We show that both RAD51 paralog complexes lack fork reversal activities. Whereas CX3 exhibits modest fork protection activity, BCDX2 significantly synergizes with RAD51 to protect DNA against attack by the nucleases MRE11 and EXO1. DNA protection is contingent upon the ability of RAD51 to form a functional nucleoprotein filament on DNA. Collectively, our results provide evidence for a hitherto unknown function of RAD51 paralogs in synergizing with RAD51 nucleoprotein filament to prevent degradation of stressed replication forks.Fork reversal is a conserved mechanism to prevent stalled replication forks from collapsing. Formation and protection of reversed forks are two crucial steps in ensuring fork integrity and stability. Five RAD51 paralogs, namely, RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3, which share sequence and structural similarity to the recombinase RAD51, play poorly defined mechanistic roles in these processes. Here, using purified BCDX2 (RAD51BCD-XRCC2) and CX3 (RAD51C-XRCC3) complexes and   Faithful and complete DNA replication is crucial for maintaining genome integrity. During DNA replication, elongating forks may encounter various endogenous and exogenous insults, leading to fork stalling. Unresolved stalled forks may give rise to fork collapse and DNA double-strand breaks (DSBs) ,2. RepliIn fork reversal, a three-way stalled fork is remodeled into a four-way junction, which actively restrains DNA replication in unfavorable conditions, thus avoiding fork collapse ,8,9. TheAlthough fork regression is a valuable mechanism for bypassing DNA lesions, reversed forks are prone to attack by cellular nucleases, including MRE11, EXO1 and DNA2 . Proteinin vitro reconstitution assays and DNA fiber analysis have revealed that RAD51 protects reversed forks  showing that depletion of RAD51 paralogs elicits severe fork degradation  that it modestly protects dsDNA from digestion by MRE11 and EXO1, and, importantly, (ii) that it significantly synergizes with RAD51 in DNA protection. Furthermore, we show that a functional RAD51 nucleoprotein filament is important for the synergistic protective effect. We analyze several disease-associated mutant variants of RAD51 and BCDX2 and find them to be defective in DNA protection. Therefore, our results provide evidence for a direct role of RAD51 paralogs in replication fork preservation. Our experimental system will be valuable for defining the impact of cancer-associated mutations in the RAD51 paralogs on replication fork protection and maintenance.6) tag added to the amino terminus of RAD51C and XRCC2, respectively. In addition, a Tobacco etch virus (TEV) cutting site was added between the tags and the genes. To express the BCDX2 protein complex, the pcDNA3.4-tetrad-BCDX2 plasmid was transfected into Expi293F cells (Thermo Fisher) according to the instruction manual from the ExpiFectamine 293 kit (Thermo Fisher). The cells were harvested by centrifugation 48 h after transfection. The subsequent purification steps were carried out at 4°C. The harvested cells were suspended in buffer A  supplemented with 1 mM β-mercaptoethanol and protease inhibitors . The cells were then lysed by sonication, followed by ultra-centrifugation at 100 000 × g for 1 h, to obtain the soluble extract containing target proteins. The supernatant was incubated with Ni-NTA agarose resin (QIAGEN) for 3 h. The resin was washed with buffer A supplemented with 10 mM imidazole and with buffer B  supplemented with 20 mM imidazole. The BCDX2 protein complex was eluted with buffer B supplemented with 200 mM imidazole. The eluate was then loaded into anti-Flag M2 affinity agarose (Sigma Aldrich), and the protein-bound resin was washed with buffer B. The BCDX2 protein complex was eluted with buffer B supplemented with 3× Flag peptide (100 μg/ml). The eluate was then loaded on a Superdex 200 Increase 10/300 GL column  equilibrated with buffer C  supplemented with 300 mM KCl. The soluble fractions (151 kDa) were pooled, concentrated, aliquoted and stored at –80°C.The RAD51B, RAD51C, RAD51D and XRCC2 cDNAs were cloned into a pcDNA3.4-tetrad vector with a Flag tag and a hexahistidine  to avoid disassembly of the protein complex.6) tag followed by a Flag tag at the amino terminus and XRCC3 cDNA were cloned into a pcDNA3.4-dual vector to obtain the CX3 expression plasmid. RAD51B and the hexahistidine (His6)- and Flag-tagged RAD51C sequence were cloned into a pcDNA3.4-dual vector to generate the BC expression plasmid. The cDNA of RAD51D and the hexahistidine (His6)- and Flag-tagged XRCC2 sequence were cloned into a pcDNA3.4-dual vector to construct the DX2 expression plasmid. To express the CX3, BC and DX2 complexes, Expi293F cells were transiently transfected with the individual plasmid and harvested by centrifugation 48 h post-transfection. The cells were suspended, lysed, and centrifuged to obtain the cell extract as described above. The cell extract was then incubated with Ni-NTA agarose resin for 3 h. The resin was washed with buffer A supplemented with 10 mM imidazole, and with buffer B supplemented with 20 mM imidazole. The target protein complexes were eluted with buffer B supplemented with 200 mM imidazole. The eluate was then loaded into anti-Flag M2 affinity agarose, and the protein-bound resin was washed with buffer B. The desired protein complex was eluted with buffer B supplemented with 3x Flag peptide (100 μg/ml). The eluate was then loaded on a Superdex 200 Increase 10/300 GL column equilibrated with buffer C supplemented with 300 mM KCl. The soluble fractions were pooled, concentrated, aliquoted, and stored at –80°C.The cDNA of RAD51C with a hexahistidine (His6) tag and a Flag tag, followed by a tobacco etch virus (TEV) cutting site added to the amino terminus of the protein. To express the SMARCAL1 protein, Expi293F cells were transiently transfected with the expression plasmid and harvested by centrifugation 48 h post-transfection. The SMARCAL1-expressing cells were then suspended, lysed, and centrifuged as described above. The cell lysate containing the His6-Flag-SMARCAL1 recombinant protein was purified by Ni-NTA agarose resin, anti-Flag M2 agarose affinity pulldown, and a Superdex 200 Increase 10/300 GL column as described above, except that the buffers contained 0.01% Igepal-CA-630. The soluble fractions were pooled, concentrated, aliquoted and stored at –80°C.The cDNA of SMARCAL1 was cloned into a pcDNA3.4 vector with a hexahistidine . The fractions containing pure MRE11 were pooled, concentrated, aliquoted, and stored at –80°C.Human MRE11 was expressed and purified as described previously . In brieThe cDNA of EXO1b  was cloned into a pcDNA3.4 vector with a Flag tag added to the carboxyl-terminus of EXO1, generating the pcDNA3.4-EXO1-Flag expression plasmid. The EXO1 expression plasmid was transfected into Expi293F cells, and the EXO1-expressing cells were harvested. The harvested cells were suspended, lysed, and centrifuged to obtain the cell extract as described above. The cell lysate was incubated with anti-Flag M2 affinity agarose for 1 h, and the protein-bound resin was then washed with buffer B. The EXO1 protein was eluted with buffer B supplemented with 3x Flag peptide (100 μg/ml). The eluate was then fractionated with a 1 ml Heparin column , using a 30 ml gradient of 100–640 mM KCl in buffer C. The EXO1-containing fractions were collected and loaded on a 1 ml HiTrap SP HP column  and fractionated with a 5 ml gradient of 100–1000 mM KCl in buffer C. The EXO1 fractions were then loaded on a Superdex 200 Increase 10/300 GL column equilibrated with buffer C supplemented with 300 mM KCl. The soluble fractions were pooled, concentrated, aliquoted, and stored at –80°C.Escherichia coli RecA-deficient strain BLR as described for the wild-type protein. The cell pellet was suspended in buffer D  supplemented with 300 mM KCl and protease inhibitors for sonication and ultracentrifugation. The cell extract was incubated with Talon resin (Clontech) for 3 h. The resin was washed first with buffer D supplemented with 300 mM KCl and 10 mM imidazole, and next with buffer D supplemented with 150 mM KCl and 10 mM imidazole. To obtain tag-free RAD51 T131P, strep-Ulp1 protease was mixed with the resin for 2 h to cleave the His-SUMO tag on RAD51 T131P. The supernatant containing non-tagged RAD51 T131P was loaded on a 8 ml Source Q column and fractionated using a 135 ml gradient of 193–715 mM KCl in buffer D supplemented with 0.5 mM EDTA. The RAD51 T131P-containing fractions were then loaded on a 1 ml Heparin column  and fractionated with a 15 ml gradient of 145–810 mM KCl in buffer D supplemented with 0.5 mM EDTA. At last, the RAD51 T131P-containing fractions were further fractionated using a 1 ml Mono Q column with a 15 ml gradient of 288–573 mM KCl in buffer D supplemented with 0.5 mM EDTA. Fractions containing RAD51 T131P were then pooled, filter-dialyzed into buffer C supplemented with 300 mM KCl, concentrated, aliquoted and stored at –80°C.Human RAD51 protein was purified as described previously . The RADSaccharomyces cerevisiae Rad51 protein (yRad51), the purification procedure was modified from a protocol described previously . Typical MS parameters included spray voltage, 1.1 kV; capillary temperature, 200°C; S-lens RF level, 200; resolution, 12 500. To avoid collisional activation of the ions before transmission into the higher-energy collisional dissociation (HCD) cell, a gentle voltage gradient , HCD energy of 5V, and a trapping voltage of 0V were applied. To dissociate protein complexes, in-source trapping was applied with desolvation voltages ranging between −50 and −250 V, and trapping gas pressure was set to a value of 3.0. RAW files were analyzed and processed manually using Thermo Xcalibur Qual Browser (version 4.4.16.14).2, 100 mM NaCl and 1 mM DTT). In parallel, oligos 3 and 4 were similarly mixed. The mixture was heated at 80°C for 3 min, then