EHBP1 is an adaptor protein that regulates vesicular trafficking by recruiting Rab8 family members and Eps15-homology domain-containing proteins 1/2 (EHD1/2). It also links endosomes to the actin cytoskeleton. However, the underlying molecular mechanism of activation of EHBP1 actin-binding activity is unclear. Here, we show that both termini of EHBP1 have membrane targeting potential. EHBP1 associates with PI(3)P, PI(5)P, and phosphatidylserine via its N-terminal C2 domain. We show that in the absence of Rab8 family members, the C-terminal bivalent Mical/EHBP Rab binding (bMERB) domain forms an intramolecular complex with its central calponin homology (CH) domain and auto-inhibits actin binding. Rab8 binding to the bMERB domain relieves this inhibition. We have analyzed the CH:bMERB auto-inhibited complex and the active bMERB:Rab8 complex biochemically and structurally. Together with structure-based mutational studies, this explains how binding of Rab8 frees the CH domain and allows it to interact with the actin cytoskeleton, leading to membrane tubulation. EHBP1 is an adaptor protein that regulates vesicular trafficking and links endosomes to the actin cytoskeleton. Here, authors show that both termini of EHBP1 have membrane targeting potential and that in the absence of its binding partner Rab8, the bMERB and CH domain of EHBP1 form an intramolecular complex which auto-inhibits actin binding. EHBP1 co-localizes with the actin cytoskeleton and overexpression of either EHBP1 or EHD2 leads to extensive actin reorganization2. Disruption of EHBP1/EHDs by siRNA leads to inhibition of transferrin endocytosis and GLUT4 transportation2. The Ras superfamily GTPase Rab10 has also been shown to regulate the translocation of GLUT4 in adipocytes3. In our previous work, we showed that EHBP1 is an effector molecule for Rab8 family members, including Rab10, and forms complexes with 1:1 stoichiometry5. Recent work has also shown that a Rab10-EHBP1-EHD2 trimeric complex plays a crucial role in lipid droplet engulfment during lipophagy in hepatocytes6. Moreover, C. elegans EHBP1 promotes endosomal tubulation by linking the membrane lipid PIP2 to the actin cytoskeleton and this interaction is enhanced upon Rab10 binding7.EHBP1 was originally identified as an Eps15-homology domain-containing protein 1/2 (EHD1/2) interacting partner that plays a central role in GLUT4 transport and couples endocytic vesicles to the actin cytoskeletonC. elegans, EHBP1 depletion leads to an endocytic recycling defect in the intestine and in nonpolarized germline cells and the phenotype was recapitulated upon Rab8/10 deletion8. Drosophila EHBP1 has been shown to play an essential role in eye development by regulating the exocytosis of Scabrous, a positive regulator of Notch signaling9. Notch signaling has been implicated in metastatic prostate cancer, and a genome-wide SNP association study shows that EHBP1 is associated with aggressive disease13. EHBP1 controls the invasiveness of PTEN-positive prostate cancer cells and is essential for the anti-invasive effect of the drug atorvastatin14.Apart from having roles in vesicular trafficking and autophagy, EHBP1 is implicated in early development and cancer. In bMERB toward its CH domain. Transient kinetic experiments show that the binding of Rab8 family members to the bMERB domain releases the CH domain. Furthermore, we have solved the crystal structure of the bMERB domain in complex with human Rab8a. Structural analyses supported by mutagenesis and biochemical experiments identify key residues for the interaction and explain why the generation of a stable CH-bMERB-Rab8 ternary complex is not possible. Our biochemical and structural data suggest that the interaction between the C-terminal bMERB domain and the central CH domain has a regulatory role in the function of EHBP1 and binding of Rab8 family members to the bMERB domain releases the CH domain, which can then interact with the actin cytoskeleton.Despite having information on EHBP1 at the functional level, convincing biochemical data on EHBP1 activation are missing. In this work, we have identified and characterized an auto-inhibited state of human EHBP1, which is mediated by an intramolecular association between the CH domain and the bMERB domain. We have elucidated the structure of the CH domain in complex with the bMERB domain, providing an explanation for the specificity of EHBP14. The UniPort database has reported three EHBP1 isoforms and for biochemical studies, the domain boundaries are taken from isoform 1 (Q8NDI1-1), whereas for cellular localization experiments, fluorescent constructs are based on isoform 3 (Q8NDI1-3)  fusion construct and showed that this construct is cytosolic as well as having the potential to associate with membranous structures  Fig. . Previoures Fig. . Thus, ores Fig. .Fig. 1Do15, and in general, most of the C2 domain family members are known to interact with phospholipids directly16. Since most of the C2 domain family members have been reported to be regulated by Ca2+ ions, we performed the experiments in the presence or absence of Ca2+ ions. The results obtained indicated that the NT-C2 domain of EHBP1 binds to phosphatidylserine in the presence of Ca2+ but also binds PI(3)P and PI(5)P in a Ca2+ independent manner  and dissociation rate (koff) constants for KD calculation (KD = koff/kon). For kon measurements, association kinetics of Rab8a loaded with fluorescent 2′3′-O-(N-methyl-anthraniloyl) mantGppNHp were monitored with increasing concentrations of the bMERB1060-1212 domain using a stopped-flow apparatus. kon for Rab8amantGppNHp is 2.79 × 107 M−1 s−1 and the intercept on the y-axis yielded the koff (13.4 s−1)  Fig. . Direct ved Fig.  and KD vT72/10T73. Phosphorylated Rabs are GDI resistant, thus increasing their lifetime on the membrane18. Further, phosphorylation also increases Rab8a/10 binding to the effector proteins RILPL1/L218. Structure-based sequence alignment shows that the switch II threonine of Rab8a/10 does not directly interact with Mical1bMERB and Mical cLbMERB4 , whereas no binding was observed for the construct lacking the N-terminal helix, clearly indicating that helices 1 and 2 constitute the CH-binding site , which usually has a lower actin-binding affinity; however, the C. elegans CHEHBP1 domain was shown to interact with actin filaments7. Using actin co-sedimentation assays, we could demonstrate an interaction with the human EHBP1 CH domain, with a KD value of 9.34 ± 1.86 µM , a tandem repeat of CH1 and CH2 is usually required µM Fig. . The affA similar interaction of the CH domain from Micals/Mical-like family members could not be detected Fig. .H1-2:CH complex and obtained crystals (SG: P 21) diffracting to 2.2 Å. The structure was solved as described in material and methods 32 , RMSD of 2.5 Å for 120 residues to Mical3bMERB (PDB 5SZG) and RMSD of 3.6 Å for 119 residues to Mical1bMERB (PDB 5LPN), whereas Rab8a is quite similar to Rab8a/10 of Mical cLbMERB:Rab8a /Mical1bMERB:Rab10 complex (RMSD 0.6 Å)  and switch II  are buried in a hydrophobic core of the interface formed by α-helices 2–3   Fig. . A conse83) Fig. . Besidesain Fig. . T72Rab84. Here, we sought to determine the contribution of essential bMERB residues that are necessary for Rab8 interaction. Similar to the CH-binding site, a robust cluster of hydrophobic residues was observed in the bMERB:Rab-binding interface   and the following loop, and then energy minimized with Macromodel (Schrodinger suite35) and PHENIX36. It is clear that the main contact site of the CHEHBP1 domain that corresponds to ABS2 is occupied by bMERB in the CH:bMERB complex structure, indicating that the bMERB domain has to dissociate to facilitate F-actin binding of the CH domain. The optimized CHEHBP1:F-actin model has a smaller buried interaction interface area (4640 Å2) between the CH domain and F-actin compared with the CH1FLNa:F-actin (6000 Å2)  cryo-EM structureαs) Fig. . To undeÅ2) Fig. . This isain Fig. . Since t4. The presence of two membrane-associating moieties can fine-tune the EHBP1 function by regulating its localization.EHBP1 has a central role in vesicular trafficking