Psoriasis is a chronic skin disease characterized by hyperproliferative epidermal lesions infiltrated by autoreactive T cells. Individuals expressing the human leukocyte antigen (HLA) C∗06:02 allele are at highest risk for developing psoriasis. An autoreactive T cell clone (termed Vα3S1/Vβ13S1) isolated from psoriatic plaques is selective for HLA-C∗06:02, presenting a peptide derived from the melanocyte-specific autoantigen ADAMTSL5 (VRSRRCLRL). Here we determine the crystal structure of this psoriatic TCR–HLA-C∗06:02 ADAMTSL5 complex with a stabilized peptide. Docking of the TCR involves an extensive complementary charge network formed between negatively charged TCR residues interleaving with exposed arginine residues from the self-peptide and the HLA-C∗06:02 α1 helix. We probed these interactions through mutagenesis and activation assays. The charged interface spans the polymorphic region of the C1/C2 HLA group. Notably the peptide-binding groove of HLA-C∗06:02 appears exquisitely suited for presenting highly charged Arg-rich epitopes recognized by this acidic psoriatic TCR. Overall, we provide a structural basis for understanding the engagement of melanocyte antigen-presenting cells by a TCR implicated in psoriasis while simultaneously expanding our knowledge of how TCRs engage HLA-C. Such plaques are characterized by infiltration of stimulated cutaneous T cells that become active in the absence of any identified etiological agent  underlying the development of psoriasis are not fully delineated. The disease displays both autoimmune and autoinflammatory characteristics . CurrentHLA-C. Approximately 60% of psoriasis patients express the HLA-C∗06:02 (HLA-Cw6) allele, and these individuals often show an early disease onset  that are ligands for αβ T-cell receptors, killer-cell immunoglobulin-like receptors, and leukocyte immunoglobulin-like receptors.Typical of systemic autoimmune disorders, an individual’s progression toward psoriatic disease likely involves multiple factors. Nonetheless, genome-wide association studies have defined a strong genetic component to psoriasis with greater than twenty risk regions characterized. Psoriasis susceptibility locus 1 (PSORS1) has the highest risk association with susceptibility subsequently mapped to the gene for se onset , 10. ThiAlthough genetic associations with autoimmune disorders are often compelling, identifying underlying molecular “triggers” or associated autoantigens for T cell–mediated autoimmune disorders has proved challenging . InsulinThe healthy and diseased peptide repertoires presented by human HLA-A and HLA-B allomorphs have historically been preferentially studied due to their higher abundance , 17. In HLA-C∗06:02–restricted autoantigens have yielded candidates derived from skin-related proteins such as antimicrobial LL-37 . Duplicate experiments produced response curves that allowed modeling of an association with a KD of ∼11 ± 0.5 μM, which falls within the range of standard TCR–peptide major histocompatibility complex interactions . Total buried surface area following binding is 1830 Å2, with ∼66% of the contact made to the HLA platform and ∼34% to the ADAMTSL5 peptide (D). TCR docking occurs at a ∼45° angle to the HLA platform’s α1 helix with the β-chain IgV centered over it and the TCR α-chain over the α2 helix (C).Binding of the TCR does not significantly alter the structure of the underlying pHLA platform (r.m.s.d. of 0.5 Å) except to the extent that some surface-exposed side chains become enveloped by the CDR loops. The psoriatic TCR-binding interface is extensive and heavily focused over the HLA-C∗06:02 α1 helix combined with interactions near the epitope midpoint C. Total  peptide D. TCR doα2 helix C.C and Fig. 1D). Binding of Vα3S1/Vβ13S1 thus occurs primarily via the three TCR β-chain loops, while the glycine-rich CDR3α wrapped around the external face of the HLA α1 helix . The ranking of contributions to the interface buried surface area are CDR3β (40%) > CDR3α (28%) > CDR2β (16%) ∼CDR1β (15%). At a global level, the TCR docking angle and CDR loop contributions all fall within ranges observed previously in other HLA-A or HLA-B structures . This charge-rich engagement mechanism is centered around the curled acidic CDR3β loop with the sequence CASSYSEGEDE101. The last germline residue from this sequence, TCRβTyr-95, inserts its aromatic ring between the extending alkyl chains of the epitope’s P5 and P8 arginine residues. This guides these relatively mobile residues to form salt bridges to acidic CDR3β loop residues (A). Alternating charge interactions then also interface with the HLA-C∗06:02 helices with TCRβGlu-99 inserting between C∗06Arg-62 and C∗06Arg-69. This yields a line of four alternate charges due to a bookended interaction with CDR3α TCRαAsp-101 and TCRαAsp-110 (B).HLA-C∗06:02 presents the ADAMTSL5 epitope such that the P4, P5, and P8 arginine residues extend upward from its presentation platform . Upon TCresidues A. AlternαAsp-110 B.Figure via an overlaying salt bridge between C∗06Lys-80 and TCRβGlu-30 (A).HLA-C allomorphs are associated with NK cell reactivity leading to clinical genotyping into two groups: C1 allotypes carry an asparagine at residue 80, whereas the C2 group contains a lysine. Both polymorphisms contact the C-terminal carboxyl group of peptide antigens. We observe this standard interaction which the TCR registers RβGlu-30 A.TCRαArg-59 (A). Overall, TCR docking does not require significant changes to the epitope structure. The aminobutyric acid residue substituted in place of the ADAMSTL5 cysteine was previously shown to be oriented toward the HLA  that associates with a complementary series of acidic-rich CDR loops (D).We next independently calculated the electrostatic potentials for the interfacing surfaces of the peptide HLA-C∗06:02 and the Vα3S1/Vβ13S1 TCR. The result clearly demonstrates that for this psoriatic TCR, docking is largely driven by a pHLA antigen-platform presenting a positive surface C that asDR loops D.B) serve to make contact with positively charged peptide or α1-helical residues , while C∗06Arg-69 from HLA-C traverses the other way into a cluster of acidic TCR residues (A and D). The interactions across the interface are thus predominantly charge-charge–based salt bridges.Acidic TCR residues derived from both chains’ CDR3 loops ; both central elements of the interleaving charge-network (A) completely abrogated binding (E). In contrast, an alanine mutation at the C-group polymorphic position, C∗06Lys-80, retained ∼0.3-fold affinity of the WT protein (E). Its salt bridge interaction with TCRβGlu-30 is at a less-central interface position.Loss of either  binding E; both c-network A complet binding E. In con protein E. Its saF). The α2 helix is not contacted by the TCR, and C∗06R151A, E154A, E166A, and R169A mutants had minimal discernible effect upon binding. Within the α1 helix the C∗06R79A mutant showed no discernible effect upon binding, this resides at the C-terminus of the helix whereas Vα3S1/Vβ13S1 binds more centrally (F).For the most part, measured affinities were equivalent or better than WT HLA for residues remaining distal from the TCR upon cocomplexation F. The α2entrally F.Our alanine scanning experiments confirm that residues near the midpoint of the α1 helix of HLA-C∗06:02 are important to docking of the Vα3S1/Vβ13S1 TCR.CLRL) and surface-exposed arginine variants at P4A (VRSARCLRL), P5A (VRSRACLRL), or P8A (VRSRRCLAL) alongside C2CD, a mimotope with sequence (LRAGRSRRL), and a negative control TRAT peptide from HCMV (TRATKMQVI).We next sought to ascertain what residues within the ADAMSTL5 epitope were required for T cell activation. Full-length Vα3S1/Vβ13S1 TCR was retrovirally transduced into the SKW3T cell line, and HLA-C∗06:02 transfected into antigen-presenting 721.221 cells. This 721.221 Cw6 cell line was pulsed with a range of self-peptides over a concentration range of 0 to 100 μg/ml. Experiments were performed with the ADAMTSL5 peptide (VRSRRA). A comparative plot of the mean fluorescence intensity of the highest 100 μg/ml antigen concentration (B) demonstrated robust activation for the WT ADAMSTL5 epitope. The R5A and R8A mutations resulted in CD69 activation levels indistinguishable from negative control, while P4A still showed a slight degree of upregulation (B). Compared to the WT ADAMSTL5 peptide, the highly charged C2CD mimotope displayed the next highest activator potential (B).After peptide pulsing and washing 721.221 cells, they were co-incubated with SKW3-Vα3S1/Vβ13S1 T cells for 2 h with subsequent staining for upregulation of CD69 and flow cytometry A. A compntration B demonstgulation B. Comparotential B.Figure Loss of any of the peptide arginine residues contributing to the electrostatic cluster reduced the activation potential of Vα3S1/Vβ13S1–expressing T cells.A). The ADAMSTL5 epitope was observed as an outlier with its presentation of three positive charges towards the TCR interface.The ADAMSTL5 epitope has four positively charged residues; thus, we next assessed the charges present in all previously characterized pHLA:TCR structures. Epitopes from all currently solved pHLA-I:TCR complexes were seen to be predominantly neutral or singly charged A. The ADB) excluding those with reversed polarity docking modes. The average number of ionic cross-interface interactions was 1.0 . With its total of seven cross-interface salt bridges, this marked the Vα3S1/Vβ13S1 recognition mechanism as the most charge-dominated to date.Next, we sought to define to what extent the cross-interface salt bridges between Vα3S1/Vβ13S1 and HLA-C∗06:02 were unique for a TCR recognition system. We enumerated all such interactions in publicly available pHLA-I:TCR structures B excludiC) and the kink in the HLA platforms’ α2 helix. In contrast, Vα3S1/Vβ13S1’s ionic contacts appear more centrally directed nearer the peptide-binding groove.We next sought to assess whether interfacial salt bridges are more likely to contribute toward binding particular regions of the HLA platform by enumerating the positions of all prior pHLA-I:TCR salt bridge interactions. Ionic contacts cluster at two points: near the epitope’s N-terminus C and theD). The interaction density for the MHC platform is highest near the midpoint of the α1 helices between HLA residues 69 and 155. For 9-mer peptides which dominate the HLA-C∗06:02 repertoire, such interactions interrogate P3-P6. For the ADAMTSL5 peptide, the P8-Arg side chain also extends backwards into this target zone.We further overlayed the surface