transferred to 65°C for 30 min, and cooled down slowly to room temperature in the water bath. The two respective mixtures (oligos 1 + 2 and 3 + 4) were subsequently mixed with an equal molar ratio and incubated at 37°C for 30 min. The annealed replication fork substrate was purified from an 8% TBE–PAGE gel by electro-elution, and the substrate was filter-dialyzed into TE buffer  at 4°C using an Amicon ultra-4 concentrator .The DNA oligonucleotides used in this study are listed in The preparation of the lagging-strand gap substrate was similar to that of the leading-strand gap substrate. Oligos 1 and 5 were mixed in a ratio of 1:1.2, and the complementary half consisting of oligos 4 and 6 were mixed separately. As described above, the two respective substrates were further annealed with an equal molar ratio, gel-purified, concentrated, and filter-dialyzed into TE buffer to generate the lagging-strand gap substrate.For nuclease degradation assays, except for the Cy3-labeled oligonucleotide, other DNA oligonucleotides were modified with phosphorothioate bonds on both ends to prevent non-specific nuclease digestion. Fluorescently-labeled oligo 7 was annealed with the phosphorothioate bond-modified oligo 8 to generate a 5′ overhang DNA substrate. Fluorescently-labeled oligo 9 was annealed with the phosphorothioate bond-modified oligo 8 to generate a 3′ overhang DNA substrate. The fluorescently-labeled reversed fork-like DNA substrate was prepared by mixing oligos 10, 11, 12 and 13 in the annealing buffer, further heated at 80°C for 3 min, then transferred to 65°C for 30 min, and cooled down slowly to room temperature in the water bath. All the annealed substrates were further gel-purified, concentrated and filter-dialyzed into TE buffer, as described above.The double-stranded DNA substrate used in the DNA mobility shift assay was prepared by mixing oligos 13 and 14 in the annealing buffer, before transferring the mixture to 65°C for 30 min and cooling it down slowly to room temperature in a water bath. The annealed substrates were further gel-purified, concentrated and filter-dialyzed into TE buffer, as described above.2) supplemented with 100 mM KCl and 2 mM ATP at 37°C for 30 min. The reaction was then mixed with 2.5 μl termination buffer  and incubated at 37°C for 15 min to stop the reaction. The reaction mixtures were resolved in a 6% TBE-PAGE gel with 1 × TBE buffer at 110 V for 70 min. Gels were analyzed using an Amersham™ Typhoon™ Biomolecular Imager with a Cy3 570BP20 560–580 nm filter. ImageJ software was then used to quantify the signal intensity of DNA species.Fluorescently-labeled fork substrate (8 nM molecules) was incubated with indicated proteins in 10 μl buffer E  was incubated with the indicated amount of BCDX2 or CX3 in 10 μl buffer E supplemented with 50 mM KCl and 1 mM ATP at 37°C for 10 min. The reaction mixtures were resolved in a 0.8% TAE-agarose gel with 0.5 × TAE buffer at 80 V for 70 min. Gels were analyzed using an Amersham™ Typhoon™ Biomolecular Imager with a Cy3 570BP20 560–580 nm filter.Fluorescently-labeled fork substrate (8 nM molecules) was incubated with RAD51 in 10 μl buffer E supplemented with 100 mM KCl and 2 mM ATP at 37°C for 10 min. The reaction mixtures were resolved in a 6% TBE–PAGE gel with 1 × TBE buffer at 110 V for 70 min. Gels were analyzed using an Amersham™ Typhoon™ Biomolecular Imager with a Cy3 570BP20 560–580 nm filter.32P]ATP in the presence or absence of single-stranded DNA  in 10 μl buffer E supplemented with 50 mM KCl and 50 μM ATP for the indicated times. Aliquots of 2 μl were withdrawn at the indicated times and mixed with an equal volume of 500 mM EDTA to stop the reaction. The level of ATP hydrolysis was determined by thin-layer chromatography on polyethyleneimine cellulose sheets  developed in 0.5 M LiCl and 1M formic acid, with phosphorimaging analysis conducted in a Personal FX phosphorimager using the Quantity One software (Bio-Rad).BCDX2 or CX3 (1.58 μM) was incubated with 1 μCi [γ-2 at 37°C for the indicated time. The reaction was then mixed with 2.5 μl termination buffer and incubated at 37°C for 15 min to stop the reaction. An equal volume of 2× denature dye  was added into the reaction mixture and incubated at 95°C for 10 min to denature the DNA substrates. The samples were analyzed on 27% denature TBE-Urea-PAGE (7 M Urea) with 1 × TBE buffer at 300 V for 40 min at 55°C. Gels were analyzed using an Amersham™ Typhoon™ Biomolecular Imager with a Cy3 570BP20 560–580 nm filter. ImageJ software was then used to quantify the intensity of DNA species. The intensity of fully protected DNA species was normalized with the intensity of DNA substrate in control experiments to obtain the percentage of protection.Fluorescently-labeled 5′ overhang DNA substrate (58 nM molecules) or fluorescently-labeled reversed fork-like DNA substrate (29 nM molecules) was incubated with the indicated amount of specified proteins and purified human MRE11 in 10 μl buffer E supplemented with 50 mM KCl, 1 mM ATP and 1 mM MnClFluorescently-labeled 3′ overhang DNA substrate (58 nM molecules) was incubated with the indicated amount of specified proteins, including EXO1, in 10 μl buffer E supplemented with 50 mM KCl and 1 mM ATP at 37°C for 40 min. Subsequent reaction termination and resolution of the various DNA species through electrophoresis were as described above.E. coli ExoIII in 10 μl buffer E supplemented with 50 mM KCl and 1 mM ATP at 37°C for 40 min. Subsequent reaction termination and resolution of the various DNA species by means of electrophoresis were as described above.Fluorescently-labeled 5′ overhang DNA substrate (58 nM molecules) was incubated with the indicated amount of the specified proteins and P value <0.05 was considered significant.All statistical tests were performed using GraphPad Prism 7 (GraphPad software) as indicated to establish statistical significance. The Shapiro–Wilk test was used to examine the normality of the data. The sample variance was confirmed to be similar between groups by the Brown–Forsythe test for multiple groups. One-way ANOVA with Tukey's post hoc test was used for comparisons of multiple groups to determine statistical significance. A 2 being included throughout the purification procedure to enhance the solubility of the protein complexes  and a mutant defective in functional filament formation (K133A), our results provide strong evidence that the DNA protection afforded by the BCDX2–RAD51 ensemble is contingent upon the ability of RAD51 to bind DNA and to form a stable nucleoprotein filament.E. coli RecA protein and yeast Rad51 (yRad51) protein, orthologs of human RAD51, with BCDX2 in a MRE11 protection assay. Both RecA and yRad51 only marginally enhanced DNA protection in association with BCDX2 (E. coli ExoIII, which, like MRE11, exerts exonuclease activity with a 3′-to-5′ polarity and serves as an excellent control in our mechanistic study (Next, we examined if enhanced BCDX2-mediated DNA protection is specific to human RAD51 protein. To do so, we tested th BCDX2 , providiic study . Taken tThe above-described findings prompted us to consider if the BCDX2-RAD51 ensemble may exert negative regulation over multiple cellular nucleases, such as the 5′-to-3′ exonuclease EXO1 that is involved in pathological fork degradation upon replication stress ,14,15. Aet al. conducted a comprehensive analysis to reveal the functional impact of various RAD51C missense mutations found in breast and ovarian cancer patients (In a recent study, Prakash patients . Importapatients . Thus, oRAD51 paralogs are indispensable for cell survival and genome stability ,51,65,66A previous cell-based electron microscopy analysis evidenced a significant decrease in the number of reversed forks in RAD51C- or RAD51D-depleted cells , indicatet al. indicated that the integrity of stalled replication forks is not compromised by knocking out RAD51C, RAD51D or XRCC3 in U2OS cells (Protection of reversed forks is a critical aspect of restarting stalled forks. Herein, our results demonstrate a synergistic effect of BCDX2 and RAD51 in protecting DNA against MRE11 and EXO1 nuclease degradation. Our study is also consistent with the notion that significant degradation of nascent DNA may be observed by knocking down RAD51 paralogs in a DNA fiber analysis . ParadoxOS cells . InteresOS cells . These cOS cells . ExtendiOS cells ,46. The E. coli ExoIII, indicating that BCDX2 regulates a fundamental attribute of RAD51-DNA nucleoprotein filaments essential for DNA protection. We propose that BCDX2 may modulate RAD51 assembly on DNA to form a non-specific roadblock against various nucleases. The broad protection effect is consistent with a study by Halder et al. showing that the DNA protective effect of RAD51 also extends beyond specific nucleases (Caenorhabditis elegans have revealed that the RAD-51 paralogs RFS-1/RIP-1 enhance the extension of RAD-51 filament in a 3′-to-5′ direction by transiently binding on the 5′ end of the RAD-51 filament (et al. reported lower and defective DNA binding activity for these mutants compared to wild-type BCDX2, respectively (Our study demonstrates that BCDX2 and RAD51 exhibit synergistic DNA protection against various nucleases, including ucleases . In addiucleases ,40,52,53filament . Single-filament . Moreovefilament ,69. Thesectively . Furtherin vivo of paralogs involved in both replication stress and DSB repair.A myriad of mutations in RAD51 paralogs have been associated with Fanconi anemia and various types of cancer ,51,65,66With regard to the CX3 complex, our biochemical assay demonstrates that while BCDX2 is the major component involved in fork protection, CX3 also contributes to RAD51-mediated fork protection. This observation aligns with a cell-based DNA fiber analysis suggesting that the CX3 complex maintains the integrity of stalled replication forks ,45. Apargkad856_Supplemental_FileClick here for additional data file.


