognate CPAGE gel A. Lane 1 studies . ACP is E. coli) B, 74% wijor ACP) C, and 858-CoA comprises a C8-chain and a phosphopantetheine arm, identical to the modifications present in C8-ACP. In addition, it contains an adenosine 3′5′-diphosphate group, not found in C8-ACP. To test whether LipB can use CoA analogs as a donor substrate, different lengths of acyl-CoA were used viz. C4-CoA, C8-CoA, C10-CoA, and C12-CoA, and the reaction progress was qualitatively monitored by following the modification of GcvH on an SDS-PAGE gel. Quantitatively, the reaction progress was followed by reverse phase HPLC chromatography. As shown in Figure 2E, LipB efficiently modified apo-GcvH using C8-CoA as acyl-chain donor (lane 5). No change in migration of the GcvH band was observed when C4-CoA, C10-CoA, or C12-CoA were used as donors, shown in lanes 4, 6, and 7, respectively in Figure 2E. MALDI-TOF mass spectrometry confirmed an increase in mass of GcvH by 128 Da after the enzyme assay, when C8-CoA was used as an acyl-chain donor. This change is mass is equivalent to the molecular mass of the C8-chain . The expected molecular mass of apo-GcvH is 15,834 Da . The terminal methionine of His tag was cleaved by E. coli MAP1, endogenous methionine aminopeptidase MAP  and B–E mass spectra of GcvH after the LipB assay using C4-CoA, C8-CoA, C10-CoA, and C12-CoA as acyl-chain donors. No change in mass was observed in the assays performed with C4-CoA, C10-CoA, and C12-CoA .Next, we probed the importance of ACP scaffold in the substrate recognition by LipB. C 127 Da, C. The exdase MAP . Fig. S5MmLipT2  to transfer octanoate from C8-CoA to E. coli GcvH was also investigated. As shown in Figure 2F, MmLipT2 also successfully converted E. coli GcvH using C8-CoA as a donor substrate, which was confirmed from the forward migration of the GcvH band in the 12% SDS-PAGE gel. Molecular weight marker is shown in lane 1, E. coli apo-GcvH (used as reference) in lane 2, and MmLipT2 assay products in lane 3.Mammalian LipT2, an octanoyltransferase is associated with vast number of neurological disorders. The ability of mice 8-CoA concentration (0–160 μM). The results were quantitatively analyzed on a C18-reversed phase column. Kinetic constants were obtained by performing hyperbolic Michaelis–Menten fits to the raw data, using GraphPad Prism version 7.0 (GraphPad Software), listed in Km value of 12.2 ± 0.8 μM and a Vmax of 1.1 ± 0.01 μmol min−1, were obtained from the graph (filled circles), illustrated in Figure 3A. In a previous study, using C8-ACP as a substrate, a Km of 10.2 ± 4.4 μM has been reported for LipB  and GcvH (25 μM) concentration constant and varying the Cfor LipB .Table 1EB. subtilis, prefers C8-ACP as a substrate and poorly utilizes C8-CoA , respectively, on a 12% native-PAGE. Lane 5 shows the assay performed in presence of equimolar concentration of C8-CoA and C8-ACP, and lane 6 displays bands for the assay carried out using C8-ACP alone as a donor substrate. It is noteworthy that in lane 5, a major proportion of C8-ACP remained unused after the assay, though GcvH displayed complete conversion. On the contrary, an intense band equivalent to holo-ACP appeared in lane 6, just above the C8-ACP band, in addition to the newly formed C8-GcvH band. Replicates of the same sample were also loaded on a 12% SDS-PAGE gel (C). Lanes 2 to 5 show bands for apo-ACP, C8-ACP, holo-ACP, and apo-GcvH, used as reference. Lane 6 corresponds to the assay carried out in presence of C8-CoA + C8-ACP, while lane 7 shows the bands for the assay with C8-ACP alone. Notably, both lanes 6 and 7 displayed complete conversion of apo-GcvH to C8-GcvH. However, based on native-PAGE (B), in lane 5, the intensity of the holo-ACP band was low compared to lane 6, suggesting less consumption of C8-ACP in presence of C8-CoA. C18-reversed phase chromatography confirmed the gel results. Figure 3D shows a chromatogram for apo-GcvH alone, while Figure 3E after the assay using C8-CoA + C8-ACP, displaying the peak for C8-GcvH formed as a product . Figure 3F displays the assay carried out using C8-ACP as a donor substrate . A distinct peak for holo-ACP appeared due to the transfer of octanoyl-chain from C8-ACP to GcvH . This peak was not observed in the assay performed in a reaction mixture containing equimolar C8-ACP and C8-CoA concentration .LipM, a LipB homolog from s C8-CoA . Does E.PAGE gel C. Lanes ive-PAGE B, in lan8-CoA contains an adenosine-3′,5′-diphosphate group, in addition to the C8-chain, and the phosphopantetheine moiety. From the biochemical assays, it was unclear whether CoA component of C8-CoA also interacts with LipB. Therefore, LipB:CoA binding studies were carried out using NMR at 283 K. The chemical structure of CoA is shown in Figure 4A, with all its nonexchangeable proton resonances labeled. Amide protons of CoA could not be observed in 90% H2O sample due to hydrogen–deuterium exchange. Proton chemical shifts of CoA were assigned based on the BMRB ID bmse000271. Figure 4B1 illustrates the 1H NMR spectra of CoA in D2O (used as a reference). The sample displays three peaks in the 6.0 to 9.0 ppm range, corresponding to 1, 2, and 1′ resonances of the adenosine component of CoA . In the 0 to 5 ppm range, 4′ and 5′ peaks from the adenosine component (colored black) and 1", 2", 3", 4", 5", 6", 7", and 8" peaks arising from the phosphopantetheine arm were also observed. Figure 4B2 displays the NMR spectra for CoA in buffer . Figure 4B3 shows the 1H NMR spectra for CoA in the same Tris–HCl buffer pH 7.8, in presence of 1 mM WT LipB (for reference). Figure 4B4 illustrates the saturation transfer difference (STD) spectra for CoA in buffer to ensure no intramolecular NOE transfer occurs. B5–B8 displays the STD spectra for CoA in the presence of WT LipB, LipBK135A/C169A , LipBG72A/G73A, and LipBR71A, respectively. All these STD experiments were acquired using a CoA:LipB molar ratio of 10:1. NOEs corresponding to position 1, 2, and 1′ were observed in B5–B8, suggesting binding of the adenosine ring. Similarly, in the 0 to 5 ppm range, peaks 2", 3", and 8" corresponding to the phosphopantetheine arm displayed NOEs in the STD spectra in presence of WT and the mutant LipB. The on resonance (red), off resonance (blue),and STD spectra (green) for the binding of LipBK135A/C169A with CoA in 50 mM Tris, 200 mM NaCl, pH 7.8 is illustrated in Fig. S6B. Fig. S6A shows the chemical structure of CoA.C31P NMR studies were also performed to understand the interaction of LipB with the phosphates of CoA. Figure 4C1 shows the 31P NMR spectra for 1 mM LipB in 50 mM Tris, 200 mM NaCl, pH 7.8 at 298 K. No peaks were observed, suggesting protein as such does not contain any phosphate group. Figure 4C2 displays the 31P spectra for CoA in 50 mM Tris, 200 mM NaCl, pH 7.8. Three prominent peaks were visible, corresponding to 3′-phosphate, 1"-phosphate, and 5′- phosphate. C3–C6 show the 31P spectra for CoA in presence of LipBK135A/C169A, WT, LipBG72A/G73A, and LipBC169A, respectively. The peak corresponding to 3′-phosphate displayed an upfield change in chemical shift upon addition of WT LipB (C4), LipBG72A/G73A (C5), and LipBC169A (C6). The new peak arising as a result of this interaction is indicated by a downward pointing arrow in the figures. No change in peak position was observed in LipBK135A/C169A sample (C3), suggesting interaction of Lys135 with 3′-phosphate of CoA, that is lost in the double mutant. A CoA:LipB ratio of 8:1 was used for these experiments. WT LipB C4, LipBG72A/G73A C5, and LipBC169A C6. The nA sample C3, sugge15N-labeled E. coli LipBK135A/C169A  was titrated with C8-CoA, and the titration was followed by NMR. Several amides in the 15N TROSY-HSQC experiment displayed significant chemical shift change (red), 1:0.2 (cyan), 1:0.4 (yellow), 1:0.8 (green), 1:1 (magenta), and 1:2 (orange) are shown as multiple overlaid spectra. The direction of chemical shift change is shown by a black arrow. Most of the peaks displaying perturbations appeared partially broadened due to chemical exchange. Unfortunately, owing to the low stability of the enzyme at high concentrations (necessary for backbone assignments), we were unable to assign the backbone amides of E. coli LipB. The pattern of amide perturbations though confirmed that E. coli LipB binds C8-CoA.t change . In the M. tuberculosis MtLipB structure (PDB 1W66) was superimposed on the catalytic domain of E. coli LplA complexed with C8-AMP , using the matchmaker option of Chimera, shown in Fig. S8A (E. coli LplA structure (PDB 3A7A), N1 and N6 of adenine form a hydrogen bond with the side chain of Asn 83 OD1, Thr 151, and the backbone O of Phe 78. Also, N7 forms a hydrogen bond with the backbone N of Phe 78. In M. tuberculosis LipB , remarkably similar residues are present at some of these sites, Gln 87 and Trp 82 (instead of Asn 83 and Phe 78 in LplA). In E. coli LipB, Gln 82 and Tyr 77 are present, while MmLipT2 possesses Gln 96 and Phe 91 at the same position in the sequence. The oxygen atoms of AMP (O4 and O5) interact with the ε amino group of Lys 133 in EcLplA. The equivalent residue is Lys 142 in MtLipB and Lys 135 in E. coli LipB. Also, in the LplA structure (PDB 3A7A), lipoyl moiety interacts with numerous hydrophobic/aromatic side chains, viz. Trp 37, Val 44, Arg 70, Val77, His79, and His149. In MtLipB, these positions are occupied by Leu 47, Thr 54, Arg 76, Thr 81, His 83, and His 157 in the structure . In E. coli LipB, Val 41, Thr 48, Arg 71, Thr 76, His 78, and His 150 are present at the same position in the sequence, shown in Fig. S8B.Notably, the lipoate scavenging enzyme LplA has adenine- and phosphate-binding sites that favor lipoic acid and ATP binding. We asked whether adenine and phosphate binding sites are also conserved in LipB, given the evolutionary relationship of the two proteins. Thus, Fig. S8A . In mostFig. S8A . In the 69RRXXGGG75 of LplA (PDB 3A7A) is also somewhat conserved in MtLipB and has a very similar conformation, shown in