footprints for all class I pHLA:TCRs D. The inIn conclusion, most currently characterized TCRs bind centrally near the epitope’s midpoint; to date, few charge-mediated TCR interactions have been observed within this zone.The majority of previously characterized pHLA TCR docking modalities derive from HLA-A– or HLA-B–presented epitopes . Thus, wC∗Arg-69. In HLA-A and HLA-B, residue 69 is a smaller Ala or Thr more often engaged in van der Waals contacts. In Vα3S1/Vβ13S1, a novel tetra-acidic “CDR-cage” actively embraces the C∗Arg-69 charge, forming a network of seven salt bridges occurring across the pHLA-TCR interface. This extensive series of ionic interactions is uncommon among TCR-HLA structures (seven times the average) and is likely to cooperatively cluster following pHLA binding. This is potentially important as the entropic cost of dehydration for isolated cross-interface salt bridges may abrogate their contribution to binding affinity . The presence of single arginine residues upon TCR-facing HLA surfaces is not uncommon ; in HLA-10 potential permutations, amply sufficient to allow TCRs to specifically expand and identify pathological states. In practice, TCR cross-reactivity arises due to reuse of key protein sequences, the potential for different amino acids to be substituted in a given interaction, and obscuration of epitope positions during HLA display. Vα3S1/Vβ13S1 largely engages only the arginines within this peptide; it could potentially have reactivity against peptides with a similar level of charge.Is ADAMSTL5 likely a unique antigen for Vα3S1/Vβ13S1? In theory, peptide-HLA presentation of a 9-mer peptide allows ∼10et al.  and 10 mg of the self-peptides (VRSRR-C-LRL) where an aminobutyric acid residue (C) is substituted in place of cysteine in the P6 position , and the α-chain and β-chain of Vα3S1/Vβ13S1 were cloned into pET30 and expressed into inclusion bodies within position . Vα3S1/VRefolds were carried out in 1 L refold buffer . The refold solution was then dialyzed into 20 mM Tris–HCL pH 8.0. Refolded protein was initially purified using DEAE-sepharose anion exchange followed by size-exclusion chromatography in 20 mM Tris pH 8.0, 150 mM NaCl. The final purification step was anion-exchange chromatography with a HiTrap Q column with a linear gradient of 20 mM Tris pH 8.0, 0 to 1 M NaCl. Samples were buffer exchanged to 20 mM Tris pH 8.0, 150 mM NaCl for storage prior to use.CLRL and incubated for 30 min at room temperature prior to purification by gel-filtration chromatography.Vα3S1/Vβ13S1 TCR was cocomplexed with a stoichiometric amount of HLA-C∗06:02-VRSRRCLRL complex at 12 mg/ml incubated over a reservoir containing 20% w/v PEG 3350, 0.2 M NaSO4. Prior to flash freezing in liquid nitrogen, crystals were cryo-protected in reservoir solution supplemented with 20% v/v ethylene glycol. All X-ray diffraction data were obtained at the Australian synchrotron MX2 beamline.Crystals were obtained by hanging drop experiments with Vα3S1/Vβ13S1:HLA-C∗06:02-VRSRR5W67 (www.rcsb.org under PDB code 8SHI.Diffraction data were processed with XDS  and then5W67 ) followe5W67  and mode5W67 . A singlExamples of class I pHLA:TCR structures filtered for redundancy were downloaded from the protein data bank and aligned based on the platform of the MHC heavy chain. Frequency distributions were determined after defining an interfacial salt bridge as being a sub 4 Å approach between two oppositely charged residues. Surface contacts were defined as within 4.2 Å, each surface overlay represented 1.5% opacity. PDBs used for the analysis are as follows: this study and 7n6e, 7n1f, 7n1e, 6vrn, 6vrm, 6vqo, 6tro, 6rsy, 6rpb, 6rpa, 6rp9, 6amu, 6am5, 5yxn, 5tez, 5nmg, 5nme, 5nht, 5men, 5jhd, 5isz, 5eu6, 5e9d, 5e6i, 5d2n, 5d2l, 5c09, 5c08, 5c07, 5c0c, 5c0b, 5c0a, 5bs0, 5brz, 3utt, 3qdm, 3qdg, 3pwp, 3o4l, 3hg1, 3h9s, 3gsn, 2bnr, 1oga, 1bd2, 1ao7, 4eup, 5wkh, 3vxm, 3vxs, 5avf, 6avg, 6vmx, 1mi5, 3ffc, 3sjv, 4qrp, 4g9f, 3mv7, 6bj2, 4jrx, 6mtm, 3dxa, 3kpr, 3kps, 4mji, 2ypi, 6uon, 3pqy, 5ivx, 5m02, 5wlg, 6g9q, 7jwj, 1fo0, 1g6r, 1kj2, 1mwa, 2ol3, 2oi9, 3tf7, 3tfk, 3tpu, 4mvb, 4mxq, 4n0c, 4n5e, 6191, 6jtn, 6uli.A BIAcore T200 was used for surface plasmon resonance affinity measurement. Two independent experiments using different batches of protein were performed at 298 K. The buffer used contained 20 mM Tris pH 8.0, 300 mM NaCl, and 0.005% surfactant P20 (TBS-300-P20). The HLA-C∗06:02 or alanine mutant was amine-coupled to flow cells of a CM5 sensorchip (BIAcore). Vα3S1/Vβ13S1 TCR was serially diluted in TBS-300-P20 and passed simultaneously over the test (HLA-C∗06:02), and empty flow cells surfaces at a flow rate of 5 μl/min. All measurements were taken in duplicate. All obtained data were analyzed using Prism (GraphPad) using steady-state equilibrium analysis.5) was assessed using cell-surface CD69 upregulation after 17 to 20 h incubation with either 721.221 parental or 721.221-HLAC∗06:02 (1:1 ratio) cells preincubated for 1 hour with different peptides. Following co-incubation, the SKW.TCR cells were co-incubated for 17-20 hours with antigen presenting cells following 1 hour preincubation with peptide. For all experiments, SKW3.TCR cells alone served as negative control. Flow cytometry data were acquired and analyzed as described previously (www.flowjo.com/).Activation of SKW3.Vα3S1/Vβ13S1 cells (1 × 10eviously . The CD6CLRL ternary complex were deposited in the Protein Data Bank (PDB) under the ID 8SHI.Atomic coordinate and structure factors of the Vα3S1/Vβ13S1: HLA-C∗06:02-VRSRRThis article contains D. G. B is an employee of Janssen Pty Ltd. All other authors declare that they no conflicts of interest with the contents of this article.


A local inversion center near 1/8, 3/4, z relates the two molecules, as is common for structures in this space group with Z′ = 2. The molecule crystallizes as the keto tautomer, and the β-diketone moieties are twisted out of planarity, with O—C⋯C—O pseudo torsion angles of −74.4 (5) and −83.9 (5)°.The title compound, C 12H15NO3, crystallizes with Z′ = 2 in space group Pca21 with the two independent molecules having almost the same conformation, differing mostly at the end of the butanamide chain. A local inversion center near 1/8, 3/4, z relates the two molecules, as is common for structures in this space group with Z′ = 2. The molecule crystallizes as the keto tautomer, and the β-diketone moieties are twisted out of planarity, with O—C⋯C—O pseudo torsion angles of −74.4 (5) and −83.9 (5)°. The N—H group of each independent molecule donates an intermolecular hydrogen bond to an amide carbonyl oxygen atom by positive or negative translations along the b axis, thus forming antiparallel chains propagating in the [010] direction.The title compound, C N-(4-Ethoxyphenyl)-3-oxobutanamide is a putative intermediate in the biotransformation of bucetin [N-(4-ethoxyphenyl)-3-hydroxybutanamide], an analgesic–antipyretic once considered to be a safer alternative for phenacetin -3-oxobutanamide, N-(4-hydroxyphenyl)-3-hydroxybutanamide, and N-(4-hydroxyphenyl)acetamide. Intravenous administration of bucetin, on the other hand, mainly resulted in the formation of the glucuronide of N-(4-hydroxyphenyl)acetamide, with a maximum yield of 98%. These findings indicate that oxidative O-de-ethylation, keto conversion, and γ-decarboxylation are involved in the biotransformation of bucetin, leading to the endogenous production of N-(4-hydroxyphenyl)acetamide, the relevant analgesic compound. However, the specific order of O-de-ethylation and keto conversion remains uncertain -3-oxobutanamide, C12H15NO3, contains a β-diketone functionality that is similar in nature to the one present in linear and cyclic 1,3-diketone compounds -peroxynitrous acid  and hypochlorite (−OCl)-hypochlorous acid  conjugate acid–base systems  distance is 2.878 (6) Å and the N2⋯O4  distance is 2.856 (6) Å. Thus, the two independent molecules form antiparallel chains in the [010] direction, as shown in Fig. 3The N—H moiety in both molecules donates an intermolecular hydrogen bond Table 1 to amideet al. -3-oxobutanamide, C12H15NO3 (CAS No. 122–87-2) was obtained from AmBeed, Arlington Heights, IL, USA and was used without further purification. Crystals in the form of colorless laths were prepared by slow cooling of a nearly saturated solution of the title compound in boiling deionized water.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314623005655/hb4435sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314623005655/hb4435Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314623005655/hb4435Isup3.cmlSupporting information file. DOI: 2276880CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Brain-directed immunotherapy is a promising strategy to target amyloid-β (Aβ) deposits in Alzheimer’s disease (AD). In the present study, we compared the therapeutic efficacy of the Aβ protofibril targeting antibody RmAb158 with its bispecific variant RmAb158-scFv8D3, which enters the brain by transferrin receptor-mediated transcytosis.AppNL−G−F knock-in mice received RmAb158, RmAb158-scFv8D3, or PBS in three treatment regimens. First, to assess the acute therapeutic effect, a single antibody dose was given to 5 months old AppNL−G−F mice, with evaluation after 3 days. Second, to assess the antibodies’ ability to halt the progression of Aβ pathology, 3 months old AppNL−G−F mice received three doses during a week, with evaluation after 2 months. Reduction of RmAb158-scFv8D3 immunogenicity was explored by introducing mutations in the antibody or by depletion of CD4+ T cells. Third, to study the effects of chronic treatment, 7-month-old AppNL−G−F mice were CD4+ T cell depleted and treated with weekly antibody injections for 8 weeks, including a final diagnostic dose of [125I]RmAb158-scFv8D3, to determine its brain uptake ex vivo. Soluble Aβ aggregates and total Aβ42 were quantified with ELISA and immunostaining.