Profilins are ubiquitous allergens with conserved structural elements. Exposure to profilins from different sources leads to IgE-cross-reactivity and the pollen–latex–food syndrome. Monoclonal antibodies (mAbs) that cross-react with plant profilins and block IgE-profilin interactions are relevant for diagnosis, epitope mapping, and specific immunotherapy. We generated IgGs mAbs, 1B4, and 2D10, against latex profilin (anti-rHev b 8) that inhibit the interaction of IgE and IgG4 antibodies from sera of latex- and maize-allergic patients by 90% and 40%, respectively. In this study, we evaluated 1B4 and 2D10 recognition towards different plant profilins, and mAbs recognition of rZea m 12 mutants by ELISAs. Interestingly, 2D10 highly recognized rArt v 4.0101 and rAmb a 8.0101, and to a lesser extent rBet v 2.0101, and rFra e 2.2, while 1B4 showed recognition for rPhl p 12.0101 and rAmb a 8.0101. We demonstrated that residue D130 at the α-helix 3 in profilins, which is part of the Hev b 8 IgE epitope, is essential for the 2D10 recognition. The structural analysis suggests that the profilins containing E130  show less binding with 2D10. The distribution of negative charges on the profilins’ surfaces at the α-helices 1 and 3 is relevant for the 2D10 recognition, and that may be relevant to explain profilins’ IgE cross-reactivity. Pollen profilins are an important cause of respiratory allergies, such as rhinoconjunctivitis and asthma. In Europe, 20–30% of allergic individuals to pollen are sensitized to profilins ,2. ProfiMonoclonal antibodies (mAbs) against specific allergens have become effective biological tools for diagnosis and IgE-epitope mapping. Moreover, they have emerged with potential clinical applications to treat allergies ,8. To daWe have also produced murine IgG monoclonal antibodies (mAbs) 1B4 and 2D10 against latex profilin (anti-rHev b 8). To define rHev b 8 epitopes recognized by these IgG mAbs and compare them with those identified by the 2F5/IgE, we performed molecular modeling of the Fv regions of both mAbs and docking simulations of the Fvs-rHev b 8 complexes. According to these analyses, the 2D10 epitope comprises α-helices 1 and 3 at the amino and carboxy-terminal regions, such as the region recognized by 2F5/IgE. In contrast, 1B4 recognized the opposite side on the profilin surface . These tThe mAbs 1B4 and 2D10 have only been studied with maize and latex profilins. Their recognition with other plant profilins has not been evaluated nor analyzed at the structural level, which may explain the bases of the cross-reactivity of profilins. Therefore, in this work, we aimed to assess mAbs 1B4 and 2D10 recognition towards allergenic plant profilins, focusing on profilins from trees , weeds , grass (rPhl p 12.0101/Timothy grass), and food (Zea m 12.0105/maize). Additionally, we used rZea m 12.0105 mutants to characterize the recognition sites of the two mAbs. Critical amino acids involved in mAbs recognition were detected through sequence alignment and comparison with the rHev b 8 epitopes reported from the structure of the complex Fab 2F5/IgE-rHev b 8 (PDB 7SBD). Specific mAbs showing high recognition for diverse profilins may represent a promising tool in the allergy field for diagnosis, immuno-localization studies, and even the development of personalized immunotherapy.Fraxinus excelsior trees in Mexico City. The pollen grains were washed with ethanol-acetone 1:1 (v/v) and centrifuged . The pellet was resuspended in 50 mM sodium carbonate-bicarbonate buffer, pH 8, with 5 mM EDTA and 1 mM PMSF, and the solution was mixed for two hours and centrifuged . Ash profilin was purified by affinity chromatography using a poly-L-proline resin in batch. The protein extract was incubated overnight at 4 °C and washed with equilibration buffer (50 mM sodium carbonate-bicarbonate). Profilin was eluted with 4 M guanidinium chloride, dialyzed against 50 mM Tris-HCl buffer pH 8, and stored at 4 °C overnight. The presence and purity of the proteins were verified by SDS-PAGE 15%. The profilin bands were cut and sent to Arizona State University for sequencing.Inflorescences were collected from E. coli Rosetta (DE3) cells. Cells were grown in Luria broth (LB) medium supplemented with 30 μg/mL kanamycin at 37 °C. Protein expression was induced with 0.5 mM isopropyl-thiol-β-D-1-galactopyranoside when it reached an optical density of 0.6 at 600 nm, and incubation was continued for 6 h at 30 °C. Cells were harvested by centrifugation  and resuspended in a lysis buffer , sonicated  and centrifuged . The supernatant was filtered and loaded onto a 5 mL Ni-NTA affinity column, equilibrated with 50 mM Tris-HCl pH 8, 300 mM NaCl, 20 mM imidazole, and washed with the same buffer. Finally, the protein was purified with anion exchange chromatography with a Mono Q HR 5/5 column in an ÄKTA FPLC , washed with 40 mM NaCl, and eluted with 160 mM NaCl. The Histag was cleaved overnight at 37 °C for 2 h using a recombinant tobacco virus protease (TEV) in 50 mM Tris-HCl pH 8, 0.5 mM EDTA, and 5 mM DTT buffer. Then, it was dialyzed against 100 mM Tris-HCl pH 8.6 and stored at −20 °C. Finally, Fra e 2.2 was loaded in an SDS-PAGE 12%. We determined the molecular mass by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry . We used 10 mg of the protein for the analysis and thaumatin (22 kDa) and hevein (4.7 kDa) as calibration standards. The matrix used was a saturated solution of sinapinic acid in 30% (v/v) aqueous acetonitrile and 0.1% (v/v) trifluoroacetic acid. The results were analyzed using Bruker’s Flex analysis 3.0 TM software.The identified peptides from pollen belonged to isoform Fra e 2.2, reported previously  ThereforArtemisa vulgaris (mugwort) (Uniprot identifier-Q8H2C9), Amb a 8.0101—Ambrosia artemisiifolia (ragweed) (Q2KN24), Phl p 12.0101—Phleum pratense (Timothy grass) (P35079), and Bet v 2.0101—Betula verrucosa (birch) (P25816) were produced using previously described protocols [The recombinant profilin allergens Art v 4.0101—rotocols ,16. Puri2. The culture supernatant was centrifuged (2000× g for 20 min) and filtered through a 0.22 µm membrane. Antibodies 1B4 and 2D10 were purified by affinity chromatography with an Affi-Gel-resin  covalently bound to rZea m 12, previously equilibrated with PBS buffer. The antibody was eluted with 0.2 M Glycine-HCl buffer pH 2.8 and 1 M NaCl buffer, and the collected fractions (800 µL) were received in 200 µL 2 M Tris HCl buffer pH 8. The purity of the antibodies was analyzed by 10% SDS-PAGE. The purified antibodies were dialyzed against PBS, concentrated to 1 mg/mL, and stored at −20 °C.Murine mAbs IgG 1B4 and IgG 2D10 were produced by hybridoma cells, as previously described . BrieflyTaenia solium, and Hev b 7 (patatin from H. brasiliensis) as negative controls. Between steps, wells were washed with 200 µL of 0.05% Tween 20 in PBS. The microplate was blocked with 150 µL, 1% BSA in PBS for two hours at 37 °C. Antibodies 1B4 and 2D10 were incubated at 2 µg/mL, diluted in PBS, and incubated at 37 °C for 2 h. The plate was then washed, and the secondary antibody, a goat anti-mouse IgG  HRP-labeled , was added per well and set for 30 min at 37 °C. ABTS substrate  was added and incubated for 30 min. Plates were read at 405 nm using a Cytation 3 plate reader , and the absorbance of the negative control wells was subtracted from each value. Each condition was assayed in triplicate. The reported absorbance values and the corresponding standard deviation were averaged from three independent experiments for each condition.We performed indirect ELISA experiments by coating microplates with 100 µL of a 10 µg/mL solution of rHev b 8, rZea m 12, rFra e 2, rArt v 4, rAmb a 8, rPhl p 12, and rBet v 2 at 37 °C using PBS, pH 7.4, incubated at 4 °C overnight. Previously, we determined the optimum mAb 2D10 concentration for recognition of rHev b 8, to be 2 µg/mL. We used albumin (BSA), glutathione S-transferase from This study used the rZea m 12 mutants to characterize mAb 1B4 and 2D10 recognition sites by indirect ELISA experiments with rHev b 8 and rZea m 12 as controls. The microplates were coated with 100 µL of a 10 µg/mL solution of rHev b 8, of rZea m 12 E128D single mutant, or of rZea m 12 G98N-E128D double mutant. The following steps were the same as described above. Plates were read at 405 nm using a Cytation 3 plate reader , and the absorbance of the negative control wells was subtracted from each value. Each condition was assayed in triplicate. The reported absorbance value and the corresponding standard deviation was averaged from three independent experiments.We performed indirect ELISA experiments to identify the relevant residues involved in IgE binding with sera of patients allergic to maize and latex profilins. Human sera from controls and profilin-allergic individuals with medical records of allergies to latex and maize were volunteers. We used a group of five individuals with an allergy to rHev b 8 or rZea m 12 . We evaluated the IgE-specificity by ELISA to rZea m 12 and rHev b 8 of the serum of maize- and latex-allergic patients and negative controls.The microplate was coated with 100 µL of a 10 µg/mL solution of rHev b 8, rZea m 12, and rZea m 12 mutants. All the following incubation steps were performed at 4 °C overnight. Between steps, wells were washed with 200 µL of 0.05% Tween 20 in PBS. The microplate was blocked with 150 µL of 1% BSA in PBS. The specific-IgE binding to rHev b 8, rZea m 12, and rZea m 12 mutants was evaluated with a pool of sera from five allergic individuals that tested positive for maize. The latter and the control (two negative sera) were prepared 1:5 in PBS. The microplate was then washed, and the secondary antibody  was added per well and incubated for 2 h at 37 °C. The following steps were the same as described for indirect ELISA. Each condition was assayed in triplicate. The reported absorbance value and the corresponding standard deviation was averaged from three independent experiments.https://www.ebi.ac.uk/Tools/msa/clustalo/ (accessed on 23 November 2022). The Alpha Fold server [The sequences of profilins Amb a 8.0101, Art v 4.0101, Bet v 2.0101, Phl p 12.0101, Zea m 12.0105, Hev b 8.0102, and Fra e 2.2 sequences were aligned in the Clustal Omega server d server  was usedd server .p-value < 0.05 was considered statistically significant [https://www.graphpad.com (accessed on 23 November 2022).The statistical analysis of the ELISAs was performed using the one way-analysis of variance (ANOVA), followed by an all-pairwise multiple comparison procedure (Šídák method). A nificant . All sumTo determine 2D10 and 1B4 mAbs recognition towards the profilins rArt v 4, rAmb a 8, rBet v 2, rPhl p 12, rFra e 2, rZea m 12, and rHev b 8, we performed ELISA experiments A. RemarkTo identify the residues on the two maize profilin mutants (rZea m 12) recognized by mAbs 2D10 and 1B4, we performed an indirect ELISA. We performed indirect ELISA experiments with the rZea m 12 mutants (rZea m 12 E128D and rZea m 12 G98N-E128D) to identify if D128 and N98 are relevant in IgE binding with a pooled sera of profilin allergic patients that cross-reacted with Hev b 8. Notably, the double mutant rZea m 12 G98N-E128D showed higher IgE binding compared to rZea m a 12 and the controls , whereasWe analyzed the profilins’ sequences to understand mAbs 1B4 and 2D10 binding towards plant profilins. We compared the theoretical epitopes of Hev b 8 recognized by 1B4 and 2D10 mAbs, reported by Mares-Mejia et al. 2020  with theTo determine relevant residues that could explain 2D10 recognition, we performed structural alignment analysis with Hev b 8 and the other reported structures for each profilin. To identify charges on the profilins’ surfaces that could be relevant for mAb recognition, we evaluated the electrostatic potential of the different profilins. They are ordered from the highest to the lowest binding with mAb 2D10 in Here, we evaluated the IgG mAbs 1B4 and 2D10 recognition towards several plants profilins, including profilins from trees (rBet v 2.0101 and rFra e 2.2), weeds (rAmb a 8.0101 and rArt v 4.0101), grass (rPhl p 12.0101), and maize (rZea m 12.0105), to identify relevant amino acids involved in mAbs recognition, and compare them with the reported IgE epitopes on rHev b 8.0102 to target residues involved in IgE cross-reactivity that could be blocked with these antibodies.The first relevant result was the different binding patterns of anti-rHev b 8 mAbs 1B4 and 2D10 towards allergenic plant profilins with amino acid sequence identities of 67 to 79%. mAb 2D10 strongly recognized weeds’ profilins (rArt v 4 and rAmb a 8), latex profilin rHev b 8, and to a lesser extent, profilins from trees (rBet v 2 and rFra e 2). Conversely, 1B4 recognized the profilins ordered from the highest to the lowest recognition: rHev b 8, maize (rZea m 12), grass (rPhl p 12), and weed (rAmb a 8) . We alsoThe second and most significant result was that the IgE from pooled sera from latex- and maize-allergic patients showed higher binding with the double mutant G98N-E128D) , compare8D , compAnother significant result was that 2D10 showed increased binding for the mutant rZea m 12 E128D . TherefoFurthermore, the sequence alignment analysis of the plant profilins suggests that the conserved D130 of rArt v 4, rAmb a 8, and rBet v 2 (D128 in Hev b 8) is essential for 2D10 recognition. In line with this, the profilins containing an E130  show less binding . The latMoreover, based on the molecular docking of both mAbs 1B4 and 2D10 of the Fvs-rHev b 8 complexes , we idenArtemisia vulgaris), ragweed (Ambrosia elatior), Timothy grass (Phleum pratense) extracts, and rBet v 2 (Betula verrucosa), which revealed that profilins from weeds and grass share common epitopes with latex, but not with Bet v 2 [Here, we also found that rAmb a 8 was highly recognized by 2D10 (88% relative to binding to rHev b 8) and was recognized by 1B4 (56%). Likewise, rArt v 4 exhibited higher recognition to 2D10 compared to rHev b 8, and rPhl p 12 was recognized (72%) by 1B4. Conversely, two profilins, rBet v 2 and rFra e 2, which were less recognized by 2D10 , might have a low ability to inhibit IgE binding. We must highlight that the mixture of these two mAbs (1B4 and 2D10) blocks the recognition of IgE and IgG4 antibodies from sera of latex-allergic and maize-allergic patients by 90% and 40%, respectively, which revealed that these mAbs have overlapping recognition sites with the specific-profilin antibodies from sera of allergic patients . Additio Bet v 2 . These dTimothy grass profilin (Phl p 12-rs) “tail-to-head”, meaning that the C terminus of Phl p 12 was at its N terminus and the N terminus was at its C terminus [The structural alignment of plant profilins indicates that Amb a 8, Art v 4, Zea m 12, Bet v 2, and Fra e 2 have a similar length in the α-helices 1 and 3 with Hev b 8 , which eterminus . This imterminus . These rThe electrostatic potential analysis indicates that the distribution of negative charge patches on the profilins’ surface at the α-helices 1 and 3 are relevant for the 2D10 recognition. These negative charge patches on profilins are suitable for their interaction with the positive amino acid of the paratope . StructuIn summary, the mAbs binding to different plant profilins and their broad cross-reactivity may represent a promising tool in allergy for both diagnoses ,10,20 anThis study has demonstrated that the mAb 2D10 tightly bound the weed profilins rArt v 4, rAmb a 8, and to a lesser extent rBet v 2, rFra e 2, and rZea m 12, while mAb 1B4 showed higher recognition to rPhl p 12 and rZea m 12, and less binding for rAmb a 8. The rZea m 12 mutant E128D demonstrated that residue D130 in plant profilins is essential for mAb 2D10 recognition. On the contrary, 1B4 did not show any recognition difference with rZea 12 mutants, demonstrating that it does not share the recognition site with 2D10. Indeed, sequence and structural alignment showed that D130 in α-helix 3 of Amb a 8, Art v 4 y Bet v 2 is relevant to 2D10 recognition. In contrast, the profilins containing E130  have less binding. The conformational differences and the distribution of negative patches on the profilins’ surface in α-helices 1 and 3 contribute to 2D10 recognition, which may be relevant to explain their IgE cross-reactivity. Altogether, these findings suggest that the development of humanized mAbs with similar characteristics to mAb 2D10 could have therapeutic implications in personalized therapies.