C and D  structurally overlaps with Arg 76 and Gly 78 of M. tuberculosis LipB (PDB 1W66). Arg 71 and Gly 72 are the equivalent residues in E. coli LipB. Interestingly, LplA Arg 70 side chain forms unique side chain to backbone hydrogen bonds with the carbonyls of Gly73, Gly75, and side chain of Ser72 in the LplA structure . These interactions have been speculated to stabilize the lipoate-binding loop of LplA. In MtLipB as well, arginine 76 side chain forms identical hydrogen bonds with the backbone carbonyls of Gly 77 and Lys 79, colored cyan in Fig. S8D. The structural similarities between LipB and LplA hint at the possibility of conservation of adenine- and phosphate-binding sites in LipB, like LplA.The lipoate-binding loop  C and D . MtLipB 8-CoA as octanoyl donor and GcvH as acceptor substrate. Figure 5A shows a 12% SDS-PAGE gel, displaying molecular weight marker in lane 1, purified apo-EcGcvH in lane 2 (used as a reference), and assays with WT LipB, LipBG72A/G73A, LipBR71A, LipBK135A, and LipBK135A/C169A, respectively, in lanes 3 to 7. Apo-GcvH to C8-GcvH conversion was ascertained from the forward migration of the GcvH band in the gel. Compared to WT LipB, GcvH conversion was remarkably reduced in lanes 4, 5, 6, and 7, suggesting loss of activity of the mutants we tested. LipBG72A/G73A displayed 39%, while LipBR71A displayed 21% activity compared to WT LipB, the values determined using C18 HPLC reversed-phase chromatography.The importance of RGG loop in LipB function was investigated by generating LipBR71A and LipBG72A/G73A mutants and performing enzyme assays using C8-ACP as an octanoate donor and GcvH as acceptor. A marked reduction in enzyme activity was observed in LipBG72A/G73A and LipBR71A mutants compared to WT LipB (B). In the 12% SDS-PAGE gel, lane 1 is molecular weight marker, lane 2 apo-GcvH (used as a reference), and lanes 3 to 5 assay performed with WT LipB, LipBG72/G73A and LipBR71A mutants, respectively. C8-GcvH band was most intense in lane 3 and relatively faint in lanes 4 and 5.Similar LipB assays were also performed using C WT LipB B. In thefilled triangles) and LipBG72A/G73A mutants (filled squares), using C8-CoA as an octanoate donor, shown in Figure 3A. A Km value of 6.7 ± 0.9 μM (a twofold decrease) and a Vmax of 0.32 ± 0.01 μmol min−1 (approximate threefold decrease compared to WT) was observed for the LipBG72A/G73A mutant. LipBR71A mutant displayed a Km value of 34.4 ± 8.8 μM (approximately threefold increase) and a Vmax of 0.26 ± 0.02 μmol min−1 . In comparison, LipBK135A mutant displayed a Km of 42.4 ± 4.6 μM (3.5-fold change) and a Vmax of 0.2 ± 0.01 μmol min−1 , MtLipBG77A/G78A, and MtLipBR76A mutants. In both WT and MtLipBG77A/G78A simulations, Arg 76 side chain was observed to flip out of the active site cavity as a function of time (A and B). During the course of simulations, capping sheet and the RGG loop were observed moving upward, away from the active site in WT LipB (A), widening the mouth of the cavity. The starting structure used for simulations is shown as a green ribbon. The direction of capping sheet and loop movement is shown by black arrows. In MtLipBG77A/G78A mutant, the capping sheet displayed restricted movement, but the loop containing C176 displayed reasonable change. In MtLipBR76A mutant, motions were restricted in both Cys176 containing loop as well as the capping sheet, with all the loops in the structures converged (C). Figure 6D shows the molecular surface of WT LipB, displaying the active site cavity tunnel, and the RGG loop masking it. We speculate that the movement of the RGG loop away would widen the mouth of the active site. Figure 6E displays root mean square fluctuation (RMSF) values as a function of residue number for WT MtLipB , MtLipBR76A (green lines), and MtLipBG77A/G78A mutant (red lines) simulations. In the WT protein, there were large magnitude RMSF fluctuations in the capping sheet and the RGG loop. The motions in the capping sheet were subdued in MtLipBG77A/G78A and MtLipBR76A mutants. Figure 6F shows the rmsd values for the three proteins as a function of time. All the structures converged within 20 ns simulation time, though MtLipBR76A converged relatively faster and at a much lower rmsd value. Overall, our results highlight the possible role of RGG loop in regulating the movement of the capping sheet and maintaining the conformation of the Cys176 loop, thus, making active site accessible to the substrate and releasing the product.The lipoate-binding ubstrate . Therefoubstrate . PDB 1W6 of time , A and B WT LipB A, widenionverged C. FigureIn prokaryotes, two different pathways for lipoic acid modification coexist: (a) an endogenous lipoic acid synthesis pathway, catalyzed by the consecutive action of LipB and lipoyl synthase (LipA) or (b) a salvage pathway carried out by LplA. Yeast and higher eukaryotes lack the scavenging pathway and derive lipoic acid exclusively from the biosynthetic pathway by the sequential action of LipT2, LIAS, and LipT1 . The thrE. coli LipB, LipA, and LplA. LipB (the biosynthetic enzyme) and the salvage enzyme LplA differ from each other in several respects: (a) C8-ACP, an intermediate of the fatty acid synthesis pathway is the only known substrate of LipB, while LplA is a lipoic acid/ATP-dependent enzyme; (b) a cysteine-lysine dyad serve as catalytic residues of LipB, whereas a lysine performs lipoate adenylation as well as transfer reactions of LplA . Despite these dissimilarities, unique parallels appear to exist between the two enzymes with regard to substrate recognition, as revealed by our studies. STD experiments and 31P NMR studies suggest binding of adenosine and phosphopantetheine moiety of CoA to LipB, similar to LplA (MtLipB (1W66) on the catalytic domain of E. coli LplA (3A7A) also hints toward conservation of residues in LipB at sites that interact with adenine and phosphate groups of AMP in LplA structures . Interestingly, the two sites are conserved in the sequence of lipoate transferase 2  as well, and the latter enzyme also converts GcvH using C8-CoA, divulging evolutionary relationships  rea to LplA . Strongeionships .via a well-defined lipoate-binding loop. An 76RGG78 loop present in the active site of (M. tuberculosis) MtLipB (1W66) has sequence and conformation fairly similar to the lipoate-binding loop 69RRSSGGGAV77 of LplA. In the LplA structure (PDB 3A7A), Arg 69 forms unique side chain to backbone hydrogen bonds with the glycine and Ala of the loop, in a manner similar to Arg 76 of LipB (M. tuberculosis). This lipoate-binding loop is crucial for lipoylation in LplA and experiences large scale conformational changes upon lipoic acid binding (M. tuberculosis LipB (PDB 1W66) suggest similar motions in the capping sheet and the RGG loop that are quenched upon mutation of Arg 76 in the sequence to alanine. Conversely, mutation of the conserved glycines in the RGG sequence to Ala increased the overall dynamics of the Cys176  loop, while the capping sheet displayed reduced motions, inducing major changes in the conformation of the active site. Interestingly, 'RGG' sequences are frequently found in RRM domains and are known to bind phosphates of nucleotides/quadruplexes  allows ACP to bind LipB with several-fold higher affinity. Addition of a Cyl chain , 29.8-CoA, which can also serve as a substrate for LipT2. This information might be of assistance while treating patients with human mitochondrial fatty acid synthase mutations; genes encoding mitochondrial trans-2-enoyl-coenzyme A-reductase (MECR)  , 44, male (MECR) . The syme (MECR) . Two cane (MECR) . Likewise (MECR) . We surmynthesis . AnotherA or ACP . Authorsplements . Our in E. coli genome and cloned in a pET28a expression vector between NdeI and HindIII restriction sites. The corresponding plasmids have been named as follows: pET28a(+)-EcACP, pET28a(+)-EcLipB, pET28a(+)-EcGcvH. Mice LipT2 gene (synthesized after codon optimization) was also cloned similarly (pET28a(+)-MmLipT2). Mutagenesis studies were performed using site-directed mutagenesis approach using Pfu turbo as the polymerase and pET28a(+)-EcLipB as a template. The PCR product was subjected to DpnI digestion overnight at 25 °C, in order to digest the methylated circular DNA. Approximately 15 μl of this product was transformed in XL-I blue competent cells. Colonies obtained were inoculated in 5 ml LB media, followed by overnight culture, pelleting, and finally plasmid isolation. For the double mutant, LipB plasmid containing lysine mutation was used as a template. All the mutations  were confirmed by DNA sequencing.Genes encoding ACP, LipB, and GcvH were PCR amplified from the EcACP and pET28a(+)-EcLipB plasmids were transformed separately into E. coli BL21(DE3) cells and plated on LB agar supplemented with 50 μg/ml of kanamycin. One colony of each was inoculated in 5 ml of LB media supplemented with kanamycin (50 μg/ml). The primary culture was incubated at 37 °C for 14 to 16 h, accompanied with shaking at 200 rpm. For large scale expression, 1 l LB medium (secondary culture supplemented with 50 μg/ml kanamycin) was inoculated with 1% of the overnight grown primary culture. The secondary culture was kept at 37 °C with shaking until its A600 reached a value of 0.6 to 0.8. For ACP and LipB expression, secondary culture was induced with 0.5 mM IPTG and incubated at 25 °C for 14 to 16 h, with shaking at 200 rpm. Post induction, cells were harvested by centrifugation at 4000 rpm at 4 °C for 30 min, and the pellet was used for protein purification.pET28a(+)-2HPO4 (3.4 g), KH2PO4 (1.5 g), NaCl (0.25 g), and NH4Cl (0.5 g) in distilled water. The media was autoclaved and cooled. Prior to culture inoculation, 1 g of unlabeled/labeled glucose (40%), 1 ml of 1 M MgSO4, 10 μl of 1 M CaCl2, 500 μl of 0.5% thiamine HCl, 500 μl of 50 mg/ml kanamycin, and 5 ml of vitamins  were added. To the 500 ml culture media, 4 ml of primary overnight grown culture was added. The secondary culture was kept at 37 °C