and lipophagy, yet its regulation is not understood. This work offers mechanistic insight into the EHBP1 activation mechanism by Rab8 family members. We have shown that both N-and C-termini of EHBP1 have the potential to interact with the membrane; the NT-C2 domain binds to PI(3)P, PI(5)P, and phosphatidylserine, lipid molecules present in the early endosomes and plasma membrane respectively. Previously, we reported that the EHBP1 CaaX box can be farnesylated24. Fremont et al., proposed a model for Mical1 activation in which an intramolecular association occurs between the C-terminal bMERB domain and the N-terminal mono-oxygenase together with the CH and LIM domains (MO-CH-LIM), forming an enzymatically dead complex. Binding of Rab35 can release the auto-inhibition, resulting in increased F-actin depolymerization20. Mical-like family members also engage in intramolecular interaction between their N-terminal CH domain  or LIM domain  with their C-terminal bMERB domain23. The present work is the first to perform biochemical characterization of the auto-inhibited bMERB complexes, but, we could not detect any interaction between the bMERB domain of Mical family members with their respective CH/LIM/CH-LIM domain , and were suggested to be inhibitoryCH_bs and helix2Rab_bs is essential for CH/Rab8 interaction and similarly CH and Rab aligned interacting residues M1116/L1179, W1119/L1182, and F1120/V1183 are for the CH/Rab interaction). Small changes in the amino acid sequence of the two halves determine the specificity toward CH or Rab binding.Here, we report the CH:bMERB and bMERB:Rab8 complex structures and demonstrate that the integrity of both N-and C-terminal hydrophobic patches is crucial for the CH and Rab interactions. We show that the CH and Rab-binding sites of the bMERB domain have significant similarities  complex with EHBP1bMERB_M1116A:Rab8a shows that the conformation of EHBP1 helix 1 and helix 2 clashes with the 2nd Rab10 molecule. For effectors displaying a 1:2 stoichiometry with Rab8 , we have previously proposed a model where the two Rab-binding sites have separate functions, with the first Rab binding leading to membrane recruitment, and subsequently to the release of auto-inhibition of the CH/LIM domains by binding of the second Rab4. In contrast, EHBP1 appears to have the intrinsic ability to be targeted to the membrane, and Rab8 only binds to the highly conserved C-terminal Rab-binding site of the bMERB domain, changing helix 3 conformation, and leading to release of auto-inhibition.Structural alignment of the EHBP137, we show that phosphomimetic mutation of the switch II threonine of Rab8/10 does not affect its interaction with the EHBP1bMERB domain and is not part of the bMERB binding interface  domain binds to F-actin26. Previously, it was shown that the C. elegans CHEHBP1 domain directly interacts with F-actin, and the authors suggested that Rab10 binding to the bMERB domain enhances the CH domain:F-actin interaction7. However, no difference was detected in the F-actin binding of the free CH domain versus a CH-bMERB fusion construct7. The authors did not consider an auto-inhibition model; instead, they proposed a model where Rab10 binding leads to multimerization of EHBP1, leading to a side by side placement of CH domains from the dimeric EHBP1, but no experimental proof is provided. Recently, Miyake et al. have reported a closed conformation mouse Mical-L1 (LIM:bMERB) and binding of Rab13 opens up the Mical-L1, allowing its interaction with F-actin23. By homology modeling, the authors built the LIM:bMERB:Rab13 tripartite complex and also docked the LIM domain onto F-actin and suggested that for the F-actin interaction, the LIM domain has to be free. However, this model could not explain how Rab13 binding to the bMERB domain releases the LIM domain. They suggested that a second competitive Rab13 binding at the N-terminal binding site is a prerequisite for the LIM:F-actin interaction. No stoichiometry is reported for the Mical-L1bMERB:Rab13 complex, and appropriate biochemical and structural studies would be needed to characterize the bMERB:Rab13 and LIM:bMERB complexes. In the current study, we clearly showed that the binding of Rab8 to bMERB releases the CH domain and further, our CH:F-actin model suggests that for F-actin interaction, the CH domain has to be free, since the bMERB-binding site is also part of the F-actin binding site  used in this paper were described previously4. Full-length human EHBP1isofrom3, ΔNT-C2-EHBP1isofrom3, and EHBP1isofrom3ΔCaaX constructs were cloned into the pEGFP (C1) vector between XhoI and SmaI sites by conventional PCR using Human EHBP1 cDNA . The NT-C2 domain was cloned into the pEGFP (N1) vector between XhoI and SmaI sites and all constructs were verified by DNA sequencing. Cos7 cells (ATCC: CRL-1651) were maintained in DMEM medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and penicillin/streptomycin at 37 °C in the presence of 5% CO2. Cells were grown on a coverslip in 6-well plates until they reached 60–70% confluency and transiently transfected using linear polyethylenimine, MW 25000 . Expression was checked 16–24 h post transfection. Cells were fixed with 3.7% paraformaldehyde in PBS for 15 min at room temperature. After washing with PBS, coverslips were mounted on glass slides with SlowFade Gold antifade reagent (Invitrogen). For EHBP1 constructs, single-plane images were taken by an EVOS fl fluorescence microscope equipped with 60x/1.42 Plan-Apochromat oil immersion objective.The full length human mCherry-tagged Rab constructs  followed by three 10 min washing steps with TBST, and strips were then incubated with HRP goat anti-mouse antibody  with a 1:5000 dilution. The washing steps were repeated and following development using the SuperSignal West Dura Substrate (Thermo Scientific) images were taken with a GelDoc system (Bio-Rad).To assess the lipid-binding properties of the EHBP1 NT-C2 domain, we performed a protein-lipid overlay assay with recombinant His6-MBP -NT-C2 domain fusion protein and His6-MBP was used as a control. 1 ng of His6-MBP-NT-C2 domain or His6-MBP as also blotted on the top of the PIP strip (Echelon Biosciences) to serve as a positive control (Antibody control). Strips were blocked for 1 h at RT in 3% (w/v) fatty acid-free BSA (Sigma) in TBST  Tween 20). PIP strips were then incubated overnight at 4 °C with gentle mixing in the blocking solution containing either 1 µg/ml of purified His6-MBP-NT-C2 domain or His6-MBP proteins. 1 mM of CaCl1-176 and Rab101-175) and phosphomimetic Rab (Rab8a1-176_T72E and Rab101-175_T73E) were expressed and purified as described previously39. Rabs were preparatively loaded with GppNHp (Guanosine-5′-[β-γ-Imido]-triphosphate) or mant GppNHp (2′/3′-O-N-Methyl-anthraniloyl)-guanosine-5′-[-imido]triphosphate) and the reaction was performed as described previously4. Nucleotide exchange efficiency was quantified by C18 reversed-phase column  with HPLC in 50 mM potassium phosphate buffer pH 6.6, 10 mM tetrabutylammonium bromide, and 12% acetonitrile (v/v) and for the mant GppNHp exchange run the buffer contains 25% acetonitrile (v/v). Protein samples were heat precipitated at 95 °C for 5 min and centrifuged at 15,700 g for 10 min and loaded  on the column. Peaks were integrated and to determine the nucleotide retention times, a nucleotide standard run was performed.Human Rab G-domains (Rab8aE. coli BL21 DE3-RIL (Agilent) cells in LB media supplemented with proper antibiotics and cells were grown at 37 °C to OD600 nm = 0.8–1.0 and stored at 4 °C for 30 min. Further expression was induced by the addition of 0.5 mM IPTG, and cells were allowed to grow at 20 °C for 14–16 h. Cells were pelleted and stored at −80 °C until ready for purification. Cells were mechanically lysed by passing through a fluidizer (Microfluidic) in a buffer  and 1 mM PMSF (Phenylmethylsulfonyl fluoride) and lysates were cleared by centrifugation at 75,600 g for 30 min. Subsequently, the proteins were purified by Ni2+-affinity chromatography . For the His6-MBP-NT-C2 domain purification, the protein was concentrated after first Ni2+-affinity chromatography