Codon deoptimization (CD) has been recently used as a possible strategy to derive foot-and-mouth disease (FMD) live-attenuated vaccine (LAV) candidates containing DIVA markers. However, reversion to virulence, or loss of DIVA, from possible recombination with wild-type (WT) strains has yet to be analyzed. An in vitro assay was developed to quantitate the levels of recombination between WT and a prospective A24-P2P3 partially deoptimized LAV candidate. By using two genetically engineered non-infectious RNA templates, we demonstrate that recombination can occur within non-deoptimized viral genomic regions . The sequencing of single plaque recombinants revealed a variety of genome compositions, including full-length WT sequences at the consensus level and deoptimized sequences at the sub-consensus/consensus level within the 3′end of the P3 region. Notably, after further passage, two recombinants that contained deoptimized sequences evolved to WT. Overall, recombinants featuring large stretches of CD or DIVA markers were less fit than WT viruses. Our results indicate that the developed assay is a powerful tool to evaluate the recombination of FMDV genomes in vitro and should contribute to the improved design of FMDV codon deoptimized LAV candidates. Aphthovirus of the Picornaviridae family. Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals, including cows, pigs, sheep, and goats that is characterized by fever, lameness, and the formation of blisters on the toes, tongue, and utters of infected animals ,2. The cpro), P1 capsid coding region , P2 non-structural coding region , and P3 non-structural coding region  . pA. pA24Cru2P3Deopt . pA24-ΔGte of 3D . Modifie6 cells/well in a 6-well tissue culture dish . Following an overnight incubation, cell media was replaced by 500 µL of complete cell culture media. Cellular monolayers were then transfected with 1.7 × 1011 copy number of the indicated RNA  in 15 µL of Lipofectamine 2000 reagent  diluted in 250 μL of Opti-MEM  for 2–3 biological repeats per reaction. Complete media (1.5 mL) was added to the wells after a 3 h incubation at 37 °C in 5% CO2. Plates were incubated at 37 °C in 5% CO2 until frozen at 24 or 48 hpt.Cell-Based Recombination Assay. BHK-21 cells were seeded at 1 × 102, which was followed by the addition of 2 mL of agarose overlay media  and 2× MEM ), supplemented with 1% Anti-Anti and 1% MEM Non-Essential Amino Acids. At 48 hpt, 200 µL of clearly detected isolated plaques were randomly chosen, quickly collected before agar solidification, diluted in 100 µL of PBS++  and frozen. Specific plaques were chosen, which was followed by passing in monolayers of LFBK-αvβ6 in T25 flasks, and they were frozen after detection of CPE (24–48 hpt). Plaque Assay and Plaque Isolation. Virus from thawed cell lysates was titered in duplicate in LFBK-αvβ6 cells. Plaques were visualized by staining cell cultures with crystal violet, which was followed by quantitation at 48 hpt. Plaque isolation was performed for each biological repeat of the ΔP1-P2P3Deopt × ΔGDD recombinant product as previously described . BrieflyViral RNA Extraction, Sequencing, and NGS analysis. FMDV full-length genome sequencing was performed as previously described . In briet test; p values of <0.05 were considered significant.Statistical Analysis. Data handling, analysis, and graphic representations were performed using Prism 9 , Microsoft Excel  and RStudio 4.1.2 (RStudio Team (2020). RStudio: Integrated Development for R. RStudio, PBC, Boston, MA, USA). Statistical significance was determined using one-tailed Student’s Picornaviridae family, poliovirus and senecavirus A, was implemented for FMDV [7 plaque-forming units (PFU)/mL ± 1.3 × 106) and 48 hpt (7.0 × 106 PFU/mL ± 7.5 × 105) and, as expected, single transfections of either non-infectious ΔP1 and ΔGDD RNAs failed to produce infectious particles (4 ± 5.1 × 103 PFU/mL) and 48 hpt (5.5 × 105 ± 2.1 × 105 PFU/mL). Viral plaque morphologies were similar for WT and ΔP1 × ΔGDD recombinants examined at 24 hpt; however, by 48 h, the WT plaques were larger than those observed in the recombinants, confirming adequate fitness for viruses derived from the WT template  and at 48 hpt (4.7 × 106 ± 1.3 × 105 PFU/mL). No infectious particles were generated when the non-infectious RNA templates  were transfected alone (p = 0.26) were detected between the number of ΔP1 × ΔGDD (1.3 × 103 ± 5.4 × 102 PFU/mL) and ΔP1-P2P3Deopt × ΔGDD (7.2 × 102 ± 2.9×102 PFU/mL) infectious recombinants generated at 24 hpt (5 ± 1.2 × 105 PFU/mL) and ΔP1-P2P3Deopt × ΔGDD (3.3 × 104 ± 1.3 × 104 PFU/mL) (p = 0.03). At 48 hpt, plaque morphologies were again similar between the ΔP1 × ΔGDD recombinants and ΔP1-P2P3Deopt × ΔGDD recombinants in both BHK-21 and LFBK-αvβ6 cells; however, WT had larger plaques than either recombinant. These results indicated that in our system, the presence of deoptimized sequences in donor RNA P2 and P3 regions fails to abolish recombination when examined at 24 or 48 hpt.Recombination efficiency of ΔP1-P2P3Deopt and ΔGDD. Previously, it was demonstrated that codon deoptimization of the conserved non-structural coding P2 and P3 regions of FMDV A24Cru gave rise to attenuated viruses in cell culture, laboratory animal models, and natural hosts including swine  and catted alone B. Unexpet 24 hpt B. Additit 24 hpt C and in t 24 hpt D. IncreaNGS analysis of recombinants. As detailed in Plaques isolated from the 48 hpt transfection did not contain any deoptimized sequence in the 2AB or 2C-3A region (data not shown). Like the plaques isolated from samples acquired at 24 hpt, multiple sites of obvious recombination were recorded in the 3CD region and, within the 3′ end of the indicated area, plaques ES08, ES10, ES11, ES14, ES16, ES17, and ES20 were composed of a deoptimized sequence at the consensus level . In contTo determine the stability of the deoptimized sequences, plaques ES08, ES10, ES11, ES14, ES15, ES16, ES17, and ES20 isolated from the 48 hpt infection were passed in cell culture. Plaques ES01 and ES02 were also included as a control. Total cell culture supernatants were collected, and viral RNA was sequenced and analyzed as detailed above. As expected, after passage, samples ES01 and ES02 remained almost exclusively WT . InteresThe work presented herein demonstrates that the in vitro recombination assay previously developed for poliovirus and senecavirus A can be adapted to quantify and evaluate recombination events for FMDV ,44,45. The co-transfection of ΔP1-P2P3Deopt × ΔGDD produced approximately the same number of viable particles as co-transfection of ΔP1 × ΔGDD RNAs at 24 hpt, indicating that recombination could still occur despite the presence of CD sequences. Furthermore, our data suggest that even if recombination occurred between a WT strain and the A24-P2P3Deopt LAV candidate in vitro, the resulting recombinant would not display a gain of fitness. Illumina sequencing of the ΔP1-P2P3Deopt × ΔGDD recombinants revealed that at 24 or 48 hpt, most recombinants were fully composed of WT sequences at the consensus level and only very few recombinants contained deoptimized sequences at the consensus level. Furthermore, even at the sub-consensus level, recombinants were much more likely to contain deoptimized sequences in the 3CD coding region, as only a few recombinants isolated at 24 hpt contained deoptimized sequences in the 2AB and 2C-3A coding regions . Thus, oDue to the molecular biology of FMDV, certain limitations were inherent to the study. In the recoding design of the original A24-P2P3deopt virus, codon deoptimization of FMDV P2 and P3 was excluded from regions that were predicted to form highly conserved secondary RNA structures. These regions have been confirmed to be critical for viral genome replication, irrespective of FMDV serotype . Within Alternatively, instead of the formation of recombinants with full-length genomes, the generation of infectious particles could be explained by the presence of defective interfering (DI) particles, which is a phenomena that has been previously described for FMDV . NoteworIn our assay, we cannot assume that all infectious particles titered at 24 or 48 hpt arose from a unique transfection event. Given the fast replication kinetics of FMDV, it is possible that infectious particles generated early post-transfection underwent multiple rounds of replication, thus inflating the total observed number of viable viruses. While plaque sequencing results indicate that all ΔP1-P2P3Deopt × ΔGDD RNA recombinants contained unique genome sequences, i.e., different breakpoints, a much larger sampling size would be needed to definitively support this hypothesis. An additional complication is that multiple viruses can contribute to a single viral plaque formation. This could result in the observation of multiple sub-consensus genomes within the purified plaques . Lastly,Additional time points and NGS that produces long reads could further refine our assay. As demonstrated in poliovirus, when imprecise recombination occurs, duplications within the genome are generated. Additional recombination events can later refine and correct the introduced errors, thus generating fitter viruses . For FMDAdditionally, in its current iteration, our assay could only quantify and sequence recombination events that should have resulted in viable virus. However, based on the reported frequency of recombination during replication of poliovirus , it is lIt is possible that the recently described RNA secondary structures within 3D contributed to the higher levels of recombination in the non-deoptimized 3D region . Indeed,Although informative, care must be taken when interpreting the results from the assay presented in this manuscript and linking it to what could occur during a co-infection of two strains of FMDV in a host. Deoptimization of the P2 and P3-coding regions resulted in attenuated phenotypes in at least two FMDV susceptible hosts of importance, swine, and cattle  and unpuTaken together, our data indicate that: (1) an in vitro assay to measure recombination for FMDV genome sequences was successfully established; (2) the majority of observed recombinants were generated by template switching that presumably occurred positionally very early during the reading of the template strand, either within the 185 nt of non-deoptimized sequence or within the first 750 nt of the 3507 nt deoptimized region in the negative strand; (3) in our system, codon deoptimization did not completely prevent recombination; (4) however, recombinants containing large regions of deoptimized regions at the consensus level were not observed and presumably displayed lower fitness than WT. Thus, our results suggest that deoptimization of the P2 and P3 coding regions of FMDV is an effective strategy to cause viral attenuation and—at least in our in vitro system—prevent the passing of DIVA markers between intra-serotype genomes. This information may be considered in the development of subsequent generations of LAVs for FMDV.