with shaking until its A600 reached a value of 0.6 to 0.8. For ACP, culture was incubated at 25 °C for 14 to 16 h, with shaking at 200 rpm. Post induction, cells were harvested at 4000 rpm at 4 °C for 30 min, and the pellet was used for protein purification.The protocol for transformation and primary culture were exactly the same as described for the unlabeled protein. However, for the secondary culture, 500 ml M9 media was prepared by dissolving NaEcACP is expressed in both apo- and holo-forms, an additional phosphodiesterase activation step was introduced. The sonicated cells were incubated with 10 mM MgCl2 and 2 mM MnCl2 at 35 °C, 120 rpm for 2 h. Both, Mg2+ and Mn2+ ions were used to activate the natural E. coli phosphodiesterase, thereby converting holo-ACP to apo-ACP, and enriching the apo-ACP concentration in the lysate (2+-NTA (Sigma) column, equilibrated with 20 mM sodium phosphate buffer, pH 7.8, containing 500 mM NaCl (washing buffer). The protein-bound resin was washed with 100 ml of washing buffer, followed by 100 ml of 20 mM sodium phosphate, pH 6.0, 500 mM NaCl wash (elution buffer) to remove the nonspecifically bound proteins. Next, the protein was eluted with imidazole in elution buffer and concentrated by 3 kDa cutoff centricons and imidazole removed. For the NMR samples, N-terminal His tag of ACP was cleaved by incubating with immobilized thrombin beads at 25 °C overnight while shaking at 120 rpm, post imidazole removal. In the subsequent step, salt concentration was decreased to 0 mM by washing the protein with 20 mM sodium phosphate buffer pH 6.0.Pellets were suspended in 30 ml of 20 mM sodium phosphate buffer (pH 7.8) and 500 mM NaCl. 1× EDTA free protease inhibitor cocktail (Sigma) was added to the suspended pellet and subjected to sonication . As e lysate . SonicatSince both apo- and holo-forms of the protein were present in the purified ACP fraction, the sample was subjected to ion-exchange chromatography using Resource Q column and eluted with a linear salt gradient of 0 to 500 mM NaCl in 20 mM sodium phosphate buffer, pH 6.0, 2 mM DTT. Separated apo- and holo-ACP were passed through size-exclusion columns to attain maximum purity, using 20 mM sodium phosphate buffer, 100 mM NaCl, pH 6.0.2+-NTA column, pre-equilibrated with resuspension buffer. The column was washed with the same buffer with 5 mM imidazole to eliminate nonspecific proteins. The protein of interest was eluted with 50 to 500 mM imidazole in resuspension buffer. The eluted fractions containing LipB were identified on a 12% SDS-PAGE gel . Suspended cells were subjected to sonication . After sonication, cells were centrifuged at 10,000 rpm, and the supernatant was passed through NiPAGE gel  and concEcLipB WT enzyme as well as the mutants. In case of EcACP, purification took nearly 2 days at 4 °C.Most of the proteins were purified within 1 day starting from pellet, specially 8-ACP in vitro, using B. subtilis phosphopantetheinyl transferase (Sfp), using a modified protocol by Lambalot and Walsh .Apo-ACP was converted to holo-ACP and Cnd Walsh . Sfp was1H,15N,13C] protein, in 50 mM Tris–HCl, 200 mM NaCl, pH 8.0, 90% H2O, and 10% D2O, 0.5% sodium azide. All the spectra were acquired on a Bruker Avance III 700 MHz NMR spectrometer, installed at the National Institute of Immunology, equipped with triple resonance TXI probe. Triple resonance NMR experiments, viz. HNCA, HNCACB, CBCAcoNH, etc. were used to assign apo-, holo-, and C8-ACP. 1H15N TROSY-HSQC spectra were acquired using 1024 data points in the t2 dimension and 256 to 512 data points in the t1 dimension. NMR data were processed on a workstation running Red Hat Enterprize Linux 5.0, using NMRDraw/NMRPipe, and analyzed using Sparky . A train of 50 ms Gaussian-shaped radio frequency pulses, attenuation level 55 dB, separated by intervals of 2 ms were used. Excitation sculpting was employed to suppress the water signal. The frequency for selective radiation for on resonance was placed at −3 ppm (for upfield shifted methyl protons) and off resonance at 35.5 ppm, respectively. A relaxation delay of 5 s was used. The acquisition time was 0.73 s. Subtracting on from off signal gave a positive STD signal from the bound CoA in the difference spectra. The ligand peaks observed in the 1D STD experiment were assigned based on 31P NMR measurements were performed on a 5-mm broad band inverse probe, at 298 K, using Bruker ZG30 parameter set. A spectral width of 400 ppm, 65,536 data points, 2.0 s recycle delay, 14,336 scans, and 0.288 s acquisition time were used. A 30° flip angle was used in the experiment, with no proton decoupling. All the spectra were processed with a line broadening of 1.0 Hz, and phase correction using Topspin 2.1pl3 software.1H chemical shifts (ΔAvgHN), derived from Equation Chemical shift perturbations have been reported as weighted average of the nitrogen and 15N-labeled C8-, apo-, or holo-ACP in 50 mM Tris and 200 mM NaCl, pH 7.8 was titrated with LipBK135A/C169A, and chemical shift perturbations were followed in a 15N TROSY-HSQC experiment. 15N-labeled holo-ACP and octanoyl-ACP were first prepared by converting apo-ACP to C8-ACP using B. subtilis phosphopantetheinyl transferase and respective CoA. For each titration point, a 1H15N TROSY-HSQC spectra were acquired. 1H15N TROSY spectra of apo-/holo-/C8-EcACP were acquired at a concentration of 0.3 to 0.4 mM and then increasing concentrations of the unlabeled protein EcLipBK135A/C169A was added starting from 0.25 M equivalents to 2 M equivalents sequentially and the spectra recorded after each addition. After each addition, the concentration of apo-/holo-/C8-EcACP was recalculated taking into account the dilution factor due to EcLipBK135A/C169A addition. Thus, after each titration point, the amount of EcACP decreased and EcLipBK135A/C169A increased. The concentration of ACP, as well as the enzyme stocks used for NMR studies and enzyme assays, were calculated using Bradford reagent in triplicates, and every time a new standard curve was prepared so as to minimize error.The buffers were prepared from a single stock solution of 250 mM Tris–HCl, pH 7.8, containing 1 M NaCl stored at 4 °C, which was diluted fivefold to obtain the working concentration  for NMR studies.Binding strength of LipB for its substrates was determined by Autolab ESPRIT SPR spectrometer and analyzed by Autolab SPR kinetic evaluation software (Metrohm Autolab B.V.). Gold surface was activated by N-hydroxysuccinimide (0.05 M)/N-ethyl-N-(diethylaminopropyl) and carboiimide (0.2 M). LipB was immobilized on the gold surface in two channels. Unoccupied sites were blocked with 100 mM ethanolamine. After each experiment, regeneration was carried out with 50 mM NaOH. Increasing concentration of the substrate was injected on the sensor surface. Association and dissociation kinetics were monitored for 300 s and 150 s, respectively, in 20 mM sodium phosphate buffer, pH 8.0, and 100 mM NaCl. All experiments were carried out at 298 K. The SDs have been obtained from global fitting of five injections using 1:1 Langmuir model.8-CoA/octanoyl-ACP as the donor substrate, and 25 μM GcvH as the acceptor substrate in 50 mM Tris–HCl, 200 mM NaCl, pH 8.0, at 30 °C. The reaction components were incubated for 2 h at 30 °C and then quenched by adding the 1× SDS loading dye and stored at −20 °C overnight. The samples were loaded on a 12% SDS-PAGE gel for qualitative estimation of the conversion of GcvH or native-PAGE gel to follow breakdown of C8-ACP. For quantitative estimation, reverse phase Symmetry C18-5 μM Waters HPLC column was used. Twenty microliter of the enzyme assay was quenched using 0.1% TFA. The sample was injected onto the C18 Waters column. Proteins were eluted by linear gradient of 0 to 100% acetonitrile, containing 0.1% TFA. For all the assays, absorbance was observed at 220 nm. The apo/unmodified protein eluted prior to holo/C8-bound form. The area under the peak was integrated to determine the concentration of apo-form and holo-form. Kinetic parameters were estimated by GraphPad Prism version 7.0.Enzyme activity was performed using 5 μM LipB, 75 μM CFor LipT2 assay, the reaction mixture was incubated at 37 °C, and the buffer comprised 50 mM Tris–HCl, 500 mM NaCl, pH 8.0. The octanoate acceptor substrate was GcvH in all the assays.All assays were done in duplicates, and in some cases, the error bars were even smaller than the symbols.4 Zip Tips to remove salt, and the final sample was eluted in 50% acetonitrile and 0.1% TFA for analysis in Applied Biosystems 4800 MALDI-TOF spectrometer. Mass Spectrometry was used only to confirm full conversion.The assay samples were passed through CM. tuberculosis LipB PDB file 1W66 (without the ligand or water) was used for WT protein simulations. For the LipBG77A/G78A and LipBR76A mutants, Chimera , volume (V), and temperature (T) (NVT) and constant number of particles (N), system pressure (P) and temperature (T) (NPT) ensembles applying a positional restraining force on the heavy atoms. NVT thermal equilibration was performed using velocity-rescaling temperature coupling for 100 ps at 300 K using a modified Berendsen thermostat with a time constant of 0.1 ps. The temperature of the system reached the target value of 300 K and was stable for the rest of the equilibration. Equilibration of pressure (1 bar) was done for 100 ps under an NPT ensemble, using the Parrinello–Rahman temperature coupling, with a time constant of 2 ps. In NVT and NPT simulations, bonds to hydrogen atom were constrained using LINCS algorithm (All MD simulations were performed using GROMACS 2021.5 software, installed on a Mac, using OPLS-All Atom force field . M. tube Chimera  was used Chimera . One Na+lgorithm . Next, Mlgorithm , coulombmonicasundd@nii.ac.in.Data are available from the authors upon request. Please send request to Monica Sundd, This article contains The authors declare that they have no conflicts of interest with the contents of this article.