+ T cell depletion was used for long-term therapy. CD4+ T cell-depleted mice, chronically treated with RmAb158-scFv8D3, showed a dose-dependent increase in blood concentration of the diagnostic [125I]RmAb158-scFv8D3, while concentration was low in plasma and brain. Chronic treatment did not affect soluble Aβ aggregates, but a reduction in total Aβ42 was seen in the cortex of mice treated with both antibodies.Neither RmAb158-scFv8D3 nor RmAb158 reduced soluble Aβ protofibrils or insoluble Aβ1-42 after a single injection treatment. After three successive injections, Aβ1-42 was reduced in mice treated with RmAb158, with a similar trend in RmAb158-scFv8D3-treated mice. Bispecific antibody immunogenicity was somewhat reduced by directed mutations, but CD4Both RmAb158 and its bispecific variant RmAb158-scFv8D3 achieved positive effects of long-term treatment. Despite its ability to efficiently enter the brain, the benefit of using the bispecific antibody in chronic treatment was limited by its reduced plasma exposure, which may be a result of interactions with TfR or the immune system. Future research will focus in new antibody formats to further improve Aβ immunotherapy.The online version contains supplementary material available at 10.1186/s13195-023-01236-3. Blood and plasma were collected 1 h, 4 h, 24 h, and 48 h post-injection. Blood, plasma, brain , and peripheral organs liver, spleen, and thyroid were extracted 3 days after [125I]RmAb158-scFv8D3 injection.One day prior to anti-Aβ immunotherapy, 7-month-old g, the supernatant was collected and divided into two separate tubes, in which one underwent an additional 100,000 × g centrifugation for 1 h. Pellets from TBS homogenization were subsequently dissolved in 70% FA and centrifuged at 16,000 × g for 1 h and supernatant was collected.Brains were homogenized at 1:10 weight/volume ratio. Tris-buffered saline (TBS) and 70% formic acid (FA) were used to isolate soluble and total Aβ, respectively. Briefly, TBS-homogenized brains were centrifuged for 1 h at 16,000 × Soluble and membrane-associated Aβ aggregates were analyzed in brain TBS extracts using a sandwich ELISA based on the Aβ N-terminal-specific antibody 3D6 (produced in-house) as both capture and detection antibody. This ELISA will detect Aβ aggregates from the size of a dimer and larger . 96-wellTotal Aβ1–40 and 1–42 were analyzed in formic acid soluble brain extracts using anti-Aβ40  and anti-Aβ42  antibodies. Briefly, 96-well plates were coated overnight at + 4 °C with 50 ng anti-Aβ40 or anti-Aβ42 antibody, then blocked with 1% BSA for 2 h at room temperature. FA brain samples were neutralized with 2 M tris buffer and incubated overnight at + 4 °C, then detected with biotinylated 3D6 and streptavidin-HRP as above.Levels of soluble triggering receptors expressed on myeloid cells 2 (sTREM2) in TBS brain extracts were analyzed with a sandwich ELISA, as previously described . 96-well+ T cell depleted, antibody-treated AppNL−G−F mice were immunostained to visualize Aβ42 or total Aβ and the microglial marker ionized calcium-binding adaptor molecule 1 (Iba-1). Sections were fixed in 4% paraformaldehyde (PFA), followed by antigen retrieval in preheated citrate buffer . After equilibration to room temperature, sections were treated with 70% formic acid for 5 min to promote Aβ immunoreactivity, then permeabilized with 0.4% Triton-X 100 in PBS and blocked with 5% normal goat serum . Sections were incubated overnight at + 4 °C with primary antibodies ,  and  in 0.1% Tween in PBS, then for 1 h with secondary antibodies; goat anti-rabbit , goat anti-mouse  and goat anti-rabbit  in 0.1% Tween20 in PBS, followed by mounting with Vectashield® Antifade Mounting Medium with DAPI (Vector Laboratories). Immunofluorescence was imaged with a Zeiss Observer Z.1 microscope and ZEN 2.6 software  and quantified with Fiji Image J RmAb158-scFv8D3 already at the second dose , 4-month-old wt mice were subjected to repeated injections of RmAb158-scFv8D  and RmAb158 (p < 0.001) compared with PBS. A similar trend was seen in the hippocampus, but a significant reduction was only observed in RmAb158 compared with PBS-treated mice (p < 0.05). Cerebellar Aβ1–42 displayed a significant reduction in mice treated with RmAb158 and low dose RmAb158-scFv8D3 compared with PBS (p < 0.05). These results were largely confirmed by Aβ42 immunostaining that showed a significant reduction in both cortex and hippocampus of RmAb158 compared with PBS-treated mice (p < 0.05) and a similar trend for RmAb158-scFv8D3 treated mice   Fig. C.Fig. 8MAPP knock-in mouse model AppNL−G−F, which is characterized by early and aggressive brain Aβ amyloidosis, with parenchymal Aβ depositions from 2 months of age RmAb158-scFv8D3 was administered i.v. after 8 weeks of chronic treatment to assess whether antibody brain retention could be used to quantify the Aβ-targeted treatment effect, as previously demonstrated with [124I]RmAb158-scFv8D3 immunoPET imaging [125I]RmAb158-scFv8D3 concentration in brain and blood revealed no correlation with treatment effect, but a dose-dependent alteration in blood and plasma in both groups that received RmAb158-scFv8D3 therapy. The higher blood concentration and lower plasma concentration of [124I]RmAb158-scFv8D3 in mice treated with bispecific RmAb158-scFv8D3 was clearly associated with a reduced brain uptake. Although the total brain uptake of [125I]RmAb158-scFv8D3 was clearly affected by treatment, ex vivo autoradiography displaying the spatial brain distribution of [125I]RmAb158-scFv8D3 showed that the antibody was accumulated in brain regions overlapping with those known to harbor Aβ pathology in AppNL−G−F mice. Thus, BBB penetration of [125I]RmAb158-scFv8D3 was only partly reduced. Biochemical and histological analysis of Aβ pathology revealed a comparable treatment effect in lowering cortical Aβ42 in animals receiving chronic treatment with RmAb158-scFv8D3 and RmAb158. Immunostainings showed abundant microglial distribution in areas of Aβ deposits, suggesting a major microglial response to Aβ pathology in this mouse model. Interestingly, the Aβ42 reduction seen in mice chronically treated with both antibodies was accompanied by increased levels of sTREM2, suggesting that microglia are further activated by the ongoing therapy and involved in clearance of Aβ from the brain. In contrast, mice subjected to acute treatment and euthanized after ten weeks showed a reduction of sTREM2. This reduction was seen only in the group that showed reduced Aβ42, i.e., RmAb158-treated mice, and likely reflects a lower level of Aβ pathology RmAb158-scFv8D3mut 3 in wt mice. Upper panel (1-3) shows naïve mice, without pretreatment, and lower panel (4-6) shows mice immunized with the same antibody. Yellow arrow points to spleen, which was readily visible in naïve mice at the end of the scan time.


Breast cancer (BC) is not only a mass of malignant cells but also a systemic inflammatory disease. BC pro-tumorigenic inflammation has been shown to promote immune evasion and provoke BC progression. The NOD-like receptor (NLR) family pyrin domain-containing protein 3 (NLRP3) inflammasome is activated when pattern recognition receptors (PRRs) sense danger signals such as calreticulin (CALR) from damaged/dying cells, leading to the secretion of interleukin-1β (IL-1β). CALR is a novel BC biological marker, and its high levels are associated with advanced tumors. NLRP3 expression is strongly correlated with an elevated proliferative index Ki67, BC progression, metastasis, and recurrence in patients with hormone receptor-positive (HR+) and triple-negative BC (TNBC). Tumor-associated macrophages (TAMs) secrete high levels of IL-1β promoting endocrine resistance in HR+ BC. Recently, an immunosuppressive soluble form of programmed death ligand 1 (sPD-L1) has been identified as a novel prognostic biomarker in triple-negative breast cancer (TNBC) patients. Interestingly, IL-1β induces sPD-L1 release. BC Patients with elevated IL-1β and sPD-L1 levels show significantly short progression-free survival. For the first time, this study aims to investigate the inhibitory impact of thymoquinone (TQ) on CALR, the NLRP3 pathway and sPD-L1 in HR+ and TNBC. Blood samples were collected from 45 patients with BC. The effect of differing TQ concentrations for different durations on the expression of CALR, NLRP3 complex components and IL-1β as well as the protein levels of sPD-L1 and IL-1β were investigated in the peripheral blood mononuclear cells (PBMCs) and TAMs of TNBC and HR+ BC patients, respectively. The findings showed that TQ significantly downregulated the expression of CALR, NLRP3 components and IL-1β together with the protein levels of secreted IL-1β and sPD-L1. The current findings demonstrated novel immunomodulatory effects of TQ, highlighting its potential role not only as an excellent adjuvant but also as a possible immunotherapeutic agent in HR+ and TNBC patients.  In 2020, breast cancer (BC) was ranked the most diagnosed cancer and the fifth-highest cause of cancer mortality worldwide . Five moInflammasomes are inflammatory signaling complexes made up of pattern recognition receptors (PRRs) that sense released DAMPs, leading to their oligomerization recruiting the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) also called PYCARD (PYD and CARD domain-containing) and the effector caspase-1 , which tIL-1β is one of the primary mediators of systemic inflammation . DistincTumor evasion has been a critical characteristic of cancer progression and poor prognosis in TNBC ,78,79. TNigella sativa seeds [Thymoquinone (TQ) is a natural pharmacologically active ingredient derived from va seeds . It has va seeds . In BC, va seeds ,95,96,97va seeds . Anotherva seeds . In addiva seeds ,100. To va seeds ,105,106.va seeds . In contp = 0.0195) and 100 µM (** p = 0.0013) concentrations significantly downregulated its expression , **** p < 0.0001; p = 0.0345) and 100 µM (** p = 0.0043) , 50 µM (* p = 0.0184), and 100 µM (* p = 0.0154) concentrations after 72 h of treatment  for 24, 48, and 72 h. CALR expression was investigated compared to that of the DMSO control, and each concentration was performed in triplicate. After 24 h of TQ treatment, the 20 µM concentration resulted in a non-significant elevation in CALR expression. In contrast, the 50 µM (* p < 0.0001), 50 µM (**** p < 0.0001), and 100 µM (**** p < 0.0001) in a dose-dependent manner  (p = 0.0357), 50 µM (** p = 0.0041), and 100 µM (* p = 0.0229)  (p = 0.0065), 50 µM (** p = 0.0033), and 100 µM (** p = 0.0029) in a dose-dependent manner  (TQ showed strong significant inhibition on the CALR expression after 24 h of treatment at 20 µM (****  0.0001) A. After  0.0054) B. TQ sho 0.0016) C. The exp < 0.0001), followed by a complete abolishment in its expression at 100 µM (** p = 0.0063)  (p = 0.0059), followed by a significant elevation at 50 µM (* p = 0.0237). Finally, its expression was significantly abolished below the detection level at 100 µM (*** p = 0.0002)—one-way ANOVA of PYCARD expression after 24 h of treatment: **** p < 0.0001.The NLRP3 and PYCARD expressions were investigated with increasing TQ concentrations  after 24 h of treatment compared