Ibrutinib, a first-in-class inhibitor of Bruton’s tyrosine kinase, is approved for the treatment of various B-cell malignancies and chronic graft-versus-host disease. Based on encouraging preclinical data, safety and efficacy of ibrutinib combined with companion drugs for advanced renal cell carcinoma (RCC), gastric/gastroesophageal junctional adenocarcinoma (GC), and colorectal adenocarcinoma (CRC) were evaluated.Ibrutinib 560 mg or 840 mg once daily was administered with standard doses of everolimus for RCC, docetaxel for GC, and cetuximab for CRC. Endpoints included determination of the recommended phase 2 dose (RP2D) of ibrutinib in phase 1b and efficacy  in phase 2.A total of 39 (RCC), 46 (GC), and 50 (RCC) patients were enrolled and received the RP2D. Safety profiles were consistent with the individual agents used in the study. Confirmed ORRs were 3% (RCC), 21% (GC), and 19% (CRC). Median (90% CI) PFS was 5.6 (3.9–7.5) months in RCC, 4.0 (2.7–4.2) months in GC, and 5.4 (4.1–5.8) months in CRC.Clinically meaningful increases in efficacy were not observed compared to historical controls; however, the data may warrant further evaluation of ibrutinib combinations in other solid tumours.ClinicalTrials.gov, NCT02599324.The online version contains supplementary material available at 10.1186/s12885-023-11539-1. By bindpression . In addipression  and incrpression . TherefoThere remains an unmet need for patients with RCC, gastric adenocarcinoma (GC), and colorectal adenocarcinoma (CRC) who have progressed on previous vascular endothelial growth factor (VEGF)-targeted and mammalian target of rapamycin (mTOR) inhibitors . In RCC,On this basis, we conducted a phase 1b/2 clinical study to explore the safety, tolerability, and preliminary activity of ibrutinib combination therapy in previously treated patients with advanced solid tumours, including RCC, GC, CRC, and urothelial carcinoma (UC) who had failed multiple lines of therapy. Here we report results from the RCC, GC, and CRC cohorts (UC cohorts to be reported separately).This was an open-label phase 1b/2 multicenter study  conducted between December 2015 and March 2020, to determine the recommended phase 2 dose (RP2D) of ibrutinib combined with everolimus in RCC, docetaxel in GC, and cetuximab in CRC for previously treated patients. The data cutoff date for this analysis was 19 April 2021.The phase 1b study followed a 3 + 3 + 3 design in each cohort to evaluate dose-limiting toxicities (DLTs) and determine the RP2D. The DLT observation period was 21 days following the initiation of combination therapy at the start of Cycle 1. A DLT was defined as any grade 3 or higher non-hematologic or grade 4 hematologic adverse event (AE) possibly related to either ibrutinib and/or drug combination that occurred during the DLT-observation period. DLTs were assessed in the first three evaluable patients at each dose level by a safety review committee and expanded to 6 or 9 patients if 1 of 3 or 2 of 6 patients experienced a DLT, respectively. The subsequent phase 2 portion of the study utilised a Simon’s 2-stage design in the GC and CRC cohorts. All patients were ≥ 18 years old with adequate hematologic, hepatic, and renal function and an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1 . Patients had advanced  disease with histologically confirmed clear cell RCC, gastric or gastro-esophageal junctional adenocarcinoma or K-Ras and N-Ras wildtype (EGFR-expressing) CRC, with one or more measurable lesions per Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 guidelines. Patients were assessed by investigator to be a suitable candidate for the treatment partner  and ibrutinib as per their tumour type. To be eligible for the RCC cohort, patients had received between one and four prior lines of therapy in the advanced setting, including a VEGF-tyrosine kinase inhibitor. For the GC cohort, patients had received between one and three prior lines of therapy in the advanced setting, including a fluoropyrimidine regimen. For the CRC cohort, patients had received at least two and no more than four prior regimens in the advanced setting, which must have included both an irinotecan and oxaliplatin-based regimen unless unable to tolerate irinotecan chemotherapy. Key exclusion criteria included prior anti-cancer therapy within 28 days of the first dose of study drug (including neoadjuvant or adjuvant therapy if disease progression occurred at ≥ 12 months of treatment completion) and prior treatment with everolimus or temsirolimus (RCC cohort), any taxane (GC cohort), or cetuximab or panitumumab (CRC cohort).All patients provided written informed consent for participation in this study as approved by the Institutional Review Board/Research Ethics Board/Independent Ethics Committee before any study-specific screening procedures were performed.2  intravenously (IV) every 3 weeks (Q3W) (GC cohort), or cetuximab 400 mg/m2 IV , then 250 mg/m2 weekly (CRC cohort) in 21-day cycles. Ibrutinib and partner agents were administered until unacceptable toxicity or disease progression at the prescribed doses in each cohort. In dose level 1 (ibrutinib 560 mg), if ≤ 22% of evaluable patients experienced a DLT during the first treatment cycle, patients were enrolled in dose level 2, in which the dose of ibrutinib was increased to 840 mg once daily in combination with the companion drug. If ≥ 33% of evaluable patients experienced a DLT at the 560 mg or 840 mg dose of ibrutinib, the dose was de-escalated to 420 mg or 560 mg once daily, respectively. Dose adjustment guidelines are described in In phase 1b, the starting dose level for ibrutinib was 560 mg orally once daily. Ibrutinib 560 mg was combined with everolimus 10 mg orally once daily (RCC cohort), docetaxel 60‒75 mg/mFollowing the determination of the RP2D by a Dose Level Review Committee (DLRC), additional patients were enrolled and treated in phase 2 at the RP2D to further evaluate the efficacy of the regimen for each specified tumour type as prespecified.The primary objectives of phase 1b were to determine the RP2D for ibrutinib in each cohort: in combination with everolimus in RCC, docetaxel in GC, and cetuximab in CRC. Secondary objectives in phase 1b of each cohort included evaluation of the preliminary safety and tolerability, overall response rate (ORR) per RECIST v1.1, disease control rate , partial response (PR), or stable disease (SD) ≥ 6 weeks), duration of response (DOR), and pharmacokinetics of the combination regimens.The primary objectives of phase 2 were to assess progression-free survival (PFS) in the RCC cohort and ORR in the GC and CRC cohorts. Secondary objectives in phase 2 included PFS in GC and CRC cohorts, ORR in RCC cohort, and DCR, DOR, overall survival (OS), safety, and tolerability in each cohort.Tumour response was assessed using computed tomography (CT) or magnetic resonance imaging (in the case of CT contraindication). Imaging was performed at baseline and every 6 weeks thereafter, per the investigator using RECIST v1.1 guidelines, including confirmation of complete and PRs at least 28 days after the criteria for response were first met . SD and Plasma samples were collected for all patients for pharmacokinetic (PK) determination of ibrutinib in all cohorts and docetaxel in the GC cohort.For phase 1b, data were summarised by dose level for each cohort separately. For Phase 2, efficacy and safety data were summarised by RP2D dose level of ibrutinib for each cohort. The safety population included all patients who received at least one dose of any study drug. The DLT-evaluable population was defined as patients from phase 1b who completed ≥ 21 days of treatment or discontinued treatment before 21 days due to a DLT event. Efficacy analyses were performed in the efficacy-evaluable population, defined as eligible patients  who received at least one dose of ibrutinib at the RP2D in combination with at least one dose of the companion drug and had at least 1 post-baseline tumour assessment, regardless of treatment duration, or had died prior to the first adequate post-baseline assessment (RCC cohort) or had measurable disease and at least 1 post-baseline tumour assessment (GC and CRC cohorts). When analysing efficacy at the RP2D, the data from patients in phase 1 treated with the RP2D phase 2 were merged prior to analysis.For the RCC cohort, a single interim analysis for PFS futility was conducted when the twenty-fifth patient of 55 total dosed at the RP2D level had completed 6 months of follow-up. The study was designed to detect a 75% increase in median PFS to 8.6 months for ibrutinib plus everolimus with a sample size of 55 efficacy-evaluable patients. Phase 2 of the GC and CRC cohorts followed a Simon’s 2-stage design for patients treated at the RP2D. For the GC cohort, 39 patients were to be enrolled in two stages; if ≥ 2 of 21 patients had a tumour response (PR or CR) in stage 1, whereupon an additional 18 patients were enrolled in stage 2. Treatment was deemed acceptable for further clinical development if ≥ 6 patients responded. The Simon’s 2-stage design provided 80% power to test the historical ORR of 7% against the target ORR of 20%. In the CRC cohort, 40 patients were to be enrolled in two stages; if ≥ 3 of 22 patients were responders in stage 1, then an additional 18 patients were enrolled in stage 2. The Simon’s 2-stage design provided 80% power to test the historical ORR of 10% against the target ORR of 25%.n = 3) or 840 mg ibrutinib (n = 7); median duration of ibrutinib exposure was 2.9 months and median everolimus exposure was 2.6 months. One patient who received 840 mg ibrutinib plus everolimus was DLT-inevaluable due to dose interruption because of a non-DLT AE. Of the nine DLT-evaluable patients in phase 1b, one at dose level 2 (840 mg ibrutinib plus 10 mg everolimus) experienced a DLT consisting of diarrhea and nausea (both grade 3) for 18 days; thus the determined RP2D was 840 mg ibrutinib plus 10 mg everolimus both orally and once daily. Best response achieved was SD in 70% (n = 7/10).A total of 10 patients were enrolled in the RCC cohort in phase 1b; patients received everolimus with either 560 mg ibrutinib  had received ≥ 3 prior lines of therapy and all had at least one metastatic site of disease (Table Median (range) time on study in phase 1b/2 was 22.5 (0.5–37.4) months. Median (range) duration of ibrutinib exposure at the RP2D was 2.8 (0.1–27.9) months. The median average daily dose of ibrutinib was 641.7 mg/day and the median relative dose intensity was 76%. The most frequent reasons for discontinuation of ibrutinib were