The cell proliferation was detected by MTT and plate clone formation assays, cell apoptosis, cell cycle, and mitochondrial membrane potential (MMP) were analyzed by flow cytometry and Hoechst 33258 staining assays, and the intracellular reactive oxygen species (ROS) accumulation were also investigated. Real-Time PCR and western blot were examined to explore the mechanism of action. The results indicated that GA suppressed HCC1806 cells proliferation and promoted HCC1806 cells apoptosis. Meanwhile, GA treatment changed the morphology of the HCC1806 cells. In addition, GA blocked the HCC1806 cells cycle in the S phase, and it induced cells apoptosis accompanied by ROS accumulation and MMP depolarization. Real-Time PCR results suggested that GA increased Bax, Caspase-3, Caspase-9, P53, JINK and P38 mRNA expression, and decreased Bcl-2, PI3K, AKT and EGFR mRNA expression. Western blotting results suggested that GA increased Bax, cleaved-Caspase-3, cleaved-Caspase-9, P53, P-ERK1/2, P-JNK, P-P38 proteins expression, and decreased Bcl-2, P-PI3K, P-AKT, P-EGFR proteins expression. Furthermore, molecular docking suggested that GA has the high affinity for PI3K, AKT, EGFR, ERK1/2, JNK, and P38. In conclusion, GA could suppress HCC1806 cells proliferation and promote HCC1806 cells apoptosis through the mitochondrial apoptosis pathway and induces ROS generation which further inhibits PI3K/AKT/EGFR and activates MAPK signaling pathways. Our study will provide some new references for using GA in the treatment of TNBC.Triple-negative breast cancer (TNBC) is a severe threat to women’s health because of its aggressive nature, early age of onset, and high recurrence rate. Therefore, in this study, we aimed to evaluate the anti-tumor effects of Gallic acid (GA) on the TNBC HCC1806 cells  Breast cancer (BC) is a malignant tumor that seriously threatens women’s life and health worldwide . AccordiBrucea javanica Seed could suppress TNBC cell proliferation and promote cell apoptosis. via NF-κB pathway activation. Therefore, the search for new anti-tumor drugs from plant extracts has attracted the attention of a wide range of researchers.As we all know, an increasing number of studies have demonstrated that plant extracts, especially traditional Chinese medicine (TCM) extracts have apoptosis-promoting effects on a wide range of cancer cells. For example, Gallic acid (GA) is a polyphenol compound with various biological activities such as antioxidant, anti-inflammatory, anti-tumor, anti-viral and anti-bacterial . In currAlthough some studies have confirmed the anti-tumor activity of GA against TNBC, for example, GA with 99% purity was purchased form Shanghai Macklin Biochemical Co., Ltd. . RPMI-1640 medium, crystal violet and Cell Cycle Assay Kit were purchased form Jiangsu KeyGEN BioTECH Corp., Ltd. . Fetal bovine serum (FBS) was purchased form Gibco (United States). MTT and Hoechst 33258 solution were acquired from Beijing Solarbio Science & Technology Co., Ltd. . Annexin V-FITC Apoptosis Detection Kit was acquired from BD Biosciences . Mitochondria Staining Kit (JC-1) was acquired from MultiSciences (Lianke) Biotech Co., Ltd. . ROS Assay Kit was acquired from Beyotime Institute of Biotechnology . The antibodies against Caspase-3, cleaved-Caspase-3, cleaved-Caspase-9, Bax, Bcl-2, PI3K, P-PI3K, AKT, P-AKT, JNK, P-JNK, ERK1/2, P-ERK1/2, P38, P-P38, EGFR, P-EGFR, GAPDH were obtained from Cell Signaling Technology . The antibodies against Caspase-9 and P53 were obtained from Proteintech Group, Inc. .2. GA was prepared into a storage solution  with FBS-free RPMI-1640 medium, then stored at -20°C. Dissolve the GA stock solution and dilute it to the appropriate concentration with FBS-free RPMI-1640 medium before using it.The human TNBC HCC1806 and MDA-MB-468 cells line were purchased form Jiangsu KeyGEN BioTECH Corp., Ltd. . HCC1806 cells were cultured with RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C in a humidified incubator with 5% CO3 cells/well) seeded into 96-well plates overnight and then treated with GA  for 24, 48, and 72 h. The MTT (5 mg/ml) reagent was added to each well (20 μL) and cultured for another 4 h. Next, dimethyl sulfoxide (DMSO) was added to each well (150 μL), and the absorbance was recorded at 490 nm by a Enzyme labeling instrument . MDA-MB-468 cells were (5 × 103 cells/well) seeded into 96-well plates overnight and then treated with GA  for 24 h. Other steps are the same as those of HCC1806 cells  for 24 h. The cell morphology was observed by an inverted microscope  .3 cells/well) seeded into 6-well plates overnight, and then treated with GA  for 24 h. Subsequently, removed the media and added to fresh media, and allowed to grow undisturbed for 10 days. Cells were fixed with 4% paraformaldehyde for 15 min and stained with crystal violet solution for 15 min. The plates were dried and photographed, and ImageJ Software counted the number of colonies  for 24 h. Subsequently, cells were digested and collected, washed once in PBS, fixed for 3 h at 4°C in 2 ml of 75% ethanol, then washed once in PBS, 500 μL PI/RNase dye solution was added and incubated for 20 min at room temperature and protected from light, and the fluorescence intensity of HCC1806 cells were detected by flow cytometry . .HCC1806 cells were seeded into 6-well plates overnight, and then treated with GA  for 24 h. Next, cells were fixed with 4% paraformaldehyde for 15 min, followed by incubation with Hoechst 33258 solution for 10 min. Cells were observed and photographed under an inverted fluorescence microscope  .HCC1806 cells were seeded into 6-well plates overnight, and then treated with GA  for 24 h. Subsequently, cells were digested and collected, then washed with cold PBS. Moreover, add 500 μL of Binding Buffer to each tube, resuspension by centrifugation, add 5 μL of Annexin V-FITC and 10 μL of PI staining solution to each tube, and incubate for 15 min at 25°C, protected from light, and the apoptosis rate of HCC1806 cells were detected by flow cytometry . .HCC1806 cells were seeded into 6-well plates overnight, and then treated with GA  for 24 h. Subsequently, cells were digested and collected, and washed with PBS. Then add 1 ml JC-1 Staining Buffer to each tube and incubated for 30 min at room temperature and protected from light, and the depolarization rate of HCC1806 cells were detected by flow cytometry  .HCC1806 cells were seeded into 6-well plates overnight, and then treated with GA  for 24 h. Subsequently, 1 ml DCFH-DA working solution was added to each well and incubation at 37°C for 30 min. Finally, cells were observed and photographed under an inverted fluorescence microscope , and fluorescence intensity was detected by ImageJ Software .−ΔΔCT method and normalized to GAPDH  for 24 h. Cells were processed and harvested according to the manufacturer’s instructions, total RNA was extracted using a TRIzol reagent kit . RNA was reverse-transcribed with cDNA reverse transcription kits . The sequences of PCR primers were design and synthesis form Sangon Biotech  Co., Ltd. . And the sequences as followed in to GAPDH .HCC1806 cells were seeded into 6-well plates overnight, and then treated with GA  for 24 h. After that, The culture solution was removed and the 6-well plates were transferred to ice and washed 3 times with PBS. Then 300 μL RIPA lysis buffer solution was added to each well, lysed on ice for 30 min and then transferred to EP tubes and centrifuged. Protein concentrations were detected by the Enhanced BCA Protein Assay Kit . The protein samples were separated by 10% or 8% SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane . BSA (5%)  was used to block the membrane for 1 h, and then the primary antibodies Caspase-3 (1:1000), cleaved-Caspase-3 (1:500), cleaved-Caspase-9 (1:1000), Bax (1:1000), Bcl-2 (1:500), PI3K (1:1000), P-PI3K (1:1000), AKT (1:1000), P-AKT (1:1000), JNK (1:1000), P-JNK (1:500), ERK1/2 (1:1000), P-ERK1/2 (1:1000), P38 (1:1000), P-P38 (1:1000), EGFR (1:1000), P-EGFR (1:500), P53 (1:1000), Caspase-9 (1:1000) and GAPDH (1:1000) were incubated overnight at 4°C, followed by incubation with the secondary antibodies  at room temperature for 1 h. Protein bands were visualized using the ECL Plus western blotting detection reagents  following the manufacturer’s instructions. The relative expression of proteins were analyzed by ImageJ Software .To further explore the molecular target of GA in TNBC cells, The molecular docking of GA with PI3K, ERK1/2, JNK, EGFR, AKT and P38 were carried out by autodock 4.2.6 software. It is generally believed that the binding energy <0 indicates that the small molecule components of the ligand and the target protein of the receptor can bind independently. The lower the binding energy, the more stable the molecular conformation, the greater the possibility of action, and the greater the absolute value of the binding energy, the better the binding between the ligand and the receptor.