to those of the DMSO control, and each concentration was performed in triplicate. TQ caused a non-significant impact on NLRP3 expression at 20 µM; then, a significant increase was noticed at 50 µM (****  0.0001) D. TQ sho 0.0001) F. At firp = 0.0006) and 100 µM (* p = 0.0266) after 48 h of treatment  (p = 0.0172) after 48 h  (p = 0.0245)   E. As for 0.0010) G. After  0.0389) H. The exp < 0.0001) followed by a completely abolished expression at 50 µM (**** p < 0.0001). Unexpectedly, TQ suddenly upregulated the NLRP3 expression at 100 µM (* p = 0.0161) after 24 h of treatment (one-way ANOVA **** p < 0.0001) (p = 0.0050), 50 µM (** p = 0.0027), and 100 µM (** p = 0.0055)  (*p = 0.0500), 50 µM (*p = 0.0500), and 100 µM (*p = 0.0500)  (TQ showed a strongly significant inhibition in NLRP3 at 20 µM (****  0.0001) D. After  0.0013) E. The NL 0.0340) F. Each Tp = 0.0070)  (p = 0.0068) and 100 µM (** p = 0.0068), while the 20 µM concentration caused a non-significant inhibition  (p = 0.0052) and 50 µM (** p = 0.0052) and completely abolished its expression after 72 h at 100 µM (** p = 0.0065)   G. PBMCs  0.0044) H. TQ sig 0.0027) I. TQ conp = 0.0195); finally, it was significantly upregulated at 100 µM (** p = 0.0013)  (p < 0.0001), whereas 20 µM caused a non-significant downregulation  (p < 0.0001)   A. After  0.0001) B. After  0.0001) C. The exp = 0.0065) and 100 µM (*** p = 0.0006) TQ concentrations after 24 h of treatment in vitro  (p = 0.0164), 50 µM (** p = 0.0015), and 100 µM (* p = 0.0254)  (p = 0.0013)   A. After  0.0010) B. PBMCs  0.0014) C. TQ conp = 0.0012)  . TQ strongly inhibited the expression of IL-1β after 72 h at 50 µM (**** p < 0.0001) and 100 µM (*** p = 0.0002), whereas the 20 µM concentration caused a non-significant change—one-way ANOVA of IL-1β expression after 48 and 72 h of treatment, **** p < 0.0001  D. Since < 0.0001 E,F. The p = 0.0020), 50 µM (*** p = 0.0010), and 100 µM (*** p = 0.0007) after 24 h of treatment  (p = 0.0005) and 100 µM (** p = 0.0071) concentrations significantly inhibited its expression  (p = 0.0013) and 100 µM (* p = 0.0123) concentrations significantly inhibited its expression  (TQ significantly downregulated the expression of IL-1β at 20 µM (**  0.0007) D. PBMCs  0.0001) E. TQ tre 0.0007) F. TQ conTo validate that the cultured CD14+ monocytes were successfully differentiated to TAMs, microscopic examination and flow cytometry (CD163 positivity) were performed to compare monocytes to TAMs. Microscopic examination revealed morphological changes. The freshly isolated monocytes showed cells that were small in size with a spherical and smooth surface A. On dayTo further confirm efficient TAMs differentiation and since CD163 is a TAMs biomarker , flow cyp = 0.0014)—one-way ANOVA, ** p = 0.0018  and 100 µM (* p = 0.0102)—one-way ANOVA, *** p = 0.0009 (p < 0.0001) and 100 µM (*** p = 0.0008) after 48 h of treatment  (p = 0.0046), 50 µM (** p = 0.0035), and 100 µM (*** p = 0.0008)—one-way ANOVA, *** p = 0.0002 (p = 0.0390)—one-way ANOVA, ** p = 0.0080  B. After = 0.0002 C. The im= 0.0080 D. The prp = 0.0296), 50 µM (* p = 0.0134), and 100 µM (*** p = 0.008)—one-way ANOVA, ** p = 0.0013 (p = 0.0067), 50 µM (** p = 0.0015), and 100 µM (** p = 0.0012) of TQ treatments significantly downregulated its protein release after 48 h  (p < 0.0001) and 100 µM (*** p = 0.005) concentrations significantly downregulated its release . (TQ significantly downregulated the protein release of IL-1β after 24 h at 20 µM (* = 0.0013 G. Simila 0.0007) H. After 0.0001). I. TQ conp < 0.0001). Data were analyzed using an unpaired t-test  (p = 0.0435), 50 µM (** p = 0.0014), and 100 µM (* p = 0.0300) significantly downregulated sPD-L1 release  (p = 0.0008), 50 µM (* p = 0.0101), and 100 µM (** p = 0.0047)—one-way ANOVA, p = 0.0037  B. After  0.0066) C. After = 0.0037 D. TQ conMore than approximately 60% of the available anticancer drugs were derived from natural sources, in one way or another . Plant-dCALR resides in the ER, acting as a chaperone in healthy cells . When ceVarious studies demonstrated that CALR is an NLRP3 activator ,37,38,39Caspase-1 is an inflammatory caspase that is activated via inflammasome complexes in response to DAMPs or pathogen-associated molecular patterns (PAMPs) . Since iTo date, the current study is the only available investigation of TQ’s impact on NLRP3 in BC. However, various research articles examined its inhibitory effect on NLRP3 in various models ,105,106.Along with the current study, various research papers showed that TQ is a possible multi-strike inhibitor of the NLRP3 pathway. It inhibited NFκB , which wp < 0.0001)  A; these  0.0001) B. AlthouThe literature showed that CALR increased PD-L1 levels . In addiThe phosphoinositide 3-kinase (PI3K)/AKT pathway is a regulator of pivotal cell functions such as cell proliferation and survival . In BC, The chemokine receptor 2 (CXCR2) has been shown to promote therapy resistance and suppress immunotherapy . The lit®, Palbociclib®, and Abemaciclib® [® in BC, as it might provide a synergistic effect.Cyclin D is frequently deregulated in human cancer and promotes cell division by activating cyclin-dependent kinase 4/6 (CDK 4/6), causing enhanced cell proliferation and BC progression ,195. In aciclib® . Studiesaciclib® . In addiaciclib® . Since tClinicalTrials.gov Identifier: NCT03208790).To date, no clinical trials have investigated the effect of TQ on cancer in general or BC patients in particular. However, according to clinical trials.gov, TQ has been evaluated for its clinical, immunohistochemical, and chemo-preventive impacts on potentially malignant oral lesions. However, the results are not published yet. Patients were divided into three equal groups: Group A was administered buccal tablets containing 10 mg TQ; Group B received buccal 5 mg TQ tablets; Group C was the placebo control group . Cell viability of PBMCs was investigated using a Trypan Blue dye exclusion test. The PBMCs of each sample were cryopreserved and stored in a freezer at −80 °C for later use . According to immunohistochemical reports, the current study categorized BC patients into luminal A and TNBC groups. PBMCs of each group of patients were thawed at room temperature, then quickly washed, and centrifuged at 1500 RPM/10 min in cell culture media  and 1% Pen-Strep . The supernatant was decanted and pellets were collected .2 and 95% humidity. The cultured cells were allowed to become 80% confluent; then, TCM was harvested and centrifuged to remove suspended cells. After that, the supernatant was collected and stored in a freezer for further use. MDA-MB 231 (purchased from Vacsera Egypt) was incubated in Dulbecco’s modified Eagle’s medium (DMEM) purchased from Lonza, Cologne, Germany (cat. no. 12-604F), supplemented with 4.5 g/L glucose, 4 mmol/L L-glutamine, 10% FBS, and 1% Pen-Strep at 37 °C with an atmosphere of 5% CO4 cells per well in a 48-well plate)  following the manufacturer’s instructions. The freshly isolated monocytes were plated in a 48-well plate . Monocytes were cultured in TCM and cell culture media with a ratio of 1:1. Additionally, 1 µg/mL of human interleukin-10 (IL-10) , 1 µg/mL of human interleukin-4 (IL-4) , and 1 µg/mL of human macrophage colony-stimulating factor (M-CSF)  were added to the culture media. Monocytes were incubated for seven days. Every other day, medium cytokines were refreshed and cells were microscopically examined; at day 7, TAMs were harvested (3 × 10l plate) .At day 7, flow cytometry was performed using anti-CD163 FITC in accordance with the manufacturer’s instructions  to ensure successful TAMs differentiation. Briefly, cells were dissociated, followed by single-cell suspension preparation . Cells were washed with 2 mL (PBS 1% FBS) and centrifuged for 5 min; then, the supernatant was discarded. After that, 1.2 µg anti-CD163 FITC was added (5 µg/1 million cells) and incubated for 30 min at 4° degrees. Finally, analysis was performed using CytoFLEX flow cytometry.First, 20, 50, and 100 mM stock solutions of TQ  were dissolved in dimethyl sulfoxide (DMSO)  and stored at −80 °C. The TQ stock solutions were diluted in fresh cell culture media into  just before use. Note that the percentage of DMSO was 0.1% in order to not be toxic to cells.6 cells per mL) at increasing concentrations  and durations , as shown in 4 cells per well) with the aforementioned concentrations for 24 h. Notably, each TQ concentration was cultured in triplicate.In a pyrogen-free 48-well plate, PBMCs were cocultured with TQ . Total RNA was then converted to cDNA by the Thermo Scientific RevertAid First Strand cDNA Synthesis Kit. The relative expressions of CALR , NLRP3 , PYCARD , caspase-1 , IL-1β , and GAPDH   were amplified and quantified on a StepOne Real-Time PCR instrument  using TaqMan Real-time PCR . For each sample, a reaction mix was prepared according to the manufacturer’s instructions, and 4 µL of the respective cDNA was added. The RT-qPCR run was performed in the standard mode, which consists of two stages: the first stage, where the Taq polymerase enzyme was activated for 10 min at 95 °C, and the second stage, which consisted of 40 amplification cycles. Notably, all PCR reactions, including controls, were run in triplicate reactions. Values were calculated as RQ represented as 2 −∆∆CT . The protein release of IL-1β and sPD-L1 in cell culture supernatants was conducted following manufacturer’s instructions using the human IL-1β ELISA kit  and the PD-L1 human ELISA kit . t-test was used . All experiments were performed in triplicate and data are expressed as the mean ± standard deviation. Statistical analysis was performed using the GraphPad Prism software version 9.1.1. The data were analyzed using one-way ANOVA and Dunnett’s multiple comparisons to compare each treated column and the DMSO control column. The column chart was plotted, RQ values were on the Y-axis, and TQ concentrations were drawn on the X-axis. To compare the DMSO control and the two BC subtypes , the unpaired The present findings showed that TQ is a novel significant inhibitor of CALR, NLRP3 complex components, as well as IL-1β and sPD-L1 in TAMs and PBMCs of HR+ and TNBC, respectively. The current study sheds light on targeting multiple pro-tumorigenic BC markers using TQ. In addition, it urgently recommends exploring TQ’s clinical impact on HR+ and TNBC patients alone and in combination with atezolizumab. Collectively, TQ might be an excellent multi-strike adjuvant and a potential immunotherapeutic in both HR+ and TNBC patients for future investigation.