progressive disease  and AEs (31%). Similarly, median (range) duration of everolimus exposure at the RP2D was 3.1 (0.1‒27.9) months. The median average daily dose of everolimus was 7.7 mg/day and the median relative dose intensity was 77%; 56% of patients discontinued everolimus due to PD and 28% due to an AE , median (90% CI) PFS was 5.6 (3.9–7.5) months. At 6 and 12 months, PFS rates were 44% and 15%, respectively  of patients and PD in 17% (n = 6/36) of patients. DCR (90% CI) was 81% (n = 29/36) (66.6%–90.5%). Response duration of the patient with PR was 3.1 months. Median (90% CI) OS was 21.0 (13.1–25.3) months , with grade ≥ 3 TEAEs occurring in 74% (n = 29/39)  due to TEAEs . The most common TEAEs of any grade included stomatitis , diarrhea and epistaxis , and anemia . The most common grade ≥ 3 events were anemia , stomatitis , hyperglycemia , diarrhea , and hypertension   had a confirmed PR, 11 (61%) had SD, and four (22%) had PD. The DCR (90% CI) was 78% (n = 14/18) (56.1%–92.0%).Twenty-one patients were enrolled in the GC cohort in phase 1b; all patients received 560 mg ibrutinib with docetaxel; median duration of ibrutinib exposure was 2.7 months and median docetaxel exposure was 1.4 months. Fourteen patients were DLT-inevaluable due to dose interruptions. Seven patients were DLT-evaluable. Two of these seven had DLTs (29%); one patient experienced grade 4 leukopenia for 3 days after receiving ibrutinib 560 mg QD plus docetaxel 75 mg/m2 docetaxel and three received starting doses between 60–75 mg/m2. Twenty-two patients (48%) had docetaxel dose reductions due to an AE during the study; 57% of patients discontinued docetaxel due to PD and 22% due to AEs  had received at least two prior lines of therapy and 15 (33%) had > 2 metastatic sites of disease , median (90% CI) PFS was 4.0 (2.7–4.2) months. At 6 and 12 months, the PFS (90% CI) rates were 26% (15.5%–38.5%) and 12% (5.1%–22.8%), respectively , amounting to a DCR (90% CI) of 74% (n = 29/39) (60.4%–85.4%). PD was best response in 26% (n = 10/39) of patients. Median (90% CI) DOR was 5.5 (3.0–10.8) months and median (90% CI) OS 7.3 (5.5–9.6) months, respectively   due to TEAEs: adenocarcinoma gastric, hematuria, and intestinal obstruction. The most common TEAEs of any grade included anemia , diarrhea , and neutropenia . The most common grade ≥ 3 events were neutropenia , anemia , febrile neutropenia , neutrophil count decreased , neutropenic sepsis, pneumonia, and white blood cell count decreased   achieved a PR and 12 patients (67%) had SD amounting to a DCR (90% CI) of 78% (n = 14/18) .Twenty patients were enrolled in the CRC cohort in phase 1b; 8 and 12 patients, respectively, received 560 mg and 840 mg ibrutinib with cetuximab. Median duration of ibrutinib exposure was 2.7 months and median cetuximab exposure was 2.3 months. Eleven patients were DLT-inevaluable, 10 due to dose interruption because of non-DLT AEs and one due to dosing non-compliance. Among the nine DLT-evaluable patients in this cohort, there were no reported DLTs. Therefore, the RP2D used in phase 2 was 840 mg ibrutinib orally daily plus 400 mg/mA total of 50 patients were enrolled in the CRC cohort in phase 1b/2. All patients had received at least two prior lines of therapy and 27 (54%) had > 2 metastatic sites of disease , median (90% CI) PFS was 5.4 (4.1–5.8) months. At 6 and 12 months, the PFS (90% CI) rates were 35% (23.7%–46.4%) and 11% (4.8%–19.8%), respectively  (71.4%–91.2%). Median (90% CI) DOR was 11.1 (4.2–12.5) months and median (90% CI) OS was 15.0 (10.5–17.2) months  and 10.6 months for those with right-sided tumours (n = 14).Among patients who received the RP2D in the efficacy-evaluable population (as 5.4 4.–5.8 montn = 34/50) , stomatitis , dry skin , and diarrhea . Common grade ≥ 3 TEAEs included dermatitis acneiform  and stomatitis   of 2.03 to 3.89 h and mean apparent terminal half-life (t1/2) ranged from 5 to 6 h. Mean ibrutinib steady-state Cmax and AUC0-24 h for RCC and CRC cohorts were 351 ng/mL and 2568 ng∙h/mL and 371 ng/mL and 2807 ng∙h/mL, respectively, at the RP2D dose of 840 mg ibrutinib in combination with everolimus or cetuximab. Ibrutinib exposures at the RP2D dose of 840 mg in patients with RCC and CRC were comparable regardless of co-medications (everolimus or cetuximab). Mean ibrutinib steady-state Cmax and AUC0-24 h for GC cohort were 164 ng/mL and 1253 ng∙h/mL, respectively at the RP2D of 560 mg. In the GC cohort, docetaxel Cmax ranged from 467 to 38,000 ng/mL following administration of 53.9 to 75.8 mg/m2 docetaxel with ibrutinib at the RP2D of 560 mg. The mean concentration (% coefficient of variation) of dose normalised docetaxel Cmax following exclusion of the high outlier concentration observed in one patient was 34.6 ng/mL (60%).Ibrutinib was rapidly absorbed with a median time to maximum concentration  than as recommended by consensus guidelines at the time of study initiation, were feasible but did not result in a meaningful increase in efficacy when compared to historical controls in each disease setting.This multicenter, open-label, phase 1b/2 study of ibrutinib combination therapy in patients with RCC (ibrutinib plus everolimus), GC (ibrutinib plus docetaxel), or CRC (ibrutinib plus cetuximab) demonstrated acceptable safety in patients with advanced tumours. Overall, results presented here suggest that administration of ibrutinib 840 mg orally with everolimus 10 mg in RCC, ibrutinib 560 mg with docetaxel 60 to 75 mg/mIn metastatic RCC, other combinations, such as sorafenib plus everolimus, have been tested. Nevertheless, excessive gastrointestinal and skin toxicity limited the translation of this combination to the clinical trials. As single agents, everolimus and sorafenib reported PFS of 4.9 and 5.5 months –22, respIn both the GC and CRC cohorts, 14 and 11 patients were DLT-inevaluable, suggesting a sick population overall at study entry. Nonetheless, in patients with refractory GC, ibrutinib plus docetaxel was associated with a higher ORR of (18%) relative to single-agent docetaxel (7%) but a similar 6-month PFS (29% and 26%)  and loweThe safety profiles for each of the drug combinations were generally consistent with the known safety profiles for the individual agents used in the study, suggesting no significant interaction between ibrutinib and partner agents and were as expected for patients with these advanced tumour types considering prior therapies and treatment duration. TEAEs related to companion drugs in this study were consistent with those reported previously in everolimus in RCC,  docetaxeThe heavily pretreated patient populations in this study may not have been the optimal contexts for evaluation of novel ibrutinib combination regimens, as evidenced by the modest efficacy observed relative to historical controls. However, the generally manageable safety profile seen across all three cohorts and the high levels of relative dose delivery of both ibrutinib and partner agents support the feasibility of the regimens studied, thereby warranting consideration of further evaluation in earlier stage settings and/or with different partner agents.Additional file 1: Supplementary Methods. Follow-up. Supplementary Methods. Maximum tolerated dose. Supplementary Methods. Dose adjustment guidelines. Supplementary Table S1. Prior therapies


Regulation of gene expression in response to various biological processes, including extracellular stimulation and environmental adaptation requires nascent RNA synthesis and translation. Analysis of the coordinated regulation of dynamic RNA synthesis and translation is required to determine functional protein production. However, reliable methods for the simultaneous measurement of nascent RNA synthesis and translation at the gene level are limited. Here, we developed a novel method for the simultaneous assessment of nascent RNA synthesis and translation by combining 4-thiouridine (4sU) metabolic RNA labeling and translating ribosome affinity purification (TRAP) using a monoclonal antibody against evolutionarily conserved ribosomal P-stalk proteins. The P-stalk-mediated TRAP (P-TRAP) technique recovered endogenous translating ribosomes, allowing easy translatome analysis of various eukaryotes. We validated this method in mammalian cells by demonstrating that acute unfolded protein response (UPR) in the endoplasmic reticulum (ER) induces dynamic reprogramming of nascent RNA synthesis and translation. Our nascent P-TRAP (nP-TRAP) method may serve as a simple and powerful tool for analyzing the coordinated regulation of transcription and translation of individual genes in various eukaryotes.  The regulation of gene expression plays a pivotal role in diverse biological and physiological processes such as cell differentiation, development, environmental responses, and immune responses. One determinant of gene expression is the amount of mature RNA, which reflects the balance between RNA synthesis and degradation . To undeThe final output of gene expression, which indicates the expression level of proteins, is significantly influenced by RNA translation. Several studies have reported that the primary set of RNA transcripts obtained by standard RNA-seq experiments may have a low quantitative correlation with the proteome ,19–21. TAlthough the regulation of RNA synthesis, degradation, and translation are distinct steps, these control interplay in gene expression ,34. The In this study, we developed a simple method for the simultaneous measurement of nascent RNA synthesis and translation at the gene level by combining the TRAP technique with 4sU metabolic RNA labeling. Since TRAP captures full-length RNAs bound to the ribosomes, it is compatible with QuantSeq, which analyzes the inherently uridine-rich 3′ UTRs of polyadenylated RNAs, facilitating the robust quantification of T>C conversions. In contrast to the conventional TRAP technique that requires genetically engineered cells, we developed an endogenous ribosome affinity purification method using a monoclonal antibody against an evolutionarily conserved ribosomal P-stalk protein. The P-stalk mediated translational ribosome affinity purification (P-TRAP) technique enables convenient access to the translatome in diverse eukaryotes, including mammals, fish, insects and nematodes, without genetic manipulations. We applied a combination of conventional TRAP or the newly developed P-TRAP with 4sU metabolic RNA labeling and defined the nascent RNA synthesis and translation at the level of individual genes in the acute unfolded protein response (UPR) within the endoplasmic reticulum (ER). This simple and versatile translatome analysis technique would enhance our understanding of the complex regulation of gene expression in various eukaryotes.A plasmid for the stable expression of enhanced green fluorescent protein (EGFP) fused to the N-terminus of the ribosomal protein L10a (EGFP-L10a) in mammalian cells was constructed using standard procedures. Briefly, a synthetic DNA fragment of EGFP-L10a was cloned into an expression vector pcDNA5/FRT/TO . Although the human ribosomal protein L10a is referred to as uL1 in standard nomenclature , we usedA homemade anti-ribosomal protein P0 monoclonal antibody (clone 9D5) was kindly provided by Dr. Toshio Uchiumi and Dr. Hiroe Sato (Niigata University) and is now available from MBL . The isotype control antibody for 9D5 (mouse IgG2a κ) was purchased from Proteintech (65208-1-IG). Antibodies against ribosomal proteins uL3 , L10a , uS2 , uS3 , uS15  and PABP4 , CBP80  and GAPDH  were purchased commercially. A homemade anti-eIF4A3 polyclonal antibody was prepared as described previously .2 and 0.33 mM MgSO4) using standard methods , supplemented with 10% fetal bovine serum and 1× antibiotic-antimycotic  at 27°C. Preparation of synchronized KH1668 smg-2 (yb979) worms were prepared as previously described  were cultured at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. pcDNA5/FRT/TO_EGFP-L10a was co-transfected with pOG44  into Flp-In T-REx 293 cells to generate a stable cell line inducibly expressing EGFP-L10a and selected in media supplemented with 50 μg/ml hygromycin according to the manufacturer's instructions. EGFP-L10a expression was induced for 24 h by the addition of doxycycline at 3 μg/ml. Zebrafish were grown at 28°C in E3 embryo medium  Triton X-100, 0.5 mM DTT, protease inhibitor cocktail, phosphatase inhibitor cocktail, 100 μg/ml cycloheximide). The cell lysates were clarified by centrifugation at 20,000 × g at 4°C for 10 min. The supernatant was mixed with 0.1 μg/μl RNase A or 1 unit/μl RNasin Plus and incubated with 1 μg of anti-ribosomal protein P0 antibody   or isotype control antibody  at 4°C for 60 min. 7 μl of Dynabeads Protein G  was added to the mixture and incubated at 4°C for 40 min. The beads were washed three times with 1 ml of wash buffer  Tween 20) and directly mixed with 30 μl of SDS-PAGE sample buffer  SDS, 50 mM DTT), heated at 95°C for 5 min, and analyzed by western blotting. All proteins were detected using an ECL select western blotting detection reagent  or ImmunoStar LD  and imaged using an ImageQuant LAS 4000 mini (cytiva).HEK293 cells were seeded in a 6-well plate at 2.0 × 10Drosophila S2 cells, 1.5 × 106 cells were harvested by centrifugation at 600 × g for 5 min. The cell pellets were lysed in 400 μl lysis buffer  NP-40, 0.5 mM DTT, protease inhibitor cocktail, phosphatase inhibitor cocktail, 100 μg/ml cycloheximide). The lysates were clarified by centrifugation at 20,000 × g for 10 min at 4°C. The supernatant was divided into two fresh microcentrifuge tubes and incubated with 1 μg of 9D5 antibody or isotype control IgG at 4°C for 60 min. The subsequent procedures were performed as described above.For immunoprecipitation of ribosomes from 2, 1% (v/v) NP-40, 0.5 mM DTT, protease inhibitor cocktail, phosphatase inhibitor cocktail, 100 μg/ml cycloheximide, 1 mg/ml heparin, 0.2 unit/μl RNasin) and homogenized by douncing on ice. The lysates were then clarified by centrifugation at 10,000 × g at 4°C for 10 min. The supernatant was divided into two fresh microcentrifuge tubes and incubated with 1 μg of 9D5 antibody or isotype control IgG at 4°C for 60 min. Subsequent procedures were performed as described.For immunoprecipitation of the ribosome from zebrafish, 30 whole zebrafish embryos (4 days after fertilization) were resuspended in 1 ml lysis buffer  NP-40, 0.5 mM DTT, protease inhibitor cocktail, phosphatase inhibitor cocktail, 100 μg/ml cycloheximide). After homogenization by sonication, the lysates were clarified by centrifugation at 20,000 × g at 4°C for 10 min. The supernatant was divided into two fresh microcentrifuge tubes and incubated with 1 μg of 9D5 antibody or isotype control IgG at 4°C for 60 min. The subsequent procedures were performed as described above.For immunoprecipitation of the ribosomes from the worms, synchronized L1 larvae were harvested by centrifugation at 600 x 5 cells per dish. The expression of EGFP-L10a was induced by 3 μg/ml of doxycycline for 24 h. The next day, the medium was quickly replaced with a freshly prepared medium containing 200 μM 4sU with either 0.02% (v/v) DMSO or 1 μM thapsigargin. The cells were incubated at 37°C for 3 h and lysed in 1 ml lysis buffer  Triton X-100, 0.5 mM DTT, protease inhibitor cocktail, phosphatase inhibitor cocktail, 100 μg/ml cycloheximide). Whole cell lysates were clarified by centrifugation at 10,000 x g at 4°C for 10 min, and the supernatant supplemented with 1 unit/μl RNasin Plus was used for further procedure. For cytosolic RNA preparation, 200 μl of the supernatant was mixed with 500 μl of ISOGEN II  supplemented with 1 mM DTT and the prepared sample solution was stored at –20°C. For P-TRAP, 400 μl of the supernatant were incubated with 4 μg of 9D5 antibody at 4°C for 60 min. Afterward, 28 μl of Dynabeads Protein G was added to the mixture and incubated at 4°C for 40 min. After the beads were washed three times with wash buffer  Tween 20), the beads were directly mixed with 500 μl of ISOGEN II supplemented with 1 mM DTT and the sample solution was stored at –20°C. For EGFP-L10a TRAP, 400 μl of the supernatant were incubated with 25 μl of GFP-Trap® Magnetic Particles M-270  at 4°C for 60 min. After the beads were washed three times with wash buffer, the beads were directly mixed with 500 μl of ISOGEN II supplemented with 1 mM DTT, and the sample solution was stored at –20°C. The manufacturer's instructions were followed for RNA extraction using ISOGEN II, and the RNA samples were assessed for quality and quantity on MultiNA capillary electrophoresis instrument (Shimadzu).The HEK293 cell line used for the inducible expression of EGFP-L10a was seeded in a 10 cm dish at 5.0 × 104 buffer pH 8.0, 50% (v/v) DMSO, 10 mM iodoacetamide) at 50°C for 15 min. The reaction was quenched by adding 1 μl of 1 M DTT, followed by ethanol precipitation. For each sample, 500 ng of cytosolic RNA or immunoprecipitated RNA was used as an input for QuantSeq 3′ mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen). The cDNA library was prepared according to the manufacturer's instructions. Libraries were assessed for quality using a MultiNA capillary electrophoresis instrument (Shimadzu), multiplexed to equimolar concentrations, and sequenced using the HiSeq 2500 system (Illumina) in PE-150 mode with a yield of 20–30 million reads per sample.Cytosolic and immunoprecipitated RNAs were processed according to the standard SLAMseq protocol described previously . In brietcount file (output from SLAM-DUNK) as input. To analyze the TcReadCount column with the DESeq2 and deltaTE method, we calculated global normalization factors using the ReadCount column. The proportion of nascent transcripts to the total transcripts was estimated by normalizing the number of T>C reads to the total reads. Gene ontology analysis was performed by over-representation analysis using a biological process database   .SLAMseq distinguishes 4sU-labeled nascent transcripts from pre-existing RNA prior to labeling by bioinformatics detection of specific T>C conversions at the sites of 4sU incorporation. The combination of SLAMseq with QuantSeq allows the robust quantification of T>C conversions . As Quan2 complex . Me. Me2 com as well . BesidesWe tested whether translating ribosomes could be immunoprecipitated from cultured human cell extracts using a monoclonal antibody (9D5) that recognizes the C-terminal IDRs of P0, P1 and P2 . The isoA C-terminal 22 amino acids sequence in the IDRs of the P-stalk recognized by the 9D5 antibody is well-conserved in diverse eukaryotes from yeast to humans Figure . TherefoWe combined SLAMseq/QuantSeq with TRAP to analyze nascent RNAs and their translation at the level of individual genes in the unfolded protein response (UPR). In the UPR, transcription and translation are dynamically reprogrammed to reduce unfolded and misfolded proteins in the ER and restore protein homeostasis ,51. To cHSPA5, DDIT3, PDIA4, DNAJC2 and HERPUD1) was significantly increased in P-TRAP-seq analysis similar to cytosolic RNA-seq and conventional L10a-TRAP-seq  that showed the highest ratio of increased RNA levels after the addition of 4sU and thapsigargin in the cytosolic RNA-seq. In these genes, most reads increased after the addition of 4sU and thapsigargin could be considered to be derived from nascent RNA. By calculating the proportion of reads detected as T>C reads out of all these reads, we estimated the sensitivity of detecting T>C reads under the present experimental conditions and determined that approximately 82–86% of the reads derived from nascent RNAs were detected as T>C reads  at serine 51, leading to global repression of translation initiation, and the whole protein synthesis rate is transiently reduced to 15–20% ,55. The We next analyzed gene expression reprogramming for the essential branches of the UPR, the ATF4 and CHOP pathway (ATF4/CHOP target) and the ATF6 and XBP1 pathway (ATF6/XBP1 target). Consistent with previous reports ,57, the In the acute phase of the UPR, the total amount of proteins synthesized on the ER membrane is reduced to decrease protein loading to the ER ,51. The HSPA5, HERPUD1, and PDIA4), and enrichment analysis showed significant enrichment of genes responsible for ER stress tolerance . TRAP-seq relies on a simple immunoprecipitation technique to extract ribosomes, including monosomes and polysomes; therefore, it cannot selectively fractionate actively translating polysomes, similar to the polysome profiling technique . Since tThere are two major advantages to the immunoprecipitation of endogenous ribosomes with C-terminal IDRs of the ribosomal P-stalk. The first is the high specificity and efficiency of immunoprecipitation. The C-terminal IDRs of the P-stalk were present in five copies on the 60S large ribosomal subunit and exposed on the solvent side Figure . AlthougSecondly, the C-terminal IDRs of the P-stalk are conserved across most, if not all, eukaryotes . Indeed,Both P-TRAP-seq and L10a-TRAP-seq captured the changes of the translatome in the UPR, but they identified several different DEGs Figure  and 5C. gkad545_Supplemental_FilesClick here for additional data file.