± standard deviation (SD). Each experiment was repeated at least six times. All the data of this paper were analyzed with SPSS 22.0 . Comparison between groups using one-way ANOVA followed by Tukey’s test. p < 0.05 was defined as statistically significant.The results were shown as the mean MTT assay was performed to detect the MDA-MB-468 and HCC1806 cells viability. As shown in After different concentrations of GA  treatment HCC1806 cells for 24 h, the changes in cell morphology could be observed under an inverted microscope. As shown in The plate clone formation assay was performed to assess the effect of GA on HCC1806 cells cytotoxic activity. As shown in 0/G1 phase was 50.47% ± 1.63%, in S phase was 23.32% ± 2.25%, and in G2/M phase was 25.88% ± 2.13%. Compared with the control group, the percentage of G0/G1 phase in GA treatment groups  were 39.18% ± 0.30%, 37.27% ± 0.22%, 36.87% ± 0.14%, respectively, the percentage of G0/G1 phase in GA treatment groups were significantly decreased. Compared with the control group, the percentage of S phase in GA treatment groups  were 31.37% ± 1.07%, 34.06% ± 1.03%, 34.16% ± 1.27%, respectively, the percentage of S phase in GA treatment groups were significantly increased. These results suggested that GA blocked HCC1806 cells cycle in S phase.The flow cytometry was performed to assess the effects of GA on HCC1806 cells cycle. As shown in Hoechst 33258 Staining and flow cytometry were performed to assess the effect of GA on HCC1806 cells apoptosis. As shown in The JC-1 staining method was used to detect the cell MMP level. As shown in The DCFH-DA fluorescent probe was used to detect intracellular ROS accumulation and assess cellular damage’s extent. As shown in To further investigate the effect of GA in promoting apoptosis and to assess whether this effect was associated with mitochondrial functional impairment, we examined the expression of related proteins and genes. As shown in via modulating PI3K/AKT/EGFR and MAPK signaling pathways.In order to investigate the possible molecular mechanisms of GA-induced apoptosis, we examined the expression of PI3K, P-PI3K, AKT, P-AKT, EGFR, P-EGFR, ERK1/2, P-ERK1/2, JNK, P-JNK, P38 and P-P38 proteins. As shown in −1, −5.61 kcal mol−1, −4.64 kcal mol−1, −9.21 kcal mol−1, −4.39 kcal mol−1 and −7.63 kcal mol−1, respectively. These molecular docking results showed that GA and each target protein had good docking activity, which could stably dock in the active capsule of the receptor protein and form hydrogen bonds. Therefore, it could be suggested that GA has a high affinity for PI3K, AKT, EGFR, ERK1/2, JNK, and P38 from the docking studies.The molecular docking results were shown in 1 phase, induce apoptosis through the P38/P21/P27 signaling axis, activate the ferroptosis pathway by inducing ROS production in MDA-MB-231 cells, and also promote cellular ROS generation and depolarization of mitochondrial membrane potential in combination with curcumin, thereby inducing MDA-MB-231 cells apoptosis  need to be explored in further studies.In conclusion, our study provided the first evidence that GA inhibited HCC1806 cells proliferation and induced HCC1806 cells apoptosis


Dioscorea caucasica Lipsky is a tertiary relict endemic plant naturally growing in the western part of the trans-Caucasus regions; it has adapted and successfully grows in the temperate region of the Baltic countries. Information about its phytochemical composition and bioactivities is rather scarce. This study reports the results of the identification of 41 compounds in D. caucasica leaf and tuber hydroethanolic extracts using UPLC-QTOF/MS. Organic acids were found in both extracts; hydroxycinnamates and flavonoids were the main phytochemicals in the leaves, while steroidal glycosides, fatty acids (mainly hydroxylated) and carbohydrates were found in the tubers. Leaf extracts inhibited enzymes in a dose-dependent manner and were remarkably stronger inhibitors of physiologically important enzymes, namely α-amylase (48.6% at 480 µg/mL), α-glucosidase (IC50 = 41.99 and 47.95 µg/mL with and without 0.1 M Na2CO3), acetylcholinesterase (45.85% at 100 µg/mL) and angiotensin-converting enzyme (IC50 = 829.7 µg/mL), most likely due to the presence of some quantified polyphenolic antioxidants. The mode of inhibition of α-glucosidase and acetylcholinesterase was assessed via kinetic studies based on Lineweaver–Burk inhibition plots. Leaf and tuber extracts acted as mixed-type and competitive inhibitors of α-glucosidase, respectively; the leaf extract demonstrated an uncompetitive inhibition mode of acetylcholinesterase. It is expected that this new knowledge of D. caucasica will serve for its valorization in developing new health beneficial ingredients for functional foods and nutraceuticals. The interest in health beneficial natural substances has been steadily increasing during the last few decades. This tendency has been fostered by the fast developments in the area of functional foods and nutraceuticals, which have contributed in shifting healthcare concepts towards the increasing role of preventive medicine. In addition, such a generic characteristic of nutritional and healthcare products as ‘naturalness’ has become very popular among consumers . Due to Plant phytochemicals exert various bioactivities, which may provide numerous health benefits, particularly in preventing and/or delaying the development of age-dependent degenerative and chronic diseases . Among tDioscorea caucasica Lipsky leaves and tubers and the evaluation of their effects on the activity of physiologically important enzymes. Dioscorea is a large genus of the Dioscoreaceae family, comprising around 715 species, which probably originates from the South-East Asia region − ion at m/z 191.0556), malic 2 ([M − H]− ion at m/z 133.0137) and citric/isocitric 4 ([M − H]− ion at m/z 191.0191) acids were detected at tR 0.4 and 1.2 min, respectively. Shikimic acid 3 ([M − H]− ion at m/z 173.0450) with tR 0.5 min was detected only in the leaf extract, while piscidic acid 5 ([M − H]− ion at m/z 255.0505) with tR 1.8 min was found only in the tuber extract. Shikimic acid was reported in D. elephantipes, D. sylvatica and D. mexicana − m/z 290.0875) at tR 0.9 min perfectly matched the fragmentation pattern of this sialic acid derivative, which has been reported in various biomaterials, including plants. The identity of pyroglutamic acid 34 ([M − H]− ion m/z 128.0347) at tR 0.9 min and glucoheptonic acid 32 ([M − H]− ion m/z 225.0610) at tR 0.3 min is also in agreement with the previously reported data − precursor ions with m/z 353.0872 were recorded for the compounds 6, 10, 12 , which indicates the presence of several CQA isomers. The first compound 6 was identified as 3-caffeoylquinic acid. The second compound 10 was assigned to 5-chlorogenic acid based on its dimer m/z 707.1823 [2M − H]−, which yielded the peaks of m/z 353.0872 [M − H]− and m/z 191.0556 [M − H]−. The spectra are in agreement with those previously reported [m/z 297.0612 [M − H]−, the compound 9 was tentatively identified as caffeoylthreonic acid. The compounds 11, 12 produced molecular ions [M − H]− with m/z 337.0923, 353.0872 and the fragments of m/z 191.0556 [M–H–146]− and m/z 191.0556 [M–H–162]−, respectively; these spectral data enabled us to assign the peaks to coumaroylquinic and 4-caffeoylquinic acids, respectively. Compound 13 (tR = 2.0 min) yielded an ion at m/z 335.0766 [M − H]− and was identified as caffeoylshikimic acid [14 (tR = 2.1 min) yielded an ion at m/z 367.1029 [M − H]− and was assigned to feruloylquinic acid. It is evident that phenolic acids and flavonoids are the most important phytochemicals in the leaf extract: based on MS and other data, eight hydroxycinnamic acid derivatives and seven flavonoids were identified . Caffeoyreported ,32 and, 15–21 were identified as flavonoids, all being quercetin derivatives ]−. Thus, in the negative ion mode, the compounds 15, 16 with molecular ions at m/z 609.1455 and 463.0876 and retention times of 2.3, 2.4 min, correspond to quercetin 3-O-rutinoside (rutin) and quercetin 3-O-glucoside (isoquercitrin). The identity was also confirmed by the reference standards. The compound 19 (tR = 2.6 min) gave m/z of 447.0927 and, based on comparison with the spectral data available in the literature − at m/z 549.0880 and m/z 505.0982, respectively. Consequently, the compounds were identified as quercetin-3-O-malonylglucoside and quercetin-3-O-acetylglucoside, respectively h − ion m/z 341.1083) [24 ([2M − H]− ion at m/z 683.2246) and 25 ([3M − H]− ion at m/z 1025.3408) correspond to the spectral characteristics of deprotonated dimer and trimer oligosaccharide ions − ion m/z 279.2324 , and those of 29 [M − H]− ion m/z 295.2273 (tR = 6.9 and 8 min) correspond to linolenic acid (C18:3). Other derivatives suggest the structures of hydroxylated fatty acids: 30 [M − H]− ion m/z 271.2273 at (tR) 8.4 min and 26 [M − H]− ion m/z 329.2328 at (tR) 4.6 min correspond to hydroxyhexadecanoic and trihydroxyoctadecenoic acids, respectively. Hydroxyoctadecatrienoic acid 27 [M − H]− ion m/z 293.2117 was detected only in the leaf extract at (tR) 5.8 min. MS data do not provide sufficient information on the exact position of the hydroxy group in the fatty acid chain. Furthermore, MS data of the compound 28, [M − H]− ion m/z 358.2593 at (tR) 6.8 min, suggest the molecular formula of C19H36NO5, which fits a fatty acid ester . The la36, 37, 38, 39, 40; however, based on their [M − H]− m/z of 1109.5379 (tR = 3.3 min), 1093.5431(tR = 3.7 min) and 1075.5325 (tR = 4.3 min), 929.4746 (tR = 6.5 min), 913.4797 (tR = 6.6 min), which correspond to formulae of C52H85O25, C52H85O24, C52H83O23, C46H73O19 and C46H73O18, respectively, most likely belong to steroidal glycosides, which are abundant in yams [41 [M − H]− ion m/z of 767.4217 (tR = 6.8 min) also indicate steroidal saponin pentandroside B [45 [M − H]− ion m/z 723.3803 corresponding to C34H59O16 detected in tubers at 6.8 min and the compound 44 [M − H]− ion m/z 721.3646 detected in leaves at 5.6 min, and corresponding to C34H57O16, most likely belong to galactolipids. The compound 35 producing [M–H]− ion at m/z 345.1186 at (tR = 1.4 min) fit well with the relevant data of an iridoid glycoside and was tentatively identified as an aucubin. This iridoid is commonly found in different anatomical parts of plants and is responsible for the defensive function.It was not possible to elucidate the exact structures of  in yams  and some in yams . SpectraD. caucasica leaf extract was 5.89, 116.6, 4.02 and 187.2 µg/mL, respectively. Thus, quercetin glycoside and isoquercitrin were the most abundant phenolic constituents recovered from the plant leaves. In general, these data agree with the UPLC-QTOF/MS analysis, which demonstrated the highest m/z intensities for the major quantified compounds. As may be judged from the peak area in the chromatographic profile  Type I, belongs to the Glycoside Hydrolase 13 (GH_13) family  = 0) to 0.005 ([I] = 15 µg/mL) and 0.004 ([I] = 25 µg/mL); in enzymatic reaction II, from 0.018 ([I] = 0) to 0.01 ([I] = 15 µg/mL) and 0.005 ([I] = 25 µg/mL) . The decD. caucasica tuber extract. At