A, purified byHPLC and analyzed by high-resolution mass spectrometry (HRMS) analysis. DBU has beenreported as a reducing agent and exhibits the potential to reduceimine intermediate A.9b,c We have furtherconfirmed the formation of reduced imine intermediate A in the presence of DBU by carrying out reactions with other peptides.Page 2, column 2, first paragraph:“However, the use ofanhydrous solvents resulted in the attachment of 9,10-phenanthrenequinoneto Arg and generated intermediate A, as analyzed by LCMS ” should be rephrasedto “However, the use of anhydrous solvents resulted in theattachment of 9,10-phenanthrenequinone to Arg, which was generatedby the reduction of imine intermediate We add the following new refs 9b,c and 12b:NyoniD.; LobbK. A.; KayeP. T.; CairaM. R.DBU-mediatedcleavage of aryl- and heteroaryl disulfides.ARKIVOC2012, 2012, 245–252. 10.3998/ark.5550190.0013.623. (c) XuS.; YangF.; FanH.; ZhaoX.; XuY.; WangS.; ZhangX.1,8-Diazobicyclo[5.4.0]undec-7-ene(DBU)-promoted reduction of azides to amines under metal-free conditions.New J. Chem.2022, 46, 9994−9998. 10.1039/D2NJ00341D(9) (b) KemnitzC. R.; LoewenM. J.Amide resonancecorrelates with a breadth of C-N rotation barriers. J. Am. Chem. Soc.2007, 129, 2521−2528. 10.1021/ja066302417295481(12) (b) Referencenumbers were changed in the revised manuscript as follows.Page1:8” should be changed to “suchas protein localizationand the formation of complexes.7,8”1. “such as protein localization and the formationof complexes.8d” should be changed to“C-terminal arginine clusters resulted in the loss of activityof the protein.8”2. “C-terminalarginine clusters resulted in the loss ofactivity of the protein.9” should bechanged to “C-terminal cleavagea point of interest in recent years.8”3. “C-terminal cleavagea point of interest in recent years.10”should be changed to “been used in deep screeningof C-terminome.8”4.“been used in deep screening of C-terminome.11” should be changed to “alongwith the formation of a fluorophore byproduct .9”5. “along with theformation of a fluorophore byproduct.Page 2:12” shouldbe changed to ”overshadow the peptide.9”6. “overshadow the peptide.11” should be changed to “HRMSanalyses .9−11”7. “HRMS analyses .Page 3:13” should be changed to “serineproteases that have been reported in previous precedent.11”8. “serine proteases that have been reported in previousprecedent.14” should be changed to “viadelocalizationof the lone pairs of the amide nitrogen with the carbonyl group ofthe amide.12,13”9. “via delocalization of the lone pairs ofthe amide nitrogen.15” shouldbe changed to “and making it moresusceptible to nucleophilic attack.14,15”10. “and making itmore susceptible to nucleophilic attack.Rewording of sentences:Page 1, column 2:2, Figure 1A).” should bechanged to “resulting in the cleavage of the peptide backbonechain to carboxylic acid .”11. “resultingin the cleavage of the peptide backbone chainto carboxylic acid ”.“Due to subpar solubility of 9,10-phenanthrenequinone in water,the solvent system was adjusted to 6:1 H2O/ACN.”We would like to add the following after“ to (6:1).Supporting Information has beencorrected to include “DBU in anhydrous ACN with peptide 1e” (HPLC and HRMS data added) and “No Basein ACN with free N-terminal Arg peptide” (HPLC and HRMS dataadded).The


Heterodera schachtii, which causes tremendous losses in crop plants, was described. Notably, rhamnolipid production in engineered Pseudomonas putida strains has already reached industrial implementation. However, the non-natural hydroxyl-decorated prodiginines, which are of particular interest in this study due to a previously described particularly good plant compatibility and low toxicity, are not as readily accessible. In the present study, a new effective hybrid synthetic route was established. This included the engineering of a novel P. putida strain to provide enhanced levels of a bipyrrole precursor and an optimization of mutasynthesis, i.e., the conversion of chemically synthesized and supplemented monopyrroles to tripyrrolic compounds. Subsequent semisynthesis provided the hydroxylated prodiginine. The prodiginines caused reduced infectiousness of H. schachtii for Arabidopsis thaliana plants resulting from impaired motility and stylet thrusting, providing the first insights on the mode of action in this context. Furthermore, the combined application with rhamnolipids was assessed for the first time and found to be more effective against nematode parasitism than the individual compounds. To obtain, for instance, 50% nematode control, it was sufficient to apply 7.8 μM hydroxylated prodiginine together with 0.7 μg/ml (~ 1.1 μM) di-rhamnolipids, which corresponded to ca. ¼ of the individual EC50 values. In summary, a hybrid synthetic route toward a hydroxylated prodiginine was established and its effects and combinatorial activity with rhamnolipids on plant-parasitic nematode H. schachtii are presented, demonstrating potential application as antinematodal agents.Bacterial secondary metabolites exhibit diverse remarkable bioactivities and are thus the subject of study for different applications. Recently, the individual effectiveness of tripyrrolic prodiginines and rhamnolipids against the plant-parasitic nematode  Furtherosin (1) , 2018. Tosin (1) . Therefo2) was reported to show remarkable plant growth promoting properties. In addition, it was not toxic for Caenorhabditis elegans at concentrations where prodigiosin (1) was lethal for this non-target organism  were investigated for the first time, demonstrating potential application as antinematodal agents.In previous work, a prodiginine bearing a terminal allylalcohol group (organism . Based oginine 3 . The mod2.2.1.1) biosynthesis, the monopyrrole MAP  and the bipyrrole MBC  are condensed by a ligase (PigC in S. marcescens) to a tripyrrolic scaffold. The mutasynthesis approach has previously been established successfully in P. putida pig-r2 ΔpigD  led to novel tripyrrolic compounds in these previous studies.In natural prodigiosin , we aimed to establish a streamlined MBC-producing chassis preferentially lacking genetic and metabolic burdens. As a new strategy, a truncated pig gene cluster specifically designed to facilitate the biosynthesis of the bipyrrole 6 was integrated in the host. To this end, the artificial pigAFGHIJKLMN gene cluster (excluding the MAP (5) biosynthesis-encoding genes pigBDE and the ligase-encoding pigC, see lacZ gene was inserted downstream of pigN , one clone (MBC17) produced a lower amount of MBC (6) than P. putida pig-r2 ΔpigD, and 14 accumulated the bipyrrole at higher levels (up to 8-fold increased). The best producer (MBC18) showed likewise elevated transcript levels (approx. 4-fold), indicating that a higher expression level could be reached in the new strain, which in turn contributed to higher bipyrrole synthesis. The enhanced MBC (6) level was verified for MBC18 in larger scale as applied in preparative mutasynthesis: Both strains, P. putida pig-r2 ΔpigD and MBC18, were cultivated in TB medium and polyurethane (PU) foam cubes were added as adsorbent for the hydrophobic compound, as previously established for prodigiosin (1) recovery (6) than those of the previously reported strain P. putida pig-r2 ΔpigD — a promising precondition for mutasynthesis.Since mutasynthesis might be limited by biosynthetic provision of the bipyrrole precursor MBC , cells were extracted and prodigiosin-specific absorption was measured at 535 nm (pigC).The next step was to evaluate the capacity of the new strains for mutasynthesis with MBC17, MBC13, and MBC18 representing a low, an intermediate, and a high MBC level producer, respectively. The ligase PigC was thus introduced into these strains by plasmid-based expression from the IPTG-inducible PT33-pigC . After st 535 nm . Howeverin vitro assays PigC does not require any additional proteins for functioning  formation, an increased signal was observed with MBC13 and highest levels were found in the high-level MBC-providing strain MBC18 .Since it is known that in ctioning  it couldetup see . Based oetup see . This suetup see , S3. Pigetup see ,D. While1) production of the newly established chassis P. putida MBC18/pVLT33-pigC-pigB was assessed in comparison with hitherto used P. putida pig-r2 ΔpigD. To this end, small scale cultivation was used to test performance at 25°C as previously established , MBC (6) was consumed to below limits of quantification, an adjustment of MAP concentrations was tested next. Hence, different MAP (5) concentrations were supplemented to P. putida MBC18/pVLT33-pigC-pigB in order to potentially match the levels of both precursors in preparative scale experiments and optimize yields (5). Isolation by soxhlet extraction of PU and column chromatography on silica yielded 17 mg prodigiosin (1) from 500 ml mutasynthesis cultures. This outcome corresponding to 34 mg/l represented an improvement to previously reported 17 mg/l, obtained with the same pyrrole via comparable procedures (6) was below 2 μM in this experiment , a hydroxylated pyrrole 11b was obtained as precursor.Starting from carboxylic acid escribed . The Troescribed . This coescribed . Observe11a was converted in P. putida MBC18/pVLT33-pigC-pigB  was used as reference in all subsequent assays. The individual effects of the hydroxylated prodiginine 3 and prodigiosin (1) against the PPN H. schachtii were determined first. To this end, the half maximal effective concentration (EC50) for the reduction of nematode numbers on the model plant Arabidopsis thaliana was assessed. Prodiginines were found to inhibit nematode infestation by up to 80%. The EC50 (nematode infection) was 31.2 μM for hydroxylated prodiginine 3 and 15.1 μM for prodigiosin (1)  did not exhibit stronger effects compared to prodiginine 3 (see 3.The main focus in this study was on hydroxylated prodiginine osin (1) . Althougne 3 see  thus jus1) and the hydroxylated prodiginine 3 was performed. To this end, fitness parameters of second-stage juveniles of H. schachtii (J2), nematode infection of A. thaliana and nematode development at the plant were investigated. These parameters were evaluated at different time points during exposure of the parasite to the above determined EC50 (nematode infection).A next aim was to pinpoint the nematode’s life stage(s) and the time during host-pathogen interaction when the effect of prodiginines on the nematode becomes obvious. Therefore a time-resolved analysis of the nematodes’ reaction to application of prodigiosin  and the hydroxylated prodiginine 3 at their EC50 concentrations for 1 h, was significantly reduced by 32 and 39%, respectively, compared to the control  and hydroxylated prodiginine 3 application, respectively  of 2.8 μg/mL  and di-rhamnolipids (4) revealed that the combination of the two compounds was more effective against nematode parasitism than the individual compounds, i.e., the combined application reduced the required concentrations for the same effect (3 and di-rhamnolipids (4) together or prodigiosin (1) and di-rhamnolipids (4) together instead of the full dose of each compound individually  this study presents an environmentally friendly way by avoiding heavy metal catalyzed synthesis of Boc-6  biosynthesis resulted in titers, e.g., ranging from 60 to 150 mg/L (6) supply, or a combination of these factors. In the present study, the MBC (6) supply was addressed by implementing expression of MBC (6) biosynthetic