Complement C1s association with the pathogenesis of several diseases cannot be simply explained only by considering its main role in activating the classical complement pathway. This suggests that non-canonical functions are to be deciphered for this protease. Here the focus is on C1s cleavage of HMGB1 as an auxiliary target. HMGB1 is a chromatin non-histone nuclear protein, which exerts in fact multiple functions depending on its location and its post-translational modifications. In the extracellular compartment, HMGB1 can amplify immune and inflammatory responses to danger associated molecular patterns, in health and disease. Among possible regulatory mechanisms, proteolytic processing could be highly relevant for HMGB1 functional modulation. The unique properties of HMGB1 cleavage by C1s are analyzed in details. For example, C1s cannot cleave the HMGB1 A-box fragment, which has been described in the literature as an inhibitor/antagonist of HMGB1. By mass spectrometry, C1s cleavage was experimentally identified to occur after lysine on position 65, 128 and 172 in HMGB1. Compared to previously identified C1s cleavage sites, the ones identified here are uncommon, and their analysis suggests that local conformational changes are required before cleavage at certain positions. This is in line with the observation that HMGB1 cleavage by C1s is far slower when compared to human neutrophil elastase. Recombinant expression of cleavage fragments and site-directed mutagenesis were used to confirm these results and to explore how the output of C1s cleavage on HMGB1 is finely modulated by the molecular environment. Furthermore, knowing the antagonist effect of the isolated recombinant A-box subdomain in several pathophysiological contexts, we wondered if C1s cleavage could generate natural antagonist fragments. As a functional readout, IL-6 secretion following moderate LPS activation of RAW264.7 macrophage was investigated, using LPS alone or in complex with HMGB1 or some recombinant fragments. This study revealed that a N-terminal fragment released by C1s cleavage bears stronger antagonist properties as compared to the A-box, which was not expected. We discuss how this fragment could provide a potent brake for the inflammatory process, opening the way to dampen inflammation. In this complex, C1s is associated to the C1r protease, and the C1r2C1s2 tetramer associates with the recognition protein C1q. Upon C1 activation on a target surface, C1s gets activated by C1r, and then triggers the CP activation by cleaving complement C4 and C2, under the control of C1-inhibitor –31. CP ieumoniae , 33, andeumoniae . Howevereumoniae . This leeumoniae . Howevereumoniae . Furthereumoniae . HMGB1 cThis study details where HMGB1 gets cleaved by C1s, at the residue level, and why this result is unexpected. It also shows examples on how the molecular environment modulates the output of this enzymatic reaction. Moreover, it reveals quite unexpectedly the potent anti-inflammatory potential of F3, a major A-box fragment released by C1s cleavage. These results will be discussed in terms of enzymatic modulation of HMGB1 function and how this might open therapeutic perspectives.22.1The C1 proteins C1s and C1r have been purified from human plasma or recombinantly according to published procedures , 29. RecSite-directed mutagenesis was performed using the QuickChange II XL kit . The sequence integrity of all constructs and mutants was confirmed by DNA sequencing analyses .2.22.2.1E. coli BL21(DE3) cells (Sigma-Aldrich), transformed with a pET-28a plasmid coding for HMGB1 or its variants. The cells were grown in an autoinduction medium  was excised from the pFast-Bac-MASP-2-RH6 plasmid (6 tag by a Flag tag (DYKDDDDK) was performed by site-directed mutagenesis . The pcDNA3.1-MASP-2-Flag construct was used for stable transfection of HEK293-F cells grown in FreeStyle 293 medium using 400 μg/ml neomycin (Thermofisher Scientific). Recombinant MASP-2 was purified from 500 ml cell culture supernatant on a 2 ml anti-FLAG M1 agarose column (Sigma-Aldrich) as described by Bally et al.  molar ratio in 50 mM Tris, 150 mM NaCl, pH 7.4, for 100 min at 37°C. The reaction products were then analyzed by SDS-PAGE in reducing conditions (see section 2.4.1). Various HMGB1 amounts, E/S ratios, incubation times were used to match appropriate conditions for the different steps of the analysis. For example, the molar E/S ratio was between 5 and 20%, 2 to 6-7 μg HMGB1 were digested by C1s, and the incubation time varied from 90 min  to 5-7 h for the MS analyses that required more complete digestion.In order to compare the C1s enzymatic activity, free or within the C1 complex, we aimed to use the same C1s molar concentration and secure C1 assembly using C1 concentrations above 0.25 μM. Serum C1q (0.25 μM), C1r (0.5 μM) and recombinant activated C1s (0.5 μM) were mixed to form the C1 complex (0.25 μM), and incubated 90 min at 37°C for its activation. Comparative cleavage experiments  were then performed on 6 μg HMGB1 with 10% C1s either free or from C1 prepared at 0.25 μM Figure 2β-mercapto-ethanol (0.35 M) and heated at 95°C for 10 min (or 85°C for 5 min for Tricine gels). The digestion mix was analysed by SDS-PAGE, further described in section 2.4.1.For the enzyme comparison, HMGB1 was mixed with C1s or Human Neutrophil Elastase (HNE) at a 2.5% E/S molar ratio in TBS and incubated at 37°C for 90 min  or 5 h at 10% . Further enzymatic comparison was performed using the C1r and MASP-2 proteases at a 10% E/S molar ratio to digest HMGB1 WT at 37°C for 90 min. For the SDS-PAGE analysis, reactions were stopped by addition of Tricine SDS Sample Buffer and reduced with 2.41%, 1 cm at 280 nm) described in Protein concentrations were estimated using the parameters . The use of Tricine SDS gels 10-20%  was implemented for this study to better distinguish HMGB1 fragments between 7 and 20 kDa.For the western blot (WB) analyses, 7 pmol of HMGB1 (full-length or fragments) was incubated with C1s (see Enzymatic Reactions) and separated on Tricine gels. Proteins were transferred on nitrocellulose membranes using the semi-wet Trans-Blot Turbo transfer system (BioRad). Membranes were blocked with TBS 5% milk (w/v). The blocked membranes were probed with primary antibodies  and anti-HIS coupled to peroxidase  for 2 h at room temperature in TBS (Euromedex) 5% milk (w/v). Membranes were washed 3 times with TBS 0.05% Tween (w/v). The anti-HMGB1-Nter probed membrane was incubated with the secondary antibody . Membranes were revealed with an enhanced chemiluminescence detection kit , scanned using a ChemiDoc apparatus (BioRad) and analyzed with ImageLab (BioRad).2.4.2m/z range. MS spectra were acquired and the data processed with MassHunter workstation software  and with GPMAW software .Liquid Chromatography Electrospray Ionization Mass Spectrometry (LC/ESI-MS) was performed on a 6210 LC-TOF spectrometer coupled to a HPLC system (Agilent Technologies). All solvents used were HPLC grade , trifluoroacetic acid (TFA) was from Acros Organics . Just before analysis protein samples were diluted in acidic denaturing conditions to a final concentration of 5 µM with solution A (0.03% TFA in water). Solvent B was 95% acetonitrile-5% water-0.03% TFA. Protein samples were firstly desalted on a reverse phase-C8 cartridge  for 3 min at a flow rate of 50 μl/min with 100% solvent A and then eluted and separated onto a RP-C8 column  at a flow rate of 50 μl/min using the following linear gradient: from 5 to 95% solvent B in 15 min, then remaining 2 min at 100% solvent B and finally re-equilibrating the column at 5% solvent B for 10 min. MS acquisition was carried out in the positive ion mode in the 300-3200 2.52.5.1RAW264.7 were sub-cultured every two days using RPMI medium supplemented with 10% of decomplemented FBS (heated at 56°C for 40 min), 10 mM of Hepes solution  and 5 µg/ml ciprofloxacin .2.5.22. For the stimulation, LPS  was used alone or in complex with HMGB1 WT, A-box or rF3. HMGB1 and its variants were produced and stored at -20°C in TBS solution, whereas LPS was stored in H2O + 30% ethanol. 24 h prior to stimulation, HMGB1 and LPS were mixed to form complexes at 4°C. The complexes were then diluted in medium to stimulate cells at a final concentration of 0.47 pmol/ml, 0.94 pmol/ml or 1.88 pmol/ml for HMGB1 and 1 ng/ml or 2 ng/ml of LPS. Cells were stimulated for 24 h at 37°C, 5% CO2. Cells supernatants were harvested and stored at -20°C for later analysis.Before stimulation, cells were seeded at 300 000 cells/ml in 6-well adherent plates  with 2 ml/well and incubated for 2 days at 37°C, 5% CO2.5.3The levels of cell viability and phagocytosis were analyzed (data not shown) by flow cytometry . The phagocytosis level was assessed by 3 h incubation with fluorescent beads . Cells were then incubated for 5 min with propidium iodide to investigate their viability levels. Finally, the cells were detached in PBS for flow cytometry analysis.2.5.4IL-6 secretion levels were measured in RAW264.7 supernatants by ELISA using a murine IL-6 kit , according to the furnished protocol. The absorbance was measured using the SpectroNano (BMG) and the data analyzed by the MARS software (BMG). Results are presented as mean +/- standard deviations of quadruplicate samples. The statistical analysis was performed according to the variance ratio (highest over lowest), with a Student (ratio < 4) or a Welch (ratio > 4) t-test and scored as follow: * p < 0.05; ** p < 0.01 and, *** p < 0.001.2.5.5Endotoxin concentration of the HMGB1 samples used to stimulate macrophages was measured with a ToxiSensor kit  according to the supplier’s instructions. Endotoxin concentrations of samples were between 0.7 - 1.5 ng/ml. Considering that HMGB1 samples were diluted 1:1000 and only 1 - 10 µl were added for stimulation, their LPS content was far too low to result in any macrophage stimulation, as was checked for control.3Following the initial description by Yeo et al. that complement C1s can cleave HMGB1 , which w3.1in vitro experiments aiming to compare HMGB1 cleavage using the same activated C1s molar concentration, either in the free state or within C1. As shown on In its serum physiological context, C1s is mainly found within the C1 complex, where it is associated to C1r and C1q. Therefore, we designed 3.2As shown above or previously , HMGB1 cAiming to identify the fragments by mass spectrometry (MS), we used conditions leading to more complete HMGB1 digestion, mainly with longer incubation times (5 h). MS experiments and analyses of HMGB1/C1s liquid mix have been performed starting from the full-length HMGB1 Figure 4MS detection and identification of the minor secondary cleavage products is quite difficult to achieve when starting from full-length HMGB1 Figure 43.3As shown on 3.4In order to confirm the identification of the primary cleavage site after K172, the double mutation K172E-K173G was introduced in HMGB1 to produce the HMGB1_KK mutant. All molecular details on HMGB1 variants are shown in The three N-terminal fragments, F1 to F3, have been recombinantly produced for further characterization, with a His-tag added for purification purpose , as deta3.5Knowing that the HMGB1 A-box is a functional antagonist of HMGB1 and provides a potential regulatory feedback mechanism  44), ou, ou44), 3.6The functional comparison between rF3 fragment (1-K65), HMGB1 and A-box was thus explored regarding the modulation of the secretion of the inflammatory IL-6 cytokine following stimulation with low concentrations of LPS , 45. Thi3.7Considering the functional interest of the F3 fragment, we started to investigate the question of a possible impact of HMGB1 molecular environment on C1s digestion outcome. As mentioned previously, the purified recombinant HMGB1 WT and its fragments contain a disulfide bridge in the A-box. What could be the impact of the disulfide bridge in the A-box regarding C1s digestion? To investigate this question, a 3S HMGB1 variant was produced, where all cysteines were mutated into serine . HNE is indeed present in inflammatory conditions where neutrophils are abundant . In thisC1s was further compared with two homologous complement proteases, C1r and MASP-2 (which activates the complement lectin pathway). MASP-2 shares the same specificity as C1s in terms of complement targets, namely C4 and C2, whereas C1r only activates C1s. As expected, 4C1S genetic deficiencies, such as severe lupus or periodontal EDS, cannot be simply explained only by considering this function, therefore suggesting auxiliary non-canonical functions for this protease. A crosstalk between the HMGB1 alarmin and the complement system components was recently evidenced at different levels .All these studies reveal the potential of some proteases to regulate the function of HMGB1. As another example, the thrombin/thrombomodulin complex cleaves off the 17 N-terminal residues of HMGB1, a process which also reduced HMGB1 inflammatory role, as measured by TNF-α and iNOS mRNA induction and TNF-α secretion in RAW 264.7 cells . Looking on RAGE . This isIn conclusion, this and previous studies highlight the impressive modularity of HMGB1 functions, which depend on its location and can be finely tuned by the oxidative or proteolytic context. Thus, by switching between different states, HMGB1 can orchestrate different steps spanning from leucocyte recruitment, inflammation signaling and resolution, as well as tissue repair , which aThe original contributions presented in the study are included in the article/ML and JK performed biochemical and cellular experiments and discussed the data. AC, IB and FD performed protein engineering and production, and initial biochemical experiments. LS performed MS experiment and their detailed analysis to precisely identify C1s cleavage sites. JV engineered the 3S mutant and the B-box fragment and analyzed their cleavage by C1s. TR and BD conceived the experiments on RAW264.7 cells and helped in their interpretation. NT supervised some protein productions and C1 cleavage experiments. VR and CG conceived the biochemical experiments and discussed the data. CG prepared the original manuscript draft and worked at the structural interpretation. All authors contributed to the article and approved the submitted version.