the concentrations of 200 and 500 µg/mL, it competitively inhibited α-glucosidase: the Km(app) values increased from 69.34 ± 0.31 to 83.33 mg/L, whereas Vmax(app) 0.99 ± 0.22 and 1.034 ± 0.38 mg/L·min were not significantly different  of α-amylase, with a remarkably lower effect than acarbose.As the tuber extract possessed remarkably weaker α-glucosidase inhibitory effects, further studies were focused on the leaf extract. It is evident that e enzyme ; at 480,AChE (IUMB enzyme nomenclature number EC 3.1.1.7), also known as acetylcholine hydrolase, choline esterase I, cholinesterase, acetylthiocholinesterase and acetyl-β-methylcholinesterase, belongs to the cholinesterase (ChEs) family, which is most widely known for hydrolyzing the neurotransmitter acetylcholine (ACh) to choline (Ch) and acetic acid in the synaptic cleft. The principle of this assay method is based on the production of tiocholine (SCh), which is formed from acetylthiocholine during enzymatic hydrolysis with Ellman’s reagent (DTNB) and produces yellow-colored chromophore 5-thio-2-nitrobenzoate. The rate of appearance of the yellow derivative is measured spectrophotometrically at 412 nm .The samples for the assay were prepared from the lyophilized powder and tested for their ability to inhibit AChE catalysis in the concentration range of 25–100 µg/mL. The results are listed in p > 0.05) differences in decreased enzyme activity. The inhibitory effect of D. caucasica solution on AChE was compared with the synthetic inhibitor Donepezil HCl. As expected, the positive control was a more potent AChE inhibitor than the extracts of D. caucasica: at a 16 µg/mL concentration, it reduced enzyme activity by 70.62 ± 2% and Vmax to 0.0593 µM/min/mg protein.It may be observed that the inhibition noticeably increased after increasing the concentration of the leaf extract from 25 µg/mL (inhibition = 27.55%) to 40 μg/mL (inhibition = 40.58%), while a further increase in the concentration to 80 µg/mL resulted in a significant increase in AChE inhibition that was, however, less remarkable. However, at 80 and 100 µg/mL extract concentrations, there were no significant , Km(app) values were remarkably lower, 0.148 ± 0.015 µM/min/mg protein and 71.56 ± 6.55 µmol/L, respectively. This profile of inhibition indicates that the inhibitor only binds to the enzyme–substrate complex -L-phenylalanine-glycyl-glycine (FAPGG), p-nitrophenyl α-D-glucopyranoside (pNPG) (N1377-1G), 5,5′-dithiobis-(2-nitrobenzoic acid) , acetylthiolcholine iodide (ATChI); and starch were obtained from Sigma-Aldrich . Acetylcholinesterase (EC 3.1.17) from Electrophorus electricus, Type VI-S; lyophilized powder, 200–1000 units mg/protein (C3389)   and donepezil hydrochloridum  were purchased from the local pharmacy. Acarbose (C25H43NO18) was from Merck KGaA ; reference compounds, neochlorogenic acid, chlorogenic acid, rutin and quercitrin were from Sigma Chemical Co. . Other chemicals and reagents used in the current study were of analytical grade and obtained from Sigma-Aldrich , unless other sources are indicated. Ultrapure water was produced using a Simplicity 185 system . All buffers were made using ultrapure water and pH measurements were performed at room temperature using an Agilent 3200P pH Meter .α-Glucosidase (EC 3.2.1.20) from —100 UN) ; α-amyla—100 UN) , lung ac—100 UN) , N--L-phenylalanine (FAP) and glycine-glycine (Gly-Gly) from the substrate FAPGG (C20H21N3O6) according to the simple, rapid and sensitive method of Vermerissen et al. (2002) [ΔA340/min inhibitor/ΔA340/min control × 100], where ΔA is the difference between the initial and final absorbance of the test sample during its incubation.ACE inhibition was estimated in vitro by measuring the release of . (2002) . Ethanolm), maximal velocity (Vmax) and kinetic constant (Ki). In this study, kinetic data were estimated by virtue of plotting the data on a double reciprocal graph, called a Lineaweaver–Burk plot [m/Vmax × 1/[S] + 1/Vmax, where V is the reaction rate, Km is the Michaelis–Menten constant, Vmax is the maximum reaction rate (mg/L·min), [S] is the pNPG concentration (mg/L). The resultant plot is a straight line, with X- and Y-axis intercepts representing −1/Km and 1/Vmax, respectively, and the slope is Km/Vmax. The inhibition type of the inhibitors was determined by analyzing the Lineaweaver–Burk plots, which were competitive, non-competitive or uncompetitive, and the inhibition constant (Ki) values for botanical extracts were estimated by fitting the equation Kmapp)( = Km (1 + [I]/Ki) for competitive inhibition and equation Vmaxapp)( = Vmax (1 + [I]/Ki) for non-competitive inhibition, where Kmapp)( and Km are the concentrations of a substrate required to produce 50% of its maximum velocity (Vmax) in the presence and absence of an inhibitor, which were determined in parallel; [I] is the compound concentration; Ki is the inhibition constant of the reactions. The application of kinetic modeling can provide important insights into how interacting components behave in biological systems. The changes in enzyme catalytic activity in a biochemical system are frequently modeled by choosing a mathematical model to evaluate the following fundamental parameters: Michaelis–Menten constant  test (p < 0.05).Data are expressed as mean ± standard error (SE) and/or standard deviation (SD). Reaction velocities and enzyme kinetics and ICD. caucasica revealed that the plant leaves are rich in phenolic compounds, mainly caffeic acid esters and quercetin glycosides, while its tubers accumulate steroidal glycosides, as the major secondary metabolites. D. caucasica leaf extracts were remarkably stronger inhibitors of α-glucosidase, α-amylase, acetylcholinesterase and angiotensin-converting enzyme than the tuber extracts. Kinetic studies of enzyme inhibition suggest that the mode of inhibition, depending on the extract origin, may be mixed-type (leaves) and competitive (tubers). Based on the results obtained in this study and previously reported data, it may be sufficiently reasonably assumed that D. caucasica leaf polyphenolics play the most important role in the enzyme inhibition, while the tuber steroidal glycosides might be remarkably weaker enzyme inhibitors or their effects may be hampered by other, antagonistically competitive constituents. In general, the new data on D. caucasica phytochemicals and bioactivities may aid in the valorization of this plant for its wider use in the development of health beneficial ingredients for functional foods and nutraceuticals.The first systematic phytochemical study of


The receptor tyrosine kinase inhibitor imatinib improves patient cancer survival but is linked to cardiotoxicity. This study investigated imatinib’s effects on cell viability, apoptosis, autophagy, and necroptosis in human cardiac progenitor cells in vitro. Imatinib reduced cell viability (75.9 ± 2.7% vs. 100.0 ± 0.0%) at concentrations comparable to peak plasma levels (10 µM). Imatinib reduced cells’ TMRM fluorescence (74.6 ± 6.5% vs. 100.0 ± 0.0%), consistent with mitochondrial depolarisation. Imatinib increased lysosome and autophagosome content as indicated by LAMP2 expression (2.4 ± 0.3-fold) and acridine orange fluorescence (46.0 ± 5.4% vs. 9.0 ± 3.0), respectively. Although imatinib increased expression of autophagy-associated proteins and also impaired autophagic flux, shown by proximity ligation assay staining for LAMP2 and LC3II (autophagosome marker): 48 h of imatinib treatment reduced visible puncta to 2.7 ± 0.7/cell from 11.3 ± 2.1 puncta/cell in the control. Cell viability was partially recovered by autophagosome inhibition by wortmannin, with the viability increasing 91.8 ± 8.2% after imatinib-wortmannin co-treatment (84 ± 1.5% after imatinib). Imatinib-induced necroptosis was associated with an 8.5 ± 2.5-fold increase in mixed lineage kinase domain-like pseudokinase activation. Imatinib-induced toxicity was rescued by RIP1 inhibition: 88.6 ± 3.0% vs. 100.0 ± 0.0% in the control. Imatinib applied to human cardiac progenitor cells depolarises mitochondria and induces cell death through necroptosis, recoverable by RIP1 inhibition, with a partial role for autophagy. Receptor tyrosine kinases are proteins critical for many cellular processes and are activated via the transfer of phosphoryl groups, driving the downstream signalling required for cell survival and proliferation . NormallAlthough IM has demonstrated substantial patient benefit, it has been linked to cardiotoxicity, particularly within the aging population . IM may + hCPCs are capable of self-renewal and development into different myocardial cell types, including vascular smooth muscle and endothelial cells [Human endogenous cardiac progenitor cells (hCPCs) have attracted significant interest since their discovery in 2003 . One endal cells ,20,22,23al cells ,22,24,25al cells ,23. Cardal cells . TherefoTo fully understand how RTKIs cause adverse effects on the myocardium, it is important to investigate their impacts on cell death pathways. Apoptosis is a tightly regulated process defined by the activation of pro-apoptotic factors such as executioner caspases 3 and 7, which eventually lead to DNA fragmentation and cell death . Necroptn = 5, p < 0.05) after the cells were treated with IM for 24 h  (n = 3)  staining. Cell viability decreased by 25% relative to the control  D. Neithe < 0.05) B,C. To r < 0.05) E, compar < 0.05) F, but on < 0.05) G or 2.82 < 0.05) H. Real-t (n = 3) I.n = 6, p < 0.05)  F.The cells exposed to 10 µM IM over periods of 24, 48, and 72 h showed a significant increase in the number of acidic organelles A–D. Quanp < 0.05), which further increased with bafilomycin to 3.5 ± 0.3- and 3.9 ± 0.9-fold increases (p < 0.05). However, 10 µM IM treatment for 48 h caused 2.6 ± 0.2- and 2.8 ± 0.3-fold changes of LC3II and P62, respectively, with no significant increases following bafilomycin co-treatment: 2.7 ± 0.1 and 2.6 ± 0.0 relative to the untreated control cells (1.0 ± 0.0)  was used to prevent protein degradation; therefore, if the cell had a functional