genes, which successfully led to reaching enhanced titers of 30–34 mg/L. Further improvements are conceivable with alternative ligase enzymes. The new strain setup of P. putida MBC18 allows their exchange more easily by use of alternative plasmids, whereas strain P. putida pig-r2 ΔpigD carried all pig genes in the chromosome. In this context, our studies uncovered, that coexpression of pigB is essential in this setup. It might be speculated that the enzymes PigN/F and PigB, that naturally deliver the pyrrole precursors, as well as PigC assemble to form a membrane-associated complex  did not lead to a likewise high amount of prodigiosin (1) and thus resulted in relatively low yields up to 17%. Interestingly, by varying the MAP (5) concentration in a range of 100–1,000 μm, the prodigiosin (1) concentrations obtained (93–167 μm) were relatively constant and did not reflect the magnitude of the fed pyrrole concentration (5) has been demonstrated in a previous study: conversion assays using different concentrations of MAP (5) at a fixed MBC (6) concentration suggested an ordered kinetic mechanism in which MBC (6) has to bind before MAP (5) (1), obtained with an adjusted MAP (5) concentration, is the highest yield achieved in a preparative scale. However, further investigation and optimization of the mutasynthesis approach may lead to even higher yields, for example by feeding the pyrrole over a period of time to overcome PigC substrate inhibition.Previous studies investigated the versatile substrate range of the condensing enzyme PigC . Accordie yields . Notablyntration . This ob MAP (5) . In the in vitro PigC assay showed acceptance for both pyrroles 11a and 11b  or normal phase column chromatography  led to an apparently inactivated prodiginine, which did not show any conversion to the hydroxylated prodiginine 3, not even under high excess of the hydroboration agent 9-BBN. Quantitative 1H-NMR spectroscopy showed, that the purity of the mutasynthesis product, which was isolated by normal phase column chromatography increased from 60 ± 3% to 92 ± 5% after an additional reversed-phase column chromatography. This finding was crucial for the success of the hybrid synthesis toward hydroxylated prodiginine 3, even if the impurities that led to an inactivation of isolated mutasynthesis products could not be identified and further investigations are necessary.As mentioned above, a strength of mutasynthesis is the substrate promiscuity of the enzymes used. Results of an  and 11b . However3.2.H. schachtii (1) and hydroxylated prodiginine 3 directly antagonize H. schachtii J2 by inhibiting motility and stylet thrusting . The frequency of this stylet movement is significantly reduced by prodiginines, as demonstrated in this study. The continuous vigorous stylet thrusting of J2 is an energy consuming process  generation . WhetherUnlike “single compound - single target” approaches, multicomponent therapeutics enable interactions with multiple targets . The disSynergistic effects between biosurfactants including rhamnolipids and various antibiotics have been described multiple times before . On the A. thaliana on alert so that the plant responds stronger to H. schachtii attack , pigFGHI  and pigJKLMN  using vector pPIG  using vector pRcExpII2-YF1-FixJ-PFixK2-LacZ  using yTREX-pig , and expression strains were identified by blue color due to β-galactosidase activity.To generate escribed . Since yr plates : The sel4.1.4.P. putida pig-r2 ΔpigD and MBC strains   were cultivated in Flowerplates. To this end, 1 mL precultures in LB medium were used for inoculation of 1,1 mL main cultures in TB medium to an OD (650 nm) of 0.05. Flowerplates were covered with breathable air sheets  and incubated for 24 h . After that, 700 μL samples were used for cell harvesting . The pellet was resuspended in 300 μL methanol and subjected to sonication in a water bath (Sonorex RH 100 H) for 10 min. After centrifugation , the supernatant was dried at 45°C under reduced pressure . For 2-phase extraction, 300 μL DCM und 300 μL MilliQ-water were added and mixed well, before centrifugation . The organic lower layer was transferred in a fresh reaction tube and the extraction repeated. The resulting pooled DCM was again dried . Samples were resuspended in 200 μL methanol, centrifuged  and finally subjected to LC–MS analysis or stored until that at 4°C. LC–MS analysis was conducted using an HP 1100 Series LC/MSD  with an Atlantis T3 column  from Waters. The eluents water (A) and methanol (B), both supplemented with 0.1% formic acid, were used for gradient chromatography at 0.6 mL/min flow rate: Starting at 90% A and 10% B, increasing to 60% B in 4 min, then in further 2 min to 100% B, which was maintained for 4 min. After that, starting conditions were implemented again (10% B) and held for 1 min. Sample volumes of 10 were injected and detection was accomplished with a G1315A DAD-detector and an G1946A mass spectrometer . After verification of specific signals corresponding to the ion [M + H]+ of MBC (191 m/z), the UV/Vis signal (364 nm) of the corresponding peaks detected at 6.7 min were evaluated for relative quantification with reference to the values obtained with strain P. putida pig-r2 ΔpigD. For subsequent routine MBC analysis, HPLC-PDA analysis using an LC-10Ai  was also applied with an SPDM10Avp photodiode array detector (PDA), and an Accucore™ C18 HPLC column  from Thermo Fisher Scientific . At a column oven temperature of 30°C, 10 μL samples were analyzed at 1 mL/min flow rate in a gradient elution using water (A) and acetonitrile (B), both with 0.1% formic acid: Starting at 95%A and 5% B for 0.5 min, increasing to 25% B at 0.9 min, to 55% at 9.5 min, and to 98% at 10 min, which was held for 1 min, before returning to 5% B at 11.5 min, which was maintained for 2 min. Chromatograms were recorded at 360 nm and detected MBC at 4.5 min (λmax 363 nm). To analyze MBC in 100 mL cultures, 1 g polyurethane (PU) foam cubes were added as described for mutasynthesis conditions, and extracted with 15 mL ethanol.For verification of MBC biosynthesis in small scale cultivation, expression cultures of 4.1.5.pigN. Precultures, grown in LB medium, were used to inoculate 1 mL TB main cultures of P. putida pig-r2 ΔpigD and MBC18 with pVLT33-pigC-pigB to an OD (650 nm) of 0.05 . These were incubated shaking  at 30°C for 4 h, before induction of the latter with 0.5 mm IPTG (10 μL of a 50 mm stock solution in water). After additional 4 h incubation, 500 μL of each culture were harvested to extract total RNA with the NucleoSpin® RNA-Kit . DNAse treatment was conducted in three steps with DNase from Macherey-Nagel, Qiagen  and Ambion (Thermo Fisher Scientific). The Maxima Reverse Transcriptase (Thermo Fisher Scientific) was used for reverse transcription of 2000 ng RNA in 20 μL volumes. The qPCR reaction mix contained 9.2 μL of the cDNA solution (diluted to correspond to 50 ng RNA), 0.4 μL containing 4 pmol of each primer (stocks: 10 pmol/μL), and 10 μL Maxima SYBR Green/ROX qPCR Master Mix . All qPCR reactions were carried out as technical quadruplicates in addition to the biological triplicates. Controls without reverse transcriptase and without template as well as monitoring of PCR product melting curves ensured signal specificity. Calibration with the pPIG plasmid allowed determination of pigN copy numbers in P. putida pig-r2 ΔpigD and MBC18, which were 177 × 104 (± 4 × 104) and 683 × 104 (± 12 × 104) per 100 ng total RNA, respectively. For direct comparison, the data was evaluated as expression levels relative to the signal of P. putida pig-r2 ΔpigD. To corroborate comparability, the transcript levels of rpoD were analyzed as internal control, which yielded similar results in both strains as expected [571 × 104 (± 16 × 104) and 750 × 104 (± 30 × 104) per 100 ng total RNA].Reverse transcription (RT) followed by quantitative PCR (qPCR) was employed to quantify mRNA levels of 4.1.6.P. putida pig-r2 ΔpigD and strains MBC13, −17, −18 with plasmids pVLT33-pigC or pVLT33-pigC-pigB  were cultivated in Flowerplates. Precultures in 1 mL LB medium were used for inoculation of 1 mL main cultures in TB medium to an OD (650 nm) of 0.05. Flowerplates were covered with breathable air sheets and incubated for 4 h , before 1 mm MAP was supplemented to the cultures (20 μL of a 50 mm stock solution in DMSO) and gene expression in strains with plasmids was induced by addition of 0.5 mm IPTG (10 μL of a 50 mm stock solution in water). Cultivation was subsequently continued for 20 h at 30°C or 25°C. After that, cultures were harvested . The pellets were pre-resolved with 20 μL milliQ-water and extracted with 500 μL acidified ethanol [4% (v/v) 1 n HCl in ethanol]. After centrifugation , 150 μL samples were either diluted by a factor of 10 or directly subjected to spectrophotometric analysis in a microplate reader “Infinite M1000 pro”  to measure characteristic absorption spectra from 400 to 700 nm. Plotting the absorption at 535 nm facilitated comparative evaluation. For quantification of prodigiosin formation via the previously published molar extinction coefficient  pET28a(+)-pigC was performed as described previously (E. coli BL21 (DE3) pET28a(+)-pigC cells, which were stored at −20°C after cultivation and harvesting. Frozen cells (1 g) were thawed and resuspended in potassium phosphate buffer . The cell suspension was disrupted using a SONOPULS Ultrasonic homogenizer  for 2 × 5 min , to obtain lysed cells which were used for the PigC substrate acceptance assay. The assay solution contained 440 μL of cell lysate in KPi buffer , 25 μL of a pyrrole 5, 11a, 11b solution in DMSO , 25 μL of an MBC (6) solution in DMSO  and 10 μL of an ATP∙Na2 solution in water . The reaction mixture was shaken in a 1.5 mL tube at 300 rpm and 30°C for 4 h. The supernatant was removed after centrifugation  and the prodiginine pellet was resuspended in 300 μL acidic ethanol [4% (v/v) 1 n HCl in ethanol]. After centrifugation , the supernatant was transferred in a new 1.5 mL reaction tube and documented photographically.The eviously . Accordi4.2.4.2.1.1H-NMR and thin layer chromatography  using an acidic solution of p-anisaldehyde for staining or UV light at 245 nm for visualization. Purification of reaction products was carried out by flash chromatography on silica gel 60 . Analytics were carried out as described in the Supplementary Information, including N-amylpyrrole , tert-butyloxycarbonyl-5′-formyl-4′-methoxy-1H,1′H-2,2′-bipyrrole  and prodigiosin (1) as chemical references were synthesized as previously described  and petroleum ether (PE) were distilled prior to use. Tetrahydrofuran (THF) was used directly. Reactions were monitored by GC–MS, escribed .4.2.2.N,O-dimethylhydroxylamine hydrochloride , N-(3-dimethylaminopropyl)-N′ethylcarbodiimid hydrochloride  and 4-(dimethylamino)pyridine . After stirring for 22 h at 22°C, the reaction mixture was quenched with a saturated solution of NaCl and extracted with dichloromethane (3 × 50 mL). The combined organic layers were first washed with 1 N HCl, afterwards with saturated NaHCO3 solution and dried over MgSO4. After removal of the solvent under reduced pressure N-methoxy-N-methyloct-7-enamide  was obtained as a yellow oil and was used for the following experiment without further purification. Rf = 0.15 (PE/EtOAc 85:15); 1H-NMR : δ [ppm] = 1.36 , 1.41 , 1.64 , 2.05 , 2.41 , 3.18 , 3.68 , 4.93 , 4.99 , 5.80 ; 13C-NMR : δ [ppm] = 24.6 (C-3), 28.8 (C-4), 29.1 (C-5), 32.0 (C-2), 32.3 (C-1′′) 33.8 (C-6) 61.3 (C-1′), 114.5 (C-8), 139.1 (C-7); IR (ATR-film): MS : m/z = 186 [(M)+], 97, 83, 55.To a solution of 7-octenoic acid  in dichloromethane (70 mL) was added 4.2.3.N-methoxy-N-methyloct-7-enamide  was dissolved in dry THF (100 mL) and methylmagnesium chloride  was added within 15 min at 0°C. The reaction mixture was stirred for 1.5 h at 0°C and afterwards quenched by the addition of a saturated solution of NH4Cl at 0°C and extracted with dichloromethane (3 × 60 mL). The combined organic layers were dried over MgSO4 and the solvent was evaporated under reduced pressure to yield non-8-en-2-one  as a yellowish oil without further purification. Rf = 0.40 (PE/EtOAc 85:15); 1H-NMR : δ [ppm] = 1.25–1.34 , 1.35–1.43 , 1.58 , 2.05 , 2.13 , 2.42 , 4.93 , 4.99 , 5.79 ; 13C-NMR : δ [ppm] = 23.8 (C-4), 28.7 (C-5), 28.8 (C-6), 30.0 (C-1), 33.7 (C-7), 43.9 (C-3), 114.6 (C-9), 139.0 (C-8), 209.4 (C-2); IR (ATR-film): MS : m/z = 123.4.2.4.9, 1.73 g, 12.4 mmol, 1.0 eq.) in ethanol (6.2 mL) was added pyridine  and grounded hydroxylamine hydrochloride (1.5 eq.). The reaction mixture was refluxed for 2 h and afterwards extracted with dichloromethane (3 × 25 mL). The combined organic layers were washed with 1 N HCl (3 × 20 mL) and dried over MgSO4. After removal of the solvent under reduced pressure, dec-9-en-2-one oxime  was obtained in a diastereomeric mixture of E:Z (1:3) without further purification. Rf = 0.34 (PE/EtOAc 80:20); 1H-NMR : δ [ppm] = 1.28–1.36 , 1.37–1.44 , 1.51 , 1.87 , 2.05 , 2.18 , 4.94 , 5.00 , 5.80 ; 13C-NMR : δ [ppm] = 13.4 (C-1), 26.2 (C-4), 28.7 (C-5), 28.8 (C-6), 33.7 (C-7), 35.8 (C-3), 114.4 (C-9), 139.1 (C-8), 159.1 (C-2); IR (ATR-film): MS : m/z = 156 [(M)+], 83; HRMS : calculated for C9H18NO [(M + H)]+ = 156.1383, found = 156.1384.To a solution of the non-8-en-2-one , potassium hydroxide , water  and DMSO (8.9 mL) was heated at 90–100°C (using previously degassed DMSO on molecular sieves (3.5 Å) could increase the yield). Over a period of 2 h, a solution of 1,2-dichloroethane  in DMSO (1 mL) was added dropwise. After 1 h of 1,2-dichloroethane addition a secondary amount of potassium hydroxide (5.0 eq.) was added. After an overall reaction time of 4 h at 90–100°C the reaction mixture was allowed to reach room temperature and subsequently ice water (20 mL) was added. The mixture was extracted with diethyl ether (3 × 20 mL) and the combined organic layers were dried over MgSO4. After the solvent was evaporated under reduced pressure the crude product was purified by column chromatography on silica gel [PE:dichloromethane (60:40) + 1% trimethylamine (v/v)] to obtain 3-(hex-5-en-1-yl)-2-methyl-1H-pyrrole  as yellowish oil. Rf = 0.37 (PE/dichloromethane 60:40); 1H-NMR : δ [ppm] = 1.44 , 1.51–1.59 , 2.02–2.14 , 2.18 , 2.39 , 4.93 , 5.00 , 5.82 , 6.01 , 6.59 , 7.70 ; 13C-NMR : δ [ppm] = 11.2 (C-1′), 25.9 (C-1′′), 28.9 (C-3′′), 31.0 (C-2′′), 33.9 (C-4′′), 109.0 (C-4), 114.3 (C-6′′), 115.0 (C-5), 119.7 (C-3), 123.4 (C-2), 139.4 (C-5′′); IR (ATR-film): MS : m/z = 164 [(M)+]; 121, 108; HRMS : calculated for C11H18N [(M + H)]+ = 164.1434, found = 164.1433.A reaction mixture of dec-9-en-2-one oxime  was added to a solution of 3-(hex-5-en-1-yl)-2-methyl-1H-pyrrole  in dry THF (11.6 mL) over a period of 15 min at 0°C. After stirring for 1 h at 0°C, the reaction mixture was heated up at 70°C under reflux for 3 h. Subsequently an aqueous solution of 3 n NaOH  and 30% H2O2  was added at 0°C. After 1 h at 0°C the reaction mixture was allowed to reach room temperature and was stirred for further 15 h at 22°C. Ice water (40 mL) was added and the mixture was extracted with dichloromethane (3 × 50 mL). The combined organic layer was dried over MgSO4, the solvent was evaporated under reduced pressure and the crude product was purified by column chromatography on silica gel [PE:ethyl acetate (70:30 to 50:50) + 1% trimethylamine (v/v)] to isolate 6-(2-methyl-1H-pyrrol-3-yl)hexan-1-ol  as orange oil. Rf = 0.20 (PE/EtOAc 70:30); 1H-NMR : δ [ppm] = 1.20 , 1.34–1.42 , 1.51–1.61 , 2.18 , 2.39 , 3.64 , 6.00 , 6.59 , 7.72 ; 13C-NMR : δ [ppm] = 11.2 (C-1′′), 25.8 (C-4), 26.0 (C-6), 29.4 (C-3), 31.4 (C-5), 33.0 (C-2), 62.7 (C-1), 109.0 (C-4′), 115.0 (C-5′), 119.7 (C-3′), 123.4 (C-2′); IR (ATR-film): MS : m/z = 182 [(M)+], 164, 108; HRMS : calculated for C11H20NO [(M + H)]+ = 182.1539, found = 182.1539.9-BBN (0.5 4.3.4.3.1.P. putida MBC18 with pVLT33-pigC-pigB in LB medium (25 μg/mL kanamycin) was incubated in a shake flask at 30°C and 130 rpm overnight. Five main cultures of 100 mL each in TB medium (25 μg/mL kanamycin) were inoculated to an OD650 of 0.05 and incubated in 1 l baffled flask with air-sheet seals for 4 h at 30°C and 130 rpm. The pyrrole precursor was dissolved in DMSO . To each culture 2 mL of pyrrole stock solution was added . Induction was performed with 0.5 mm IPTG . After an additional hour at 30°C and 130 rpm, 1 g of polyurethane (PU) foam cubes  were added to each culture and the cultures were incubated for further 23 h at 30°C and 130 rpm. After a total of 28 h of cultivation, the foam cubes were wrung out, washed with dH2O, and then extracted with diethyl ether (250–500 mL) in a soxhlet extractor. After evaporation of the solvent, the crude product was dissolved in diethyl ether (20 mL), washed with water, and the aqueous layer was extracted with dichloromethane (3 × 15 mL). The combined organic layers were washed with saturated NaCl (20 mL) and dried over MgSO4. After evaporation of the solvent, the crude product was purified by flash column chromatography on silica gel (CH2Cl2 + 1.0–1.5% NH3 in MeOH). For subsequent hydroboration, the product was further purified by reversed-phase chromatography  to obtain the mutasynthesis product as red solid.A preculture of 4.3.2.11a  as precursor prodiginine 12  was obtained as red solid. Rf = 0.15 (dichloromethane); 1H-NMR : δ [ppm] = 1.43 , 1.53–1.59 , 2.08 , 2.41 , 2.54 , 4.01 , 4.95 , 5.01 , 5.80 , 6.08 , 6.36 , 6.68 , 6.92 , 6.96 , 7.24 , 12.58 , 12.75 ; 13C-NMR : δ [ppm] = 12.6 (C-12′′), 25.3 (C-6′′), 28.6 (C-8′′), 29.7 (C-7′′), 33.7 (C-9′′), 58.9 (C-7), 93.0 (C-3), 111.9 (C-4′), 114.7 (C-10′′), 116.1 (C-8), 117.2 (C-3′), 120.9 (C-5), 122.4 (C-2′), 125.3 (C-2′′), 127.1 (C-5′), 128.3 (C-4′′), 128.4 (C-3′′), 138.8 (C-9′′), 146.9 (C-5′′), 147.9 (C-2), 165.9 (C-4); IR (ATR-Film): MS : m/z = 336 [(M)+], 266, 163; HRMS : calculated for C21H26N3O [(M + H)]+ = 336.2070, found = 336.2076.According to the general procedure for preparative mutasynthesis and the use of pyrrole 4.3.3.H,1′H-2,2′-bipyrrole  in dry THF (2 mL) over a period of 15 min at 0°C. After stirring for 1 h at 0°C, the reaction mixture was heated up at 70°C under reflux for 3 h. Subsequently an aqueous solution of 3 N NaOH  and 30% H2O2  was added at 0°C. After 1 h at 0°C the reaction mixture was allowed to reach room temperature and was stirred for further 15 h at 22°C. Ice water (10 mL) was added and the mixture was extracted with dichloromethane (3 × 15 mL). The combined organic layer was dried over MgSO4, the solvent was evaporated under reduced pressure and the crude product was purified by column chromatography on silica gel [dichloromethane + 0.5–4.0% trimethylamine (v/v)] to isolate 6--5-yl)methylene)-5-methyl-2H-pyrrole-4-yl)hexan-1-ol  as red solid. Rf = 0.16 (PE/EtOAc 50:50); 1H-NMR : δ [ppm] = 1.28 , 1.37 , 1.50–1.63 , 2.40 , 2.54 , 3.64 , 4.00 , 6.08 , 6.35 , 6.67 , 6.92 , 6.94 , 7.23 , 12.56 , 12.72 ; 13C-NMR : δ [ppm] = 12.6 (C-13′′), 25.0 (C-6′′), 25.7 (C-8′′), 29.1 (C-9′′), 30.2 (C-7′′), 32.9 (C-10′′), 58.9 (C-7), 63.1 (C-11′′), 93.0 (C-3), 111.9 (C-4′), 116.3 (C-8), 117.3 (C-3′), 120.9 (C-5), 122.4 (C-2′), 125.3 (C-2′′), 127.2 (C-5′), 128.3 (C-4′′), 128.4 (C-3′′), 147.0 (C-5′′), 148.0 (C-2), 166.0 (C-4); IR (ATR-film): MS : m/z = 354 [(M)+], 279, 157; HRMS : calculated for C21H28N3O2 [(M + H)]+ = 354.2176, found = 354.2179.9-BBN  was added to a solution of 4-Methoxy-5-((5-methyl-4-hex-5-en-1-yl-2H-pyrrole-2-yliden)methyl)-14.4.4.4.1.Arabidopsis thaliana Columbia (Col-0) seeds were surface-sterilized by soaking in 0.7% sodium hypochlorite for 5 min and submerging them in 70% (v/v) ethanol for 1 min. Subsequently, the seeds were rinsed with sterile distilled water 5 times, dried at room temperature for 4 h and stored at 4°C for further experiments. H. schachtii cysts were harvested from the roots of mustard , which was grown aseptically on modified Knop agar medium, and submerged with sterile 3 mm ZnCl2 in the Baermann funnel (n funnel . After 74.4.2.50 determination of selected compounds was performed in an in vitro agar system: Petri dishes (90 mm diameter) were filled with modified Knop medium . EC50 was determined by using software ‘CompuSyn’  or hydroxylated prodiginine 3 at the determined EC50, which is 15.1 and 31.2 μM, respectively. Modified Knop medium alone or supplemented with 0.5% (v/v) DMSO served as controls.The time-resolved analysis consisted of four assays investigating nematode motility, stylet thrusting, infection, and development. Petri dishes (90 mm diameter) were filled with modified Knop medium supplemented with prodigiosin  in order to count the number of stylet movements for 5 min. The experiment was performed independently in quadruplicate.Two surface-sterilized 4.4.4.3.A. thaliana seeds were germinated aseptically on the medium containing the test compound. At 12 days post seeding, approximately 60 H. schachtii J2 were inoculated to each plant. At 2, 4 and 10 days post inoculation, the number of males and females was counted, and the size of male and female nematodes was measured under a LeicaS4E Stereo Microscope  equipped with Leica Application Suite (LAS) software. Four independent biological replicates with 14 plants per variant and biological replicate (n = 56) were conducted for the nematode infection assay. Three independent biological replicates with in total n = 70 technical replicates (nematodes) were performed for the development experiment.Two surface-sterilized 4.4.5.p < 0.05; SIGMAPLOT 12.5, Systat Software, Inc., San Jose, CA, United States).All data are expressed as mean ± standard error (SE). Statistical analysis was performed by using one-way analysis of variance . Parts of this work were funded by the state of NRW in the project RhamnoLizer. This work was supported by the Open Access Publication Fund of the University of Bonn.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Correction: Molecular Medicine (2023) 29:49https://doi.org/10.1186/s10020-023-00638-1Following publication of the original article  Representative Micro-CT images of 3-month-old caRhoA and RCre;caRhoA mice tibias. (b) The parameters of tibias growth plate trabecular bone, n = 6. (c) Representative H&E and TRAP staining images of mice femurs. Scale bars, 200 µm (H&E) and 20 µm (TRAP). (d) The parameters of femur osteoclasts, n = 6. (e) Representative TRAP staining images of BMMs from caRhoA and RCre;caRhoA mice. Scale bars, 200 µm. (f) SEM analysis and quantification of bone resorption area of bone slides, n = 6, Scale bars, 20 µm. (g) Representative Phalloidin staining images and quantification of F-actin ring per well and nuclei per osteoclast, n = 3, Scale bars, 500 µm. Mean ± s.d., *P < 0.05, Student’s t-test.Additional file 1: Figure S4. The authors confirm all results, and conclusions of this article remain unchanged. The authors apologize for this error and any confusion it may have caused.