and subjected to gel filtration  in the final buffer .All other proteins were recombinantly expressed in 2+ affinity chromatography . The His6-tag was cleaved by Tobacco Etch Virus (TEV)-protease, and a second Ni2+-affinity purification was performed to remove the TEV protease and His6-tag. The final purification step was achieved by gel filtration  . The purified protein was collected and concentrated; flash-frozen in liquid N2, and stored at −80 °C.For the human EHBP1 CH, bMERB domain and other protein purifications, cells were lysed in a buffer  having 1% CHAPS and lysates were cleared by centrifugation at 25,000 rpm for 30 min. Subsequently, the proteins were purified by Ni1-176 complex formation was analyzed by aSEC. 110 µM of bMERB domain and 121 µM of GppNHp Rab81-176 protein (Effector: Rab stoichiometry of 1:1.1) was mixed in a buffer containing 20 mM Hepes 7.5, 50 mM NaCl, 1 mM MgCl2 and 2 mM DTE (Rab buffer) and centrifuged for 15 min at 15,700 g at 4 °C. 40 µl of the mixture was injected into a Superdex 75 10/300 GL gel filtration column  pre-equilibrated with the Rab buffer with a flow rate of 0.5 ml/min at room temperature and absorption at 280 nm was recorded.The bMERB:GppNHp Rab8aSimilarly, the complex formation between the bMERB (110 µM) domain and the CH (121 µM) domain was analyzed in the buffer containing 20 mM Hepes 7.5, 150 mM NaCl, and 2 mM DTE (CH buffer) and centrifuged for 15 min at 15700 g at 4 °C. 40 µl of the mixture was injected onto a Superdex 75 10/300 GL gel filtration column  pre-equilibrated with the CH buffer with a flow rate of 0.5 ml/min at room temperature and absorption at 280 nm was recorded.2, and 1 mM Tris (2-carboxymethyl) phosphine (TCEP) whereas bMERB: CH interactions were performed in buffer containing 20 mM Hepes 7.5, 150 mM NaCl and 1 mM TCEP at 25 °C. Wild types and mutant proteins were dialyzed overnight in their respective buffer. Samples were centrifuged at 15,700 g for 30 min at 4 °C and protein concentration was determined by Bradford assay (Bio-Rad). 500 µM of GppNHp Rab8a1-176 was titrated into the cell containing 50 µM bMERB domain and for bMERB:CH interaction 600–800 µM of CH domain was titrated into the cell containing 40–60 µM bMERB domain. For the control experiments, the buffer was titrated into the cell containing the bMERB domain and in the second control experiment, the CH domain was titrated against buffer. The binding isotherms were integrated and the data were fitted to a one-site-binding model using Origin 7.0 . The reported ITC result is the representative one of at least three independent measurements.Protein–protein interaction measurements were conducted by ITC using an ITC200 microcalorimeter . bMERB:Rab interaction measurements were performed in buffer containing 20 mM Hepes 7.5, 50 mM NaCl, 1 mM MgCl100 µM the CH domain is incubated with 300 µM Cy3-thioester (Jena bioscience) in the buffer containing 50 mM Hepes pH 7.5, 150 mM NaCl and 50 mM MPAA  at RT for 1 h. Free dye was removed by passing through a PD10 column  in buffer containing 50 mM Hepes pH 7.5, 150 mM NaCl, and 2 mM DTE. To check the extent of labeling, samples were analyzed by mass spectrometry (LC/ESI-MS)  using a SX-20 stopped-flow apparatus (Applied Photophysics) at 25 °C. For bMERB:mantGppNHp Rab8a1-176 kinetic measurements, the experiments were performed using the signal from the methylanthraniloyl group of mantGppNHp, the mant group was excited with a 360 nm LED, and emission was detected through a 420 nm cutoff filter.bMERB: Rab kinetic measurements were performed in Rab buffer  was used. All stopped-flow results that were analyzed are averages of 6–8 individual traces. Single exponential functions were fit using the Origin9 software (OriginLab).H1-2:CH (300 µM of 1:1 complex) was crystallized in 0.18 M Tri-ammonium citrate and 20% (w/v) PEG 3350. The complex of bMERBM1116A:Rab8a1-176_GppNHp (400 µM of 1:1 complex) was crystallized in 0.1 Mes pH 6.0, 5% (w/v) PEG 3000, and 30% (v/v) PEG 200. The complex of bMERBF1120A:Rab81-176_GppNHp was crystallized in 0.1 Mes pH 6.5, 10% (w/v) PEG-MME 5000 and 12% (v/v) 1-Propanol. Crystals were fished directly from the crystallization drop and flash-cooled in liquid nitrogen. Diffraction data were collected at 100 K on beamline X10SA at the Swiss Light Source . For the bMERBH1-2:CH complex crystal, a native data set was collected at a wavelength of 1.000010 Å whereas two data set from a single crystal was taken for the bMERBM1116A:Rab8a1-176_GppNHp complex at a wavelength of 0.919532 Å. A native data set was collected for bMERBF1120A:Rab8a1-176_GppNHp complex at a wavelength of 0.919550 Å. Data were integrated and scaled with XDS40.Initial crystallization condition screens for all protein complexes described in the paper were performed with the JSG Core I-IV, Pact, and Protein Complex suites (Qiagen). The sitting-drop vapor diffusion method was used, with a reservoir volume of 70 μl and a drop volume of 0.1 μl protein  and 0.1 μl reservoir solution at 20 °C. The best conditions were then optimized using the sitting-drop vapor diffusion method varying drop sizes in order to obtain well diffracting crystals. The complex of bMERBH1-2:CH complex diffracted to a resolution of 2.2 Å  and two copies of the complex is present in the asymmetric unit of the crystal. The initial model for bMERBH1-2:CH complex was obtained by molecular replacement using PHASER41 with the NMR structure of the EHBP1 CH domain (PDB 2DK9) as a search model32. The partial model was completed with PHENIX AutoBuild42 and manual building in Coot43. For the bMERBM1116A:Rab8a1-176_GppNHp complex, the crystal diffracted to a resolution of 1.914 Å  and two copies of the complex constitute the asymmetric unit of the crystal. The partial model was obtained by MR using PHASER41 and the Rab8a (PDB 5SZI) was used as a search model4. The initial model was completed with PHENIX AutoBuild42 and by manual building in Coot43. The final models were refined to convergence with phenix.refine36 or Refmac544 of the CCP4 package45. The final model of the bMERBM1116A:Rab81-176_GppNHp complex was refined with phenix.refine using refined Translation/Libration/Screw tensors, which further lowered the Rfree by 9%. For the bMERBF1120A:Rab8a1-176_GppNHp complex, the crystal diffracted to a resolution of 2.0 Å  and bMERBM1116A:Rab8a1-176_GppNHp complex was used as a model for molecular replacement.The Crystal of bMERBhttp://www.pymol.org).Data collection and refinement statistics are summarized in Supplementary Table 2, 15 mM KCl, 1 mM DTT, and 1.8 mM NaN3. Samples were centrifuged at 100,000 g for 1 h at 4 °C. The supernatant and pellet were subjected to 18% SDS-PAGE, followed by Coomassie Brilliant Blue staining. The quantitative analyses were performed using the Bio-Rad image analysis software in the ChemiDoc system (Bio-Rad).Rabbit skeletal muscle G-actin (AKL99) was purchased from Cytoskeleton and polymerized into F-actin according to the manufacturer’s protocol. F-actin (10 μM) was incubated for 1 h at RT with different concentrations of the EHBP1 CH domain (5–60 µM) in the buffer containing 5 mM Tris-HCL pH 7.5, 0.18 mM CaClFor comparative actin co-sedimentation assays, 10 μM F-actin was incubated with 40 μM CH domain from EHBP1, Mical1, Mical3, and Mical-L1, and co-sedimentation was performed as described above.46. The protein interaction interfaces from the asymmetric unit were examined in detail using the PDBePISA server 47. DALI server was used for structural comparison29.Multiple sequence alignments were generated using Clustal OmegaFurther information on research design is available in the Supplementary informationPeer Review FileReporting Summary