autophagic flux, there would be an increase in protein levels after bafilomycin inhibition. Western blot analysis of 10 µM IM treatment for 24 h caused 2.7 ± 1.3- and 1.9 ± 0.0-fold increases in LCII and P62, respectively (0 ± 0.0) A,B.p < 0.05). The puncta were significantly decreased in number after 10 µM IM treatment for 48 h: 2.7 ± 0.7 compared to levels after 24 h .These data were reaffirmed using the PLA assay: this enables a resolution of 50 nm, giving more detailed images to confirm the colocalisation between LC3II and p62. Cells were left untreated A or treater 24 h E (n = 10p < 0.05 relative to untreated control), whereas in cells co-treated with IM and wortmannin for 24 h, cell viability decreased to 91 ± 3.7% , which prevents the initial formation of autophagosomes and is, therefore, an upstream autophagy inhibitor. Western blotting was carried out to ensure that wortmannin was preventing autophagosome formation, by analysing LC3II expression A. Densit1 ± 3.7% C.p < 0.05), whereas 10 µM IM treatment for 24 h co-treated with nec-1 reduced the cell viability to 88.7 ± 3.1%   C.This study demonstrates that IM causes significant impacts on the hCPC population. Only a few previous studies have fully investigated the cell death pathways induced by IM, with a very small number addressing IM effects on hCPCs ,34,35,36The number of acidic organelles increased after IM application, which was accompanied by increased expression of the lysosomal structural protein LAMP2. This is similar to findings in previous studies in the mouse neuroblastoma cell line N2a, with one study suggesting IM-induced autophagy is due to inhibition of c-Abl rather than of c-kit or PDGF . There aDue to the impairment of autophagic flux and an increase in the p62 protein, this study investigated the involvement of necroptotic cell death in IM-induced toxicity. A previous study by Goodall et al. showed the presence of the p62 protein to be vital for MLKL phosphorylation; if p62 was removed, the cell death pathway would switch to an apoptotic cell death . This cuAs this study was aimed at characterising the mechanisms underlying the toxicity of IM to CPCs, following our previous study of the broad impacts of IM and two other RTKIs on the CPC phenotype , we alsoIn summary, this study demonstrated a range of impacts caused by the clinically used cardiotoxic drug IM on human endogenous cardiac progenitor cells. The study identified two possible targets to overcome IM-induced toxicity on hCPCs, through either the inhibition of upstream autophagy or inhibition of necroptosis .v/v), basic-FGF (10 ng/mL), EGF (20 ng/mL), and human leukaemia inhibitory factor (10 ng/mL). Solution B comprised Neurobasal medium supplemented with Glutamax (2% v/v), B27 supplement (2% v/v), and N2 supplement (1% v/v). To prepare growth medium: 45% of solution A and 45% of solution B were combined and supplemented by: embryonic-stem-cell-qualified FBS (10% v/v); penicillin-streptomycin (1% v/v); Fungizone (0.1% v/v); gentamicin (0.1% v/v), and then sterilised through a 0.22 μm pore filter. ‘Medium’ is defined as above, unless otherwise stated.All cell culture techniques were performed under aseptic conditions. Growth medium was composed using two solutions. Solution A comprised DMEM-F12-Ham containing insulin-transferrin-selenium  at 37 °C: first, for 5 min, followed by 7–9 repeat digestions for 3 min, with each post-digest suspension passed through a 100 µm filter. After tissue sample digestion, all collected cells were strained through a 40 µm filter to isolate the smaller cell population. The collected cell fraction was spun at 400× g for 10 min, prior to elimination of debris through Optiprep solution . Cells were pelleted by centrifugation through a dual-layered Optiprep suspension: lower layer of 36% (16 mL) and upper layer of 16% (16 mL) at 800× g for 20 min. The cells were then negatively selected for CD45 and positively selected for CD117 (c-kit) using magnetic bead separation . After selection, the hCPCs were re-suspended in medium and plated onto a coated 6-well plate for conditioning. Cells were cultured in a humidified tissue culture incubator at 37 °C, 5% CO2, and 1% O2 and passaged upon reaching 70–80% confluency. All reagents were sterilised through a 0.22 µm filter.Adult hCPCs were isolated from myocardial tissue obtained during the routine course of cardiac surgery. Briefly, biopsies were weighed and dissected into 0.5–1 mmImatinib  was either added at the peak-plasma-level-equivalent concentration of 10 µM , or in a dose-dependent manner of 1–100 µM according to experimental conditions . This study used RTKI concentrations chosen after review of relevant study data for clinical plasma concentrations at peak and trough ,48 level3 hCPCs were plated into each well of a 96-well plate and treated with IM in medium for 24 h. After treatment, medium was removed and replaced with 5 µg/mL and FDA , and incubated for 10 min at 37 °C. Fluorescent readings were taken using a Varioskan Flash plate-reader  at excitation/emission wavelengths of 485/520 nm. Cell viabilities of treatment groups were calculated as a percentage of the untreated control group. For cell viability with the autophagy inhibitor, cells were left to attach for 24 h before 10 µM of IM treatment and co-treatment with 200 nM wortmannin for 24 and 48 h time points, before applying FDA as described.The FDA cell viability assay was performed as previously described . BrieflyLive cell staining for high-content image analysis: cells were added to a CellCarrier-96 Ultra Microplates , 1000 cells/well. Fam/Cas dye (1:150) was added and incubated for 1 h and washed with apoptosis wash buffer . Hoechst 1:200, ToPro-3 1:1000, and TMRM 1:100,000  were added and incubated for 10 min, then replaced with medium (without indicators) and analysed by an Operetta platform (Perkin Elmer). Results were interpreted using the Columbus™ software (Perkin Elmer). Annexin staining was carried out with an Annexin V FITC labelling kit (Miltenyi Biotec), analysed by a Cytoflex S flow cytometer  using a 488/525 nm filter. g for 5 min and then re-suspended in a PBS wash; the cells were then pelleted at 300× g for 5 min. Finally, cells were re-suspended in incubation buffer: PBS ; 2.5 g of bovine serum albumin (Sigma A9418); 1% (v/v) Penicillin/Streptomycin (Invitrogen 15140-122); 0.1% (v/v) Fungizone (Invitrogen 15290-018); 0.1% (v/v) Gentamicin (Sigma G1397), and analysed using a Cytoflex S flow cytometer using a 561/585 nm filter. Data were interpreted using Cytexpert and Excel. The mitochondrial membrane potential was calculated using the fluorescence intensity of TMRM of treated cells relative to untreated cells. To evaluate the effect of IM on mitochondrial membrane potential, flow cytometry was used to analyse the retention of a cell-permanent cationic dye called tetramethylrhodamine methyl ester perchlorate (TMRM). Cells were seeded onto 100 mm tissue culture dishes until 80% confluent; TMRM  was added for 10 min and the cells were then washed with PBS before being detached using Accutase (Sigma). Following this, the cells were centrifuged at 300× To determine the role of autophagy in IM-induced cell death, acridine orange  was used to determine if the drug increased acidic properties within the cell (acidic organelles), indicating the presence of increased lysosome content. Cells were plated into a 12-well plate and allowed to reach confluency. Following this, 5 µM acridine orange was added to medium for 30 min. Cells were then washed once with PBS and imaged using confocal microscopy . The number of positive cells were counted using Image J .v/v) Penicillin/Streptomycin (Invitrogen 15140-122); 0.1% (v/v) Fungizone (Invitrogen 15290-018); 0.1% (v/v) Gentamicin (Sigma G1397)) and centrifuged at 1500 rpm for 5 min. Briefly, RNA was then isolated using the QIAshredder and RNeasy mini kit , according to the manufacturer’s guidelines. The RNA sample purity was tested using a Nanodrop 2000 (Thermo Fisher). The iScript cDNA synthesis kit and BioRad  CFX96 Real-Time system were used to generate 100 µL of cDNA from 2000 ng of RNA. mRNA sequences for all primers were obtained from the NIBC nucleotide database and assessed using Primer-BLAST software . All PCR primers  for 30 min in a humidified chamber. Cells were washed 3 times with PLA wash buffer A and incubated with amplification reaction  for 100 min in a humidified incubator at 30 °C. Finally, cells were washed 3 times with PLA wash buffer A and 0.01% wash buffer B for 10 min. Results were visualised by confocal microscopy . Following image acquisition, PLA puncta staining was counted manually for each condition. The proximity ligation assay (PLA) gave visualisation of two proteins within 40 nm of each other (indicating higher probability of direct binding). The Duolink kit was used  with cells cultured on 8-well glass chamber slides (Thermo Fisher 155409) and treated with 10 µM IM for 24 or 48 h, or 400 nM rapamycin for 4 h. Cells were fixed with 2% paraformaldehyde for 10 min at room temperature, permeabilised with 0.1% Triton X-100, and blocked with goat serum  for 1 h at room temperature. Cells were then incubated with primary antibodies: 1:200 LAMP2 (Novus) and 1:200 MAP1LC3B Antibody (CSB- Cusabio PA887936LA01HU), overnight at 4 °C. For secondary incubation, PLA (+) and PLA (−) probes were incubated for 1 h in a humidified incubator. For each treatment, 8 µL of PLA (+) and 8 µL of PLA (−) were added in 24 µL of blocking buffer and placed onto treatment wells. Cells were washed with PLA wash buffer A and incubated with ligation reaction . For the necroptosis-positive control, cells were incubated with 20 ng/mL of TNF-α , 100 nM SMAC mimetic Birinapant , and 20 µM V Z-VAD(OMe)-FMK  for 8 h. After treatment, cells were lysed using RIPA buffer (Sigma) and quantified using the Pierce™ Rapid Gold BCA Protein Assay Kit (Thermo Scientific). Protein was loaded  and transferred using a semi-dry turbo blotter (Bio-Rad). Blots were placed into 5% milk solution  containing primary antibodies and were incubated overnight at 4 °C . The bloBlots were developed using West Pico plus (Thermo Fisher) and imaged on the automated developer G-box . Densitometry was analysed using Image J and Excel, with treatments normalised against control groups.t-test on ∆∆Cq values was employed to compare the target gene expression between each treatment and the respective control values.One-way ANOVA was used for comparison of mean data between groups for analyses of cell viability, live cell staining, mitochondrial membrane potential staining, and PLA assay, followed by the post hoc Tukey test for comparisons between groups. A two-tailed, unpaired Student’s