The binuclear title compound consists of two iron(0) centres, each of them surrounded by a cyclopentadienone moiety and three carbonyl ligands in a three-legged piano-stool geometry. 2(C28H48N2O2Si4)(CO)6], consists of two central iron(0) atoms, each of them surrounded by a cyclopentadienone moiety and three carbonyl ligands in a three-legged piano-stool shape. Furthermore, the bis(cyclopentadienone) ligand acts as a bridge between the two metal atoms.The binuclear title compound, [Fe Next, the tetrayne N1,N1,N2,N2-tetrakis[3-(trimethylsilyl)prop-2-yn-1-yl]ethane-1,2-diamine  was dissolved in 13 ml of dry toluene in another flask. The tetrayne solution and the iron precursor were then mixed in the Schlenk tube and heated to 383 K for 16 h. The resulting black mixture was filtered through Celite. The filtrate was further purified via column chromatography over silica gel with pentane/ethyl acetate (90:10 v/v) as eluent to finally afford a brown solid . Crystals suitable for X-ray analysis were obtained by slow diffusion of pentane into a solution of the complex in dichloromethane. 1H NMR : δ (ppm) 3.92 , 3.42 , 3.06 , 0.27 . 13C NMR : δ (ppm) 208.69 (C≡O), 182.39 (C=O), 113.06 (CCp), 69.33 (CCp), 54.36 (–CH2—CH2–), 53.72 (Cpyr—N), −0.69 (SiMe3). HR–MS (ESI): theoretical mass for C34H48Fe2N2O8Si4: 836.11811; found: 836.11579.The iron precursor FeCrystal data, data collection and structure refinement details are summarized in Table 110.1107/S2414314623003462/bh4073sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2414314623003462/bh4073Isup2.hklStructure factors: contains datablock(s) I. DOI: 2256807CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Nature Communications 10.1038/s41467-023-37743-y, published online 13 April 2023Correction to: The original version of this Article contained an error in Fig. 2 and supplementary Figures 1, 2, 3 and 48, in which the structure of PIM-1 is displayed wrongly. The correct version of Fig. 2 and Supplementary Figures Fig. 2: The quantitative post-modification of PIM-1 through conventional and non-solvent methods with a volatile Grignard reagent, methylmagnesium bromide (CH3MgBr). Ketone-PIM−1 (K-PIM-1) was further reacted by the same reagent to make Alcohol-PIM−1 (OH-PIM-1) with remarkably high retention of porosity, considering the two-step reaction. Solvent: (i) CH3MgBr in tetrahydrofuran (THF), THF solvent at 0 °C → RT for 24 h (ii) 0.5 M hydrochloric acid (HCl) in methanol (MeOH), H2O at 60 °C for 4 h. Non-solvent: (i) CH3MgBr in diethyl ether (Et2O) at 0 °C → RT for 24 h (ii) 0.5 M HCl in MeOH, H2O at 60 °C for 4 h. K-PIM−1 was further functionalized by phenylhydrazine (PhNHNH2) and polyethylenimine (PEI) to show versatility in the reactive portfolio and to create CO2 adsorbents.Supplementary Figure 1: Synthesis of PIM-1 via low temperature approach.Supplementary Figure 2: Synthesis of PIM-1 via high temperature approach.Supplementary Figure 3: Control reaction using PIM-1 as a starting material to react with phenylhydrazine (PhNHNH2) and polyethylenimine (PEI), refluxed in the presence of ethanol solvent (EtOH) under mild acidic condition.Supplementary Figure 48: a Mechanism and b FT-IR spectra revealed the reversible reaction of HZ-PIM-1.which replace the previous incorrect versions:Fig. 2:The quantitative post-modification of PIM-1 through conventional and non-solvent methods with a volatile Grignard reagent, methylmagnesium bromide (CH3MgBr). Ketone-PIM−1 (K-PIM-1) was further reacted by the same reagent to make Alcohol-PIM−1 (OH-PIM-1) with remarkably high retention of porosity, considering the two-step reaction. Solvent: (i) CH3MgBr in tetrahydrofuran (THF), THF solvent at 0 °C → RT for 24 h (ii) 0.5 M hydrochloric acid (HCl) in methanol (MeOH), H2O at 60 °C for 4 h. Non-solvent: (i) CH3MgBr in diethyl ether (Et2O) at 0 °C → RT for 24 h (ii) 0.5 M HCl in MeOH, H2O at 60 °C for 4 h. K-PIM−1 was further functionalized by phenylhydrazine (PhNHNH2) and polyethylenimine (PEI) to show versatility in the reactive portfolio and to create CO2 adsorbents.Supplementary Figure 1: Synthesis of PIM-1 via low temperature approach.Supplementary Figure 2: Synthesis of PIM-1 via high temperature approach.Supplementary Figure 3: Control reaction using PIM-1 as a starting material to react with phenylhydrazine (PhNHNH2) and polyethylenimine (PEI), refluxed in the presence of ethanol solvent (EtOH) under mild acidic condition.Supplementary Figure 48:a Mechanism and b FT-IR spectra revealed the reversible reaction of HZ-PIM-1.This has been corrected in both the PDF and HTML versions of the Article.Updated Supplementary Information


Page 16, line 17 from the bottom: “a significant positive correlation” should read “a significant negative correlation.”Volume 14, no. 2, e00418-23, 2023,


The results showed that podophyllotoxin-imidazolium salts and podophyllotoxin-1,2,4-triazolium salts a1–a20 exhibited excellent cytotoxic activity. Among them, a6 was the most potent cytotoxic compound with IC50 values of 0.04–0.29 μM. Podophyllotoxin-1,2,3-triazole derivatives b1–b5 displayed medium cytotoxic activity, and podophyllotoxin-amine compounds c1–c3 has good cytotoxic activity with IC50 value of 0.04–0.58 μM. Furthermore, cell cycle and apoptosis experiments of compound a6 were carried out and the results exhibited that a6 could induce G2/M cell cycle arrest and apoptosis in HCT-116 cells.Three series of podophyllotoxin derivatives with various nitrogen-containing heterocycles were designed and synthesized. The antitumor activity of these podophyllotoxin derivatives was evaluated  According to the data from the International Agency for Cancer Research (IARC), there would be around 19.3 million new cancer diagnoses and nearly 10 million cancer-related deaths in 2020 . TherefoPodophyllotoxin is a natural product with anticancer activity belonging to the lignans cyclolignolide family . PodophyB591 . Then, treatment of a1–a3 with various bromides generated the podophyllotoxin imidazolium/triazolium salts a4–a21 (9%–91% yields). Secondly, as shown in S2 was obtained by commercial podophyllotoxin reacting with thionyl chloride. Next, a nucleophilic substitution reaction with sodium azide was conducted to obtain compound S3 . Then, azide S3 reacted with various terminal alkynes under Click reaction condition to get the podophyllotoxin-1,2,3-triazole derivatives b1–b5 (31%–47% yields). Finally, as shown in S1, which then underwent a nucleophilic substitution reaction with commercial cyclic amines  to furnish the podophyllotoxin-amines c1-c3 . To summarize, the structures and yields of all new podophyllotoxin nitrogen-containing heterocycle derivatives were shown in As shown in in vitro antitumor cytotoxic activity screening by MTS method (in vitro cytotoxic activity. DDP (Cisplatin), Etoposide, and Paclitaxel were chosen as positive controls. The results were listed in The synthesized twenty-nine podophyllotoxin nitrogen-containing derivatives were evaluated S method . Four huAs presented in a1/a3/a4-a12/a20/a21) and podophyllotoxin-1,2,4-triazole and its salts (a2/a13–a19) exhibited excellent cytotoxic activity with IC50 values of 0.04–1.53 μM except a20. Among them, a6 was the most potent cytotoxic compound and its IC50 values for HepG2, A-549, MDA-MB-231 and HCT-116 were 0.07, 0.29, 0.11 and 0.04 μM, respectively. Secondly, the introduction of 1,2,3-triazole derivatives b1–b5 by Click reaction showed medium cytotoxic activity with IC50 values of 0.04–9.64 μM except b5. Finally, while compounds c1–c3 introduced with cyclic amines also showed excellent cytotoxic activity with IC50 values of 0.04–0.58 μM.For pharmacophores of nitrogen-containing heterocycles, podophyllotoxin-imidazole and its salts (a4–a21), the cytotoxic activities of most substituted benzyl groups were superior to those of substituted phenacyl groups. Among them, 2-naphthylmethyl substituent at position-3 of the imidazole ring (a6 and a15) showed excellent cytotoxic activity with IC50 values of 0.04–0.75 μM and a6 was the most powerful compound. Similarly, 4-bromobenzyl, 4-methylbenzyl, 4-methoxybenzoyl and 2-bromobenzyl groups at position-3 of the imidazole ring exhibited good cytotoxic activity with IC50 values of 0.04–0.65 μM.For the groups at position-3 of imidazolium and triazolium salts (b1–b5), when the substituent was replaced with electron donating groups (R2 = OMe), b3 exhibits higher inhibitory activity with IC50 values of 0.49–3.48 μM. In contrast, when the substituent was charged with electron-withdrawing groups , b1 and b2 were decreased slightly with IC50 values of 0.04–7.31 μM. When the substituent group was pyridine, b4 exhibited poor inhibitory activity with IC50 values of 1.49–9.64 μM, due to the electron-withdrawing effect of pyridine. When the substituent was a naphthalene ring, b5 did not exhibit any inhibitory activity.For the groups at position-4 of 1,2,3-triazole ring (c1–c3), piperidine derivative of podophyllotoxin (c2) displayed excellent cytotoxic activity with IC50 values of 0.10–0.39 μM, which was superior to pyrrole derivative (0.04–0.58 μM) and morpholine derivative (0.06–0.39 μM). Notably, compound c1 has selective inhibitory against MDA-MB-231 cell lines with an IC50 value of 0.04 μM.For the cyclic amines  of the derivatives were summarized in a6 induced proliferation inhibition, cell cycle and apoptosis analysis were performed with flow cytometry. Firstly, HCT-116 cells were treated with indicated concentrations of compound a6 for 24 h and the cell cycle phase distribution of a6-treated cells was determined with propidium iodide (PI) staining. As shown in a6 exposure caused G2/M phase arrest in HCT-116 cells when compared with the control group, indicating that compound a6 inhibited cell proliferation through inducing G2/M cell cycle arrest.To determine the possible mechanism of compound a6 induced cell apoptosis was also determined with Annexin V-FITC/PI staining. As shown in a6 at 25, 50 and 200 nM for 48 h, the apoptotic rate of HCT-116 cells remarkably elevated to 5.37 ± 0.37%, 10.45 ± 0.20% and 64.98 ± 2.40%, respectively. The results suggested that compound a6 inhibited cell proliferation through induction of G2/M cell cycle arrest and apoptosis of HCT-116 cells.The compound a6 was the most potent cytotoxic activity with IC50 values of 0.04–0.29 μM. It has an obvious selective inhibitory against HCT-116 cell lines with an IC50 value of 0.04 μM and could induce G2/M cell cycle arrest and apoptosis in HCT-116 cells. Podophyllotoxin-imidazolium salt a6 could be employed as a promising lead compound for further structural modification and in-depth activity research to identify new starting points for more effective anticancer agents.In conclusion, a series of novel podophyllotoxin nitrogen-containing heterocycle derivatives with potential antitumor activity were prepared using a straightforward synthetic approach. The results showed that the imidazole-substituted derivatives demonstrated more effective inhibitory activity than 1,2,4-triazole-substituted and 1,2,3-triazole-substituted equivalents. The biological activity was significantly improved when the imidazole or imidazolium salt group was introduced into the structure of podophyllotoxin. Among them, imidazolium salt