Phaseolus vulgaris L. (Fabaceae) (the common bean), Vicia faba L. (Fabaceae) (the broad bean) and Glycine max L. (Fabaceae) (the soya bean), against 4th instar larvae of Trogoderma granarium Everts (Coleoptera: Curculionidae). The flours were tested at different concentrations. They all demonstrated significant effects on larval mortality, as well as they all induced a decrease in the number of larvae reaching the pupal stage. The flours of P. vulgaris and V. faba were highly insecticidal against T. granarium larvae, especially at the highest concentrations (86.7% for PV100 and 90% for VF100). Our results enrich previous findings on the entomotoxic effect of leguminous plant lectins and highlight P. vulgaris and V. faba lectins as potential alternative control agents against T. granarium.Chemical storage pest control is interlinked with many challenges such as environmental pollution and toxicity to humans and animals. Alternative tools are thus being increasingly researched and applied to supplement and/or substitute old-fashioned chemical means. Entomotoxic proteins, such as the lectins of leguminous seeds, have been shown to be effective alternative control agents against many serious insect pests. The objective of this work was to evaluate the effect of the flour of three leguminous seeds,  Trogoderma granarium Everts (Coleoptera: Dermestidae), also known as the khapra beetle, has been attributed the status of a quarantine organism [Insects constitute the major pests of stored products whereby insect-related damage impacts the quality, quantity, and commercial and agronomic value of these food products. Many stored product pests are Coleopterans, and one of the most destructive and hard-to-kill species belong to the genus Trogoderma . Trogodeorganism  and has organism . It can organism .The challenges involved in the control of the khapra beetle are reinforced by the fact that chemical applications for pest control are problematic due to environmental pollution, emergence of pest resistance, toxicity to humans and related concerns, which account for the strict requirements imposed on their application on or near food, for safety reasons . ChemicaCallosobruchus maculatus F. (Coleoptera: Chrysomelidae) by lectins from Koelreuteria paniculate Laxm.  seeds, in the order of 0.7 and 0.3% (w:w), when added in artificial diets. The same entomotoxic lectins caused LD50 against Ephestia kuehniella , albeit at 0.65% (w:w) [Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) larvae reached the pupal instar stage when they were fed with the Gleheda solution, indicating that the entomotoxic Lamiacae lectin, which is structurally similar to the classical legume lectins, caused complete mortality [Meligethes aeneus larvae F. (Coleoptera: Nitidulidae) when fed Brassica napus L. (Brassicacae) anthers which had been soaked in 1% solution containing the lectins, with excellent results. Plant lectins can also be detrimental to insects with chewing and sap-sucking mouthparts such as aphids [Plant lectins, which are carbohydrate-binding proteins, are such non-chemical control agents with proven insecticidal action against many insect taxa (for an extensive overview of the role and classification of plant lectins ). They a5% (w:w) . Zero Leortality . Melandeortality  investigs aphids ,32.Phaseolus vulgaris L. (Fabaceae: Faboideae), Vicia faba L. (Fabaceae: Faboideae) and Glycine max L. (Fabaceae: Faboideae) against 4th instar larvae of T. granarium. Legume lectins have provided encouraging output in terms of their insecticidal potential [V. faba, or any plant lectins, have been tested against the khapra beetle. Our results are discussed in terms of enhancing the use of these alternative chemicals in stored-product Integrated Pest Management (IPM).The objective of this study was to test the lectin insecticidal properties of three leguminous flours derived from seeds of otential . They maotential ,35,36,37T. granarium was obtained from infested wheat. Insects were established in lab culture in a controlled environment chamber at 27 ± 3 °C and 73 ± 5% r.h., with alternating light ± cycles of 12 h. Larvae were kept in glass jars (0.25 l capacity) covered with muslin cloth. For their diet, they were provided with pesticide-free sterilized corn flour (200 g). Every two weeks emerged adults were carefully removed by sieving to use in experiments. Experimental insects were kept in a growth chamber , in controlled environmental conditions . These experimental insect larvae L4 were identified with their morphological traits of robust and hairy body, body length and dark brown color.The initial stock of P. vulgaris, V. faba and G. max were reduced to flour in EMBIA Laboratory, Department of Pharmacy, University of Patras. For each of the three plants, the seeds (2000 g) were ground to obtain the flour, using the Waring Blender CB15TP . We then eliminated large particles by further sifting the flours with the Retch Jaw Crusher BB 50 sieve (mesh-size < 0.5 mm) . The different flour fractions were weighted using the Sartorius 126,400 scales , with precision of 0.01 g. All the flours were used directly in the biological assays.The seeds of T. granarium. Prior to the experiment, the larvae had been starved for a 24 h period. Daily observations of the Petri dishes were carried out for 16 days, by emptying the contents onto sterilized white paper to identify dead individuals. Forty-five days after the applications, we checked all Petri dishes and recorded the number of T. granarium pupae. Finally, after 60 days, we documented adult emergence.The tested formulations were prepared by first placing the flours of the insecticidal legume seeds in sterile Petri dishes, in quantities which incrementally ranged every 2.5 g . To the leguminous seed flours, we also added corn flour to further supplement them. Corn flour quantities also ranged every 2.5 g, but they were provided in a decremental fashion  so that each Petri dish had the same overall content (10 g). The content of each Petri dish was homogenized by stirring it 20 times with a spatula. The Petri dish which contained only the corn flour constituted the control. In total, 10 Petri dishes were assembled, and 10 insects were placed in each Petri dish with 10 g of flour. Each Petri dish underwent 10 replications. Each treatment involved ten 4th instar larvae of T. granarium individuals in each applied concentration, per treatment (flour) and exposure time. Comparison of median survival time was obtained using the Breslow test  (SPSS v.25.0).Corrected percent mortality was calculated using Abbott’s formula  and, priThe survival probability , S(t), is the probability that an individual survives from the original time  to a specified future time, t. The following equation calculates the proportion of initial individuals which are still alive at time t:hazard function [t while evaluating the effect of several simultaneous factors on survival. The hazard function is estimated as follows:We also used the Cox regression model to calculate the function . The latT. granarium larvae. However, the flours tested were not equally toxic to the beetles. Toxicity increased as the concentration in seed flour increased. The flours of P. vulgaris and V. faba were the most effective against T. granarium. More specifically, with the flour of V. faba, the median lethal time values varied from 8.87 (VF100) to 12.2 days (VF25)  to 12.7 days (PV25)  to 13.1 days (GM25) ). The control median lethal time was 15.6 days.All the flours derived from the seeds of the three edible leguminous crops demonstrated noteworthy insecticidal action against the s (VF25) , with ths (PV25) , and, fis (GM25)  (Chi-squp < 0.001), for exposure time , and for flour × exposure time . After 16 days, control mortality was 3.3%. On the G. max flour, it ranged between 30 (GM25) and 83.3% (GM100)  and 90% (VF100)  and 86.7% (PV100)  A; on the (VF100) B; on the (PV100) C.T. granarium pupae 45 days post treatment. The number of pupae was also dependent upon the applied concentrations. Indeed, there was a significant difference between the number of treated pupae  and the number of control pupae. After 60 days, there was also a significant difference between the number of treated and untreated adults .T. granarium larvae had positive β-values .The objective of this work was to evaluate the effect of the flour of three leguminous seeds, Canavalia brasiliensis (Fabaceae: Faboideae), Dioclea grandiflora (Fabaceae: Faboideae), Dioclea rostrate (Fabaceae: Faboideae), Cratylia loribunda (Fabaceae: Faboideae), and P. vulgaris plants contain lectins which have successfully been assayed for their protection of seeds against the beetle Callosobruchus maculatus F. (Coleoptera: Chrysomelidae) [Entomotoxic lectins have been reported plentiful in the seeds of legumes, including bean and soybean.  