Robotic surgery may improve surgical performance during minimally invasive pancreatoduodenectomy as compared to 3D- and 2D-laparoscopy but comparative studies are lacking. This study assessed the impact of robotic surgery versus 3D- and 2D-laparoscopy on surgical performance and operative time using a standardized biotissue model for pancreatico- and hepatico-jejunostomy using pooled data from two randomized controlled crossover trials (RCTs).Pooled analysis of data from two RCTs with 60 participants  from 11 countries (December 2017–July 2019) was conducted. Each included participant completed two pancreatico- and two hepatico-jejunostomies in biotissue using 3D-robotic surgery, 3D-laparoscopy, or 2D-laparoscopy. Primary outcomes were the objective structured assessment of technical skills (OSATS: 12–60) rating, scored by observers blinded for 3D/2D and the operative time required to complete both anastomoses. Sensitivity analysis excluded participants with excess experience compared to others.p < 0.001], as compared to the laparoscopic group. Robotic surgery resulted in higher OSATS ratings  and shorter operative time , as compared to 3D- and 2Dlaparoscopy, respectively, which remained in the sensitivity analysis.A total of 220 anastomoses were completed . Participants in the robotic group had less surgical experience [median 1 (0–2) versus 6 years (4–12), In a pooled analysis of two RCTs in a biotissue model, robotic surgery resulted in better surgical performance scores and shorter operative time for biotissue pancreatic and biliary anastomoses, as compared to 3D- and 2D-laparoscopy. Minimally invasive pancreatoduodenectomy (MIPD) is becoming increasingly popular . RecentlRobotic surgery aims to overcome the compromises made with 2D-laparoscopic surgery by improving dexterity, 3D vision, and ergonomic comfort –7. OutcoIn recent years, use of artificial organs (biotissue) to improve surgical training in MIPD has gained popularity –20. SincThe aim of the present study is to assess surgical performance with robotic surgery, 3D-, and 2Dlaparoscopy for the pancreatico- and hepatico-jejunostomy anastomoses of MIPD by pooling data from two previous randomized controlled crossover trials using the same standardized biotissue model.This study was reported in accordance with the Consolidated Standards of Reporting Trials (CONSORT) . Data fri.e., robotic or laparoscopic. Participants were excluded if they had no 3D-vision abilities, as evidenced by < 200 seconds of arc on Randot Test   or 95% confidence intervals (95%CI). Categorical  data are presented as frequencies and percentages. Likert-Scale ordinal data are also presented in means and standard deviations, as this allows more insight into the effect size . A two-tBaseline demographics were compared with Student’s t-test for normally distributed data, Chi-squared test for frequencies in one or more categories, and Mann–Whitney U test for non-normally distributed data. The primary outcomes were analyzed according to Mann–Whitney U test because the samples were independent. To assess the impact of surgical experience, a sensitivity analysis excluded participants with > 7 years experience (upper limit of experience in the group with the lowest experience) and experience in MIPD. Lastly, a sensitivity analysis was performed excluding those who participated in both trials.A total of 34 surgeons and 26 residents participated and subsequently performed anastomoses between December 2017 and June 2019. Three participants only completed one anastomosis, one participant left to perform an emergency procedure, and one participant performed the anastomoses in a running fashion. These five participants were excluded for the analysis of the primary and secondary outcome. Eight participants participated in both the robotic and laparoscopic trials. Figure p < 0.001), including a lower number of annual advanced minimally invasive procedures , compared to the experience of the laparoscopic group with laparoscopic surgery. In total, 15 participants from the laparoscopic group had more than 7 years experience vs none in the robotic group. Table The 55 included participants originated from 11 countries . Their mean age was 38 years (SD 9), 45 were male (80.0%). The groups for robotic surgery and laparoscopy were comparable for age, sex, hand dominance, the number of MIPD performed in clinical practice, and stereopsis abilities. Participants in the robotic surgery group had less experience with robotic surgery  and 11 points , as compared to 3D- and 2Dlaparoscopy, respectively. Figure In the robotic surgery group, higher OSATS ratings were obtained (attainable range 12–60) as compared to 3D- and 2Dlaparoscopy, median 50 [44–55] vs 43 [38–50] vs 39 [32–46]. Robotic surgery resulted in higher OSATS ratings by 7 points  and 30.7% (p < 0.001), as compared to 3D- and 2D-laparoscopy. Figure In the robotic surgery group, operative time was shorter as compared to 3D- and 2D-laparoscopy: 56.5 min [52.4–67.5] vs 65.0 min [57.0–83.0] vs 81.5 min [68–97.8], i.e., eye strain (minor n = 2), headache , dizziness . In the 3D-laparoscopy group, 14/39 (36%) participants reported one or more side-effects. No significant differences were found between the two groups. Table Of the 59/60 participants  who completed the survey on side-effects and preferences, 57/59 (96.6%) preferred 3D-vision over 2D-vision . In the 3D-robotic group, 4/20 (20%) participants reported one or more side-effects, p = 0.004) and 27.8% (p = 0.174), as compared to 3D- and 2Dlaparoscopy. Robotic surgery resulted in shorter operative time compared to 3D- and 2D-laparoscopy: 37.5 min [30.5–43.8] vs 39.5 min [35.5–50.8] vs 50.0 min [38.0–59.0]. Relatively, in the robotic surgery group, operative time was shorter by 5.1% (p = 0.176) and 22.0% (p = 0.001), as compared to 3D- and 2Dlaparoscopy.For the PJ, robotic surgery resulted in better OSATS ratings compared to 3D- and 2D-laparoscopy: 24 points [20–27] vs 22 points [18–25] vs 19 points [15–24]. Relatively, in the robotic surgery group, OSATS ratings were higher by 11.1% (p < 0.001) and 42.9% (p = 0.002), as compared to 3D- and 2Dlaparoscopy. Robotic surgery resulted in shorter operative time compared to 3D- and 2D-laparoscopy: 19.5 min [16.4–27.2] vs 25.0 min [19.0–34.0] vs 32.0 min [23.0–44.0]. Relatively, in the robotic surgery group, operative time was shorter by 22.0% (p = 0.061) and 39.1% (p < 0.001), as compared to 3D- and 2D-laparoscopy.For the HJ, robotic surgery resulted in better OSATS ratings compared to 3D- and 2D-laparoscopy: 27 points [26–29] vs 21 points [18–27] vs 18 points [16–24]. Relatively, in the robotic surgery group, OSATS ratings were higher by 28.6% (p < 0.001). However, number of MIPDs performed was significantly higher (p = 0.023) in the robotic group, even though the median number was 0 in both groups.The sensitivity analysis excluded 15 participants with experience > 7 years , all in the laparoscopy group. Table p = 0.009) and 19 points . The robotic approach still significantly resulted in shorter operative times: 56.5 min [52.4–67.5] vs 69.0 min [57.8–82.3] vs 81.0 min [65.0–95.5]. In the robotic surgery group, operative time was shorter by 12.4% (p = 0.003) and 30.2% (p < 0.001).In the sensitivity analysis, the robotic approach still had a superior OSATS as compared to 3D- and 2D-laparoscopy, median 50 [44–55] vs median 35 [27–45] vs median 31 [26–36]. Robotic surgery resulted in higher OSATS ratings by 15 points . Finally, we excluded participants who performed in both the LAELAPS-3D2D and LAEBOT-3D2D trial (n = 8). The results for the primary and secondary outcome remained consistent.In an additional sensitivity analysis, we also excluded participants with experience in MIPD. After excluding participants with more than 7 years experience and experience in MIPD, the primary outcomes remained significantly better in the robotic group vs the 3D-laparoscopy group  laparoscopic surgery and 64% of the participants stated that the reason for superiority was, among other reasons, 3D-vision . Since t, i.e., with further implementation [By looking at expert and fellow performance using the same biotissue model, Tam et al. determined that the biotissue operative time can be generalized to clinical surgical performance . Similarentation , 39.Robotic instrumentation provided a major contribution to surgical performance in this experimental setting. However, as reviewed by Anderson et al. elements of the robotic platform can be applied to laparoscopic surgery as well, this may be relevant in order to reduce the high cost associated with robotic surgery . One stuThe results of this study should be interpreted in light of several limitations. First, the randomized trials were performed in an experimental setting using a biotissue model and not in clinical practice. In clinical practice, clearly much more situational variation would have been observed. However, the previously mentioned studies , 42 cleaStrengths of this study include the pooling of data from two randomized controlled crossover trials, thus controlling for known and unknown confounders.In conclusion, the present study demonstrated that robotic surgery provides additional benefits over 3D- and 2D-laparoscopy when creating pancreatic and biliary anastomoses in a biotissue model. Future randomized studies should confirm these benefits in the clinical setting.