They havmelidae) .Lectins can interfere with important physiological insect functions either by binding to sugars on the surface of glycoprotein-rich epithelial gut cells, thus affecting nutrient absorption, or by becoming internalized and finding targets intracellularly, thereby disturbing metabolic pathways. A significant prerequisite is that lectins first bypass being degraded by the digestive enzymes of the insect gut. Most insecticidal lectins appear resistant to proteolysis as well as tolerant in a broad pH spectrum. The cellular pathways affected will vary in relation to insect species and lectin type ,42,43.Hypera postica Gyllenhal (Coleoptera: Curcuniolidae) larvae died swiftly after feeding on bean flour [Sitophilus oryzae L. (Coleoptera: Curculionidae) [Bactrocera cucurbitae (Coquillet) (Diptera: Tephritidae) in terms of pupal weight, number of pupae and number of emerging insects, as lectin concentration increased [Callosobruchus maculatus which is detrimental to the chickpea Cicer arietinum (Fabaceae: Faboideae) could not develop in the seeds of Phaseolus spp. In fact, the bean meals disrupted female fecundity, adult emergence, and the developmental timespan [The array of plant lectin-induced effects involves significant mortality, delays in insect development, reduced pupation, fecundity and/or adult emergence, susceptibility to natural enemies and other ,22,23,44an flour . The blaionidae) ,46. SBA ncreased . Callosotimespan .The different degrees of insecticidal efficiency between the three leguminous flours of the present study must be linked to their botanical group. Observed differences in their entomotoxicity can be attributed to their taxonomy as well as to the variability in the chemical and biochemical constituents ,45,46,49T. granarium larvae could be considered in the framework of storage infestation control.Edible legumes containing entomotoxic lectins should be considered in pest management strategies. They could also provide an alternative to introducing entomotoxic factors in the genome of plants which is otherwise prohibitive considering the cost of seeds. Based on our experimental results, the bean flour and the fava bean flour which caused the highest mortality to T. granarium larvae could be considered in the framework of storage infestation control.Edible legumes containing entomotoxic lectins appear promising for use in pest management strategies. They could also provide an alternative to introducing entomotoxic factors in the genome of plants which is otherwise prohibitive considering the cost of seeds. Based on our experimental results, the bean flour and the fava bean flour which caused the highest mortality to


The deformation measurement of a specular surface is of great importance during the quality inspection and installation of optical elements or wafers, especially those with large apertures. We propose a deflectometry method with speckle digital image correlation (DeSDIC) to realize the dynamic and high-accuracy measurement of the deformation on specular surfaces, with a simple system structure and robustness to noises and environmental vibrations. Random speckle pattern displayed on liquid crystal display is reflected by the original surface under test (SUT), and the distorted pattern is recorded by a camera. This originally distorted pattern is taken as the reference image, and the patterns captured afterwards are digitally correlated with the reference image to calculate the gradient change and deformation of the SUT. The theoretical relationships and an experimental one-step calibration scheme are proposed. Both static and dynamic deformations of a deformable mirror were experimentally measured to demonstrate the feasibility and accuracy of DeSDIC, which is comparable to phase-measuring deflectometry and interferometry. Deformation of the solid surface is the consequence of various dynamic physical events, including the strain from stress or temperature change. For example, when mounted with an improper mechanism or subjected to excessive force, optical elements will suffer from surface deformation or non-uniform in the refractive index, which will greatly affect the performance of the elements. The measurement of such deformation is an effective approach for digging into the corresponding physical process and furthermore guaranteeing fine surface quality, appropriate mounting and environment control.Deformation is the change in the surface shape; thus, various methods for surface shape or figure error measurement can help solve the problem. Interferometry or deflectometry is among the effective approaches for surface measurement of optical elements. Commercial interferometers from Zygo in the US, XONOX in Germany, and other manufacturers have been popularly used in both laboratory and the industrial field. However, interferometers are generally expensive and complicated, requiring careful environmental control. As a typical kind of delectometry, phase-measuring deflectometry (PMD)  with sinIf we do not pay attention to the absolute surface shape at each moment but focus only on the deformation, we can adopt techniques in mechanical testing, e.g., digital image correlation (DIC) ,19,20,21We report a combination of deflectometer setup and speckle DIC algorithm (DeSDIC for short) to realize high-accuracy dynamic deformation measurement of specular surface with a simple and low-cost system. The theoretical relationship between the deformation and speckle displacement is deduced and an experimental one-step calibration method is proposed. The static deformation of a deformable mirror (DM) was measured and the result was compared with that obtained by a commercial interferometer. Finally, dynamic measurements of DM deformations demonstrated the feasibility and accuracy of the proposed method.f = 35 mm and F number is 1.4. The CCD chip has a size of 1/2 inch, a pixel size of 4.65μm, and a resolution of 1392 × 1040 pixels. The field of view is about 40 mm × 30 mm in the DM plane, and the pixel number along the diameter of the DM is around 520. The distance between the cell phone and the DM d1 and that between the DM and the camera d2 are about 280 and 320 mm, respectively. The included angle β between the normal of the DM and the optical axis of the camera is about 30°. The interferometer in The setup of the DeSDIC system is shown in Unlike PMD, DeSDIC does not measure the phase but uses DIC to calculate the pattern distortion caused by the local slope change of the DM. Thus, the deformation is reconstructed in four steps: (1) quantifying local pattern displacement from two speckle images with DIC; (2) measuring the rotational angle of a facet from the local distortion; (3) calculating the gradient of the deformation from the rotational angle; and (4) reconstructing the deformation from the gradient. The detailed mathematical analysis is proposed as follows.u, Δv) of the speckle. In detail, we denote the intensity distribution of the recorded speckle pattern image before and after the deformation to be S0 and S1. S0 and S1 in the full field of view of the camera. Little information can be retrieved by the naked eye. Thus, from the two images, we sample two square subsets denoted as g and h centered at  with a size of (2m + 1) × (2m + 1) pixels, where m is an integer. In m = 12. Obvious shifts along the arrows can be noticed. For the quantitative displacement, we adopt an established DIC method: the zero-normalized cross-correlation of the two subsets [Step 1: DIC is adopted to calculate the 2D displacement (Δ subsets (1)CZNCCu, Δv) of the speckle pattern between the two subsets can be obtained from the correlation peak shift. The center of the subset scans over the whole image and the displacement distribution over the whole aperture can be obtained. The immediate result from DIC is in pixel unit and can be converted to linear displacement with the pixel size of the detector. The size of the subsets should be compromised between the accuracy and the computation time.A sub-pixel interpolation scheme can be utilized before the similarity between the two subsets is evaluated . Finallyxθ, yθ) of a facet M is measured from the image displacement. A, e.g., a point in the speckle displayed by the cell phone, is virtually imaged to A1 by the mirror M. Then, A1 is imaged to A2 on the CCD sensor by the objective lens L. O is the optical center of L. We set a Cartesian coordinate system at the center of the mirror to describe its rotation, with the normal as z-axis and the direction perpendicular to the plane ACO as x-axis. An image coordinate system uv is set at the CCD image plane with u-axis parallel to x-axis.Step 2: The rotation angle , then we haveIf L as f, the image distance l between O and the CCD plane can be calculated from the object distance (d1 + d2) asGiven the focal length of v due to the rotation xθ equalsk is a calibrated coefficient because the distance and focal length are estimated. If M is rotated around y-axis, then yθ will have a similar relationship with the image shift Δu. The detailed calibration method is shown in the experimental result section. Moreover, because of the off-axial configuration, the circular aperture is distorted into a standing ellipse in β in Then, the image shift Δxθ, yθ). If we consider M a facet on the DM, then its rotation is caused by the deformation of the mirror, and then the angle xθ or yθ will be equal to the local normal direction change in x or y-direction. With the surface shape of the measured DM before and after the deformation denoted as W1 and W2, respectively, then ΔW = W1 − W2 is the deformation we want to measure, where  is omitted for concision. Considering the physical property of a deformation mirror, W1 and W2 are continuous and differentiable. The normal vector of W1 and W2 can be formulated asStep 3: The gradient change W isThe normal vector of Δxθ, which is equal to the angle between N1 and N2 in the x-direction, is related to the components of the vectors asAccording to basic geometry, the angle If the rotation angle is small enough, we have Together with Equation (6) we haveSimilarly,W1 is required here, which can be obtained from its nominal shape or rough measurement in advance. Foreknowledge of the surface shape W is constructed from the partial derivatives with Tikhonov deconvolution in Ref. [F{∙} and F−1{∙} are the Fourier and inverse Fourier transform, respectively, λ and μ are frequency coordinates, and γ is the regularization number.Step 4: The deformation Δ in Ref. ,32 for ik, and verify the angular measurement linearity and accuracy. Second, a static deformation was measured and quantitatively compared with the result from the interferometer to demonstrate the accuracy. Finally, dynamic measurement of the deformation was performed to show the potential in dynamic inspection.We conducted three experiments to verify the system and method. First, the rotation angle of the DM was measured to calibrate the system parameter, especially k in Equation (6) because the distance and focal length are estimated. Completely calibrating the internal and external parameters of the imaging system is not necessary. We believe this simple calibration method can contribute to simple structure and fast measurement of small deformation on specular surface.Deformation measurement focuses more on the local slope change rather than the absolute slope; thus, we deduce the approximately linear relationship between slope change (rotation) and the speckle pattern shifting in Equation (6). Consequently, we propose a one-step calibration method for the correction coefficient k. In the first experiment, the DM in xθE, which is treated as the true value and plotted as the horizontal axis in k is preset to be 1, are denoted as xθD, the average of which is denoted as The rotational angle measured with the encoder is denoted as xθE increases from 56” to 278”, the average Δv changes from 1.62 pixels to 8.46 pixels, and STD varies from 2.05” to 4.90”, corresponding to 0.07 pixels and 0.18 pixels. The accuracy of DIC decreases with the increment of the rotational angle because of the decorrelation of the speckles, which has been an important problem in DIC and will be focused in our future work. The calibration results are plotted in 2 of nearly 1. This finding indicates that a systematic error exists, and we calibrate the linear coefficient in Equation (6) as k = 1/0.86. The corrected The blue solid line in xθD when xθE = 278” is plotted against the subset size in The subset size in DIC was optimized for low measurement error and yet relatively short computational time. The average value and STD of W1 was measured with the interferometer, and it was a bowl shape with a peak-to-valley (PV) value of 8.34 λ (λ = 632.8 nm) corresponding to 5.28 μm. Thus, In the second experiment, the DM was controlled to change the surface shape. The deformation was simultaneously measured by an interferometer and the proposed system. The surface shape of the DM before the deformation Finally, the DM was controlled as follows to change the surface shape at 5 Hz. The 37 actuators were set to the maximum voltage one by one with all the other actuators set to 0. The changing time was shorter than 1 ms; thus, the surface was almost static between two changes. A video of the deformed speckles was recorded at a frame rate of 15 fps, and the images were extracted from the video for deformation reconstruction. As long as the exposure time is short enough to record clear speckle images without any motional blur, the dynamic deformation, i.e., the instantaneous off-plane deformation of the surface related to the original shape, can be correctly measured with DeSDIC. The deformation is dynamic and cannot be continuously measured with the phase-shifting interferometer; thus, we recorded only the corresponding interferograms for comparison. Four frames of typical interferograms and measured deformation results with DeSDIC are shown in Considering the error analysis of the system and method, we have to mention three main error sources. −4. If the local slope angle change increases to 5°, the maximum principle relative error will grow up to 2 × 10−3. Deflectometry measurement relies on the accuracy of slope measurement and DeSDIC inherits this feature. Second, the size of the subset in DIC guaranteed the accuracy, as shown in First, the theoretical model in Equations (3) and (11) introduced the principle error. The small angle approximation limited the local slope angle change measurement range to less than 1° or 17.5 mrad, with a maximum principle relative error of 1 × 10According to the above analysis, possible improvements are as follows: When applied in a large deformation situation, a more accurate model should be derived, and a telecentric lens should be adopted. Distortion should be corrected with a better lens or a software algorithm. Co-axis configuration, smaller speckle size , and betWe proposed a combination of deflectometer setup and speckle DIC algorithm to realize high-accuracy dynamic deformation measurement of specular surface with a simple and low-cost system. A linear model of the local slope change and the speckle displacement is deduced. A one-step calibration scheme based on the model is proposed for measuring the deformation with a maximum local slope change less than 1°, which makes completely calibrating the internal and external parameters of the imaging system not necessary. The current system realized a slope measurement precision of 5.75” (27.9 μrad) and deformation RMS measurement accuracy of 0.1 μm, which is comparable to traditional PMD and interferometry. Continuous deformation of the DM was also measured to demonstrate its potential in dynamic measurement. This method and system are applicable in the measurement of fast and small deformation on specular surfaces with a very simple system structure and calibration procedure.The authors have been authorized a Chinese patent ZL201510810141.4 resulting from the work reported in this manuscript.


Streptomyces and Aspergillus that are well known to have a great capacity for secreting a wide range of these polysaccharide degrading enzyme. The potential of the CBM and other ancillary domains for the design of improved polysaccharide decomposing enzymes is discussed.Enzymatic degradation of abundant renewable polysaccharides such as cellulose and starch is a field that has the attention of both the industrial and scientific community. Most of the polysaccharide degrading enzymes are classified into several glycoside hydrolase families. They are often organized in a modular manner which includes a catalytic domain connected to one or more carbohydrate-binding modules. The carbohydrate-binding modules (CBM) have been shown to increase the proximity of the enzyme to its substrate, especially for insoluble substrates. Therefore, these modules are considered to enhance enzymatic hydrolysis. These properties have played an important role in many biotechnological applications with the aim to improve the efficiency of polysaccharide degradation. The domain organization of glycoside hydrolases (GHs) equipped with one or more CBM does vary within organisms. This review comprehensively highlights the presence of CBM as ancillary modules and explores the diversity of GHs carrying one or more of these modules that actively act either on cellulose or starch. Special emphasis is given to the cellulase and amylase distribution within the filamentous microorganisms from the genera of  Plant biomass contains lignocellulose and starch which represent the most abundant carbohydrate biopolymers in nature. Lignocellulose is a complex polymer mix composed of lignin, an aromatic polymer and two carbohydrate polymers, cellulose and hemicellulose. The main component is cellulose consisting of β-1,4-linked D-glucose. In plant cell walls, cellulose chains interact with each other through hydrogen bonds to form microfibrils . Like ce-binding modules (CBM).The degradation of the renewable biopolymers cellulose or starch into sugar monomers by industry has a great economic value for its use as a feedstock for the production of various value-added products, such as fuels, chemicals and foods . In geneIn the CAZy classification system, the CBMs are a large group of protein domains that are frequently found attached to GH enzymes . The CBMTrichoderma reesei  because of their affinity for cellulose. The first of these domains was found appended to the cellobiohydrolase CBHI and cellobiohydrolase CBHII in the fungus a reesei . Thereafnas fimi . As seve and glutamic acid (Glu) residues . For gluIn the sequence-based classification of GHs in the CAZy database, α-amylases are represented by four different families, GH13, GH57, GH119, GH126. The main α-amylase family, GH13, is found in a broad range of organisms, including bacteria, archaea, eukaryota, and even viruses. GH57 is found in bacteria, archaea, and very limited in eukaryotes. The other two families, GH119 and GH126 have been found in bacteria only  which is a highly specific substrate for endo-acting cellulase. CMC is decrystallized cellulose and thus contains more amorphous sites that are ideal for access to the cellulose chain by endoglucanases that cleave internally. Whereas, the exoglucanase GH6 PaCel6A showed higher activity on the insoluble microcrystalline cellulose Avicel than on CMC, as measured by the liberation of cellobiose from the non-reducing end of the cellulose chain  are associated with CBM20. In Aspergillus amylases, CBM20 was found as the only starch binding domain associated with GH13 α-amylases and GH15 glucoamylases (Streptomyces. CBM20 was found as the most frequently domain (68%), followed by CBM25 (21%), leaving the rest (around 4% each) to CBM41 and amylases with a combination of CBMs: CBM20&CBM25 and CBM41&CBM48. Thus, in amylases from these genera, CBM20 is the most dominant CBM  and bifunctional α-amylase-pullulanases with two catalytic domains, one from α -amylase and one from pullulanase . Accordius tauri . CBM41s us tauri . Interese domain , and evee domain .Streptomyces, the Fn3 domain is found in combination with CBM25. The CBM25-Fn3 domain combination is also present in Microbacterium aurum α-amylase and suggested to improve binding on starch indicated by the alteration of starch granule morphology  families do contain a CBM domain in ptomyces .Aspergillus are absent in Streptomyces. According to Aspergillus encodes two GH7-cellobiohydrolases which can be present with or without a CBM. Meanwhile, three families, GH9, GH44, and GH48, containing a CBM present in Streptomyces are completely lacking in Aspergillus. The GH12 family proteins are present in both genera, but only in Streptomyces the GH12 have a CBM. The GH5 and GH6 cellulases with a CBM are found in both genera.Interestingly, members of two GH families (GH7 and GH45) containing a CBM in Aspergillus and Streptomyces genomes, 1946 sequences of cellulases with CBM domain were identified. Among these sequences 188 were annotated as cellulase with a single CBM in Aspergillus, 1256 as cellulase with a single CBM in Streptomyces and 502 as cellulases with multiple CBMs in Streptomyces.Streptomyces cellulases are not only higher in their diversity of the CBM type, but also more varied in the domain organization compared to Aspergillus. In Streptomyces, four different CBMs associated with the GH cellulase catalytic domain were identified, including CBM2, CBM3, CBM4, and CBM46. Moreover, each CBM is not only present as a single module, but also appears in combination with other accessory domains including another CBM (CBM2&CBM4 and CBM2&CBM3). CBM2 is the most frequently found CBM (67%) among the four different CBMs in Streptomyces cellulases . A similar architecture was also found in GH5s of Bacillus halodurans which are associated with CBM46 and CBM_X2  (Another CBM present in d CBM_X2 . The CBMd CBM_X2 . Moreove modules . Besides(Avicel) .Streptomyces GH cellulases are the Fn3 domain and the variants of the X2 module CelD-N and Big7. According to the Pfam database  domain in GH cellulases, was performed by querying the genomes of both radation . Indeed,Streptomyces, in correspondence to the suggestion that it plays a role as stable linker between the enzymatic and substrate binding domain (Aspergillus GH3 is positioned at the C-terminal end, and not followed by a CBM. Several other fungal β-glucosidases carry Fn3 domains, such as those from Aspergillus aculeatus and Trichoderma reesei (Rasamsonia emersonii (As can be seen from g domain . Howevera reesei , as wellmersonii .Cellulomonas fimi cellulase disrupted the surfaces of cellulose fibers (Cellulosimicrobium sp. strain HY-13 (Flavobacterium johnsoniae (Marinifilaceae bacterium strain SPP2 (Streptomyces CBM2 domain does not result in a functional GH3 enzyme (Although Fn3 is suggested as a stable linker , many ste fibers , indicate fibers . As othein HY-13 , Flavobahnsoniae , a Marinain SPP2  and cellain SPP2 . Deletioain SPP2  and GH9 ain SPP2 . Likewisain SPP2  and GH5 ain SPP2 . Surpris3 enzyme . Another3 enzyme . KataevaIn the field of enzyme engineering, the design of chimeric enzymes has been developed as one of the promising approaches to obtain an enzyme with the desired characteristics like improved hydrolysis efficiency. A chimeric enzyme is commonly generated by fusing the GH catalytic domain obtained from one species with another protein domain such as a CBM from another species. In several studies, chimeric enzymes have been shown to improve the catalytic efficiency, thermostability, as well as substrate specificity .Streptomyces species display a much higher diversity in cellulase domain architecture than Aspergillus. This observation led us to review the Streptomyces domain architecture in more detail as the basis for design of novel chimeric enzymes with cellulolytic activity. As an example, we selected Streptomyces leeuwenhoekii C34 (Aspergillus niger.As shown above ekii C34  and AspeS. leeuwenhoekii is presented in S. leeuwenhoekii, to proteins associated with a CBM2, other than cellulases, a considerable group of enzymes such as xylanases, chitinases, LPMOs and an esterase showed up from the genome sequence, in addition to a GH3 family β-glucosidase (A. niger, resulted in only two other proteins, both related to polysaccharide degradation, being a cellulose active LMPO and a GH74 xyloglucanase (The variation in the domain organization of cellulases tethered to a CBM in cosidase . The samlucanase .Streptomyces domain architecture, a new concept to design chimeric enzymes could be studied by fusing a CBM2 domain from a Streptomyces GH3 β-glucosidase to a well-known A. niger GH3 β-glucosidase. However, it should be kept in mind that the design of new chimeric enzymes is not as simple as replacing one domain for another as shown by the example of replacing a Fn3 domain by CBM2 (In fungal cellulolytic enzyme cocktails, the β-glucosidases are considered to be the limiting factor in obtaining full conversion of cellulose to glucose. The GH3 family β-glucosidase in filamentous fungi do not have native CBM domains see . TherefoAspergillus and Streptomyces is presented in this review. The Streptomyces species harbor cellulases and amylases with a more diverse domain organization, including CBMs and other accessory domains, than found in Aspergillus. This remarkable feature provides inspiration to design chimeric enzymes with a potential for substrate degradation improvement.As functional domains, the CBMs have attractive characteristics like promoting substrate binding and thus supporting the catalytic function of enzyme. So far, CBMs have been classified in more than 80 families and more CBM families will undoubtedly be discovered. CBMs appear in association with a wide range of proteins in particular the CAZymes and are assembled in various domain organizations in numerous microorganisms. As an example, the diversity in the domain organizations in Aspergillus GH3 enzyme that does not have a CBM but does have a C-terminal Fn3-like domain, was redesigned to replace the Fn3 with a CBM2 of Streptomyces. To our surprise these studies revealed that the Fn3-like domain of the fungal GH3 seems to play a pivotal role in the enzymatic activity (Fusions of enzyme domains have been developed in many scientific studies to create improved chimeric enzymes. In one of our studies, the activity , This obStreptomyces. GV performed the bioinformatic analysis for the graphical domain annotation. AR presented ideas and edited the review. EV and PP involved in the review outline, ideas, planning, supervision, writing, and editing. All authors reviewed and agreed on the final manuscript.AS analyzed the data, wrote the review, performed the literature data mining, and designed the figures. EA conducted the cellulases analysis of EA was employed by company Sun Pharmaceutical Industries Europe BV. GV and PP were employed by company Dutch DNA Biotech B.V. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sordaria macrospora is a model organism for studying fruiting-body development. The genome of S. macrospora encodes one Lon protease with the C-terminal peroxisomal targeting signal (PTS1) serine-arginine-leucine (SRL) for import into microbodies. Here, we investigated the function of the protease SmLON2 in sexual development and during growth under stress conditions. Localization studies revealed a predominant localization of SmLON2 in glyoxysomes. This localization depends on PTS1, since a variant without the C-terminal SRL motif was localized in the cytoplasm. A ΔSmlon2 mutant displayed a massive production of aerial hyphae, and produced a reduced number of fruiting bodies and ascospores. In addition, the growth of the ΔSmlon2 mutant was completely blocked under mild oxidative stress conditions. Most of the defects could be complemented with both variants of SmLON2, with and without PTS1, suggesting a dual function of SmLON2, not only in microbody, but also in cytosolic protein quality control.Microbodies, including peroxisomes, glyoxysomes and Woronin bodies, are ubiquitous dynamic organelles that play important roles in fungal development. The ATP-dependent chaperone and protease family Lon that maintain protein quality control within the organelle significantly regulate the functionality of microbodies. The filamentous ascomycete  In fungi, microbodies comprise single membrane bound organelles, including peroxisomes, glyoxysomes and Woronin bodies, which play important roles during developmental processes ,2. Woron2O2 as well as catalases decomposing the toxic hydrogen peroxide. Glyoxysomes harbor the key enzymes of the glyoxylate cycle, but typically no catalases .,25.T. laproteins .P. chrysogenum, the absence of SmLON2 seemed not to interfere with the import of matrix proteins into glyoxysomes, since the import of DsRED-SKL into glyoxysomes was similar in the ΔSmlon2 mutant and the WT  that Smlon2 (SMAC_00912) contains six edited sites with four editing sites leading to nonsynonymous codon changes. RNA editing of the Smlon2 transcript does not affect the PTS1 motive or the catalytic dyad of the protease domain. One site K245E is located in the N-terminal LON domain, one site N594D in the ATPase domain and two, I740M and S753G, in the protease domain (Smlon2 might alter protein properties, leading to dual targeting of the protein. Previously, it has been reported that posttranscriptional A-to-I RNA editing occurs during sexual development in udogenes ,59,60. We domain . How theIn plants, two functions of LON2 were dissected. The chaperone function of LON2, but not its protease activity, suppresses autophagy. Furthermore, the protease function of LON2 was shown to interfere with its chaperone function, suggesting that intramolecular modulation between both functions of LON2 regulates degradation of peroxisomes by autophagy .S. macrospora. Our data suggest that SmLON2 might be a dually targeted protease having functions in glyoxysomes and in the cytoplasm. Which specific functions the two isoforms of SmLON2 have and whether RNA editing plays a role in fulfilling its functions remains so far elusive, but will be addressed in the future.In conclusion, SmLON2 is involved in fruiting-body development, ascosporogenesis and stress resistance in


G protein-coupled estrogen receptor (GPER), or G protein-coupled receptor 30 (GPR30), is reported to mediate non-genomic estrogen signaling. GPR30 associates with breast cancer (BC) outcome and may contribute to tamoxifen resistance. We investigated the expression and prognostic significance of GPR30 in metachronous contralateral breast cancer (CBC) as a model of tamoxifen resistance.TOT) and plasma membrane-localized GPR30 expression (GPR30PM) were analyzed by immunohistochemistry in primary  and contralateral BC , and in lymph node metastases . Death from BC (BCD), including BC death or death after documented distant metastasis, was used as primary end-point.Total GPR30 expression . In BC1 and BC2, GPR30PM associated with estrogen receptor (ER)-negativity  and progesterone receptor (PR)-negativity . The highest GPR30TOT and GPR30PM were observed in triple-negative BC. GPR30PM associated with high Ki67 staining in BC1 (p<0.0001) and BC2 (p<0.0001). GPR30TOT in BC2 did not associate with tamoxifen treatment for BC1. However, BC2 that were diagnosed during tamoxifen treatment were more likely to express GPR30PM than BC2 diagnosed after treatment completion (p = 0.01). Furthermore, a trend was observed that patients with GPR30PM in an ER-positive BC2 had greater benefit from tamoxifen treatment.GPR30PM-localized GPR30 staining is associated with increased risk of BC death when expressed in BC2 and LGL2. Additionally, PM-localized GPR30 correlates with prognostic markers of worse outcome, such as high Ki67 and a triple-negative subtype. Therefore, PM-localized GPR30 may be an interesting new target for therapeutic exploitation. We found no clear evidence that total GPR30 expression is affected by tamoxifen exposure during development of metachronous CBC, or that GPR30 contributes to tamoxifen resistance. Tamoxifen reduces the incidence of CBC, but CBC evolving during tamoxifen treatment is assumed to have intrinsic resistance. Efforts aimed to further understand resistance mechanisms have led to a number of important discoveries, including pathological epigenetic changes or mutations in the ESR1 gene, and interference with other growth stimulatory signaling pathways. These mechanisms subsequently result in augmented receptor activity, ligand-independent growth and transcription, or reduced drug sensitivity [Metachronous contralateral breast cancer (CBC) is a second, presumably independent primary tumor (BC2) developed in the contralateral breast after the first breast cancer BC1). The lifetime risk of a breast cancer (BC) patient developing CBC has been estimated at 2–20%, depending on factors such as family history, prior endocrine treatment, and age at BC1 diagnosis . The lif. Similarsitivity , 9, 10. in vitro studies have shown that GPR30 is pro-apoptotic in the ER-positive BC cell line MCF-7, but proliferative in the ER-negative cell line SkBr3 [in vitro study showed that PM localization of GPR30 is important for receptor stimulation of ERK1/2 activity [G protein-coupled estrogen receptor (GPER), originally named G protein-coupled receptor 30 (GPR30), is a receptor involved in rapid, non-genomic responses to estrogen . In contne SkBr3 . Thus, Gne SkBr3 . Consistactivity , a celluin vitro data suggest that tamoxifen directly stimulates cell growth via GPR30 [Studies have reported that GPR30 may contribute to tamoxifen resistance –24. Someia GPR30 . This siThe aim of this study was to further understand how GPR30 expression relates to BC progression, patient outcome, and previous tamoxifen treatment. To this end, we used a unique retrospective cohort of patients with CBC, either naïve or exposed to tamoxifen following BC1, with paired expression data from primary tumors and lymph node metastases, as a stepwise model of tamoxifen resistance.A previously constructed tissue-microarray (TMA) from 728 patients diagnosed with CBC between 1977 and 2007 at 14 hospitals within the Southern Swedish Healthcare Region was used. Details of TMA construction have been previously described . Patient™ Mycoplasma Detection System (ThermoFisher Scientific). HeLa TET-On/Off cells were grown in Dulbecco’s modified Eagle’s medium , 10% fetal calf serum , and 1% penicillin/streptomycin (Sigma-Aldrich), using blastidine and zeocin as clone selection markers. MCF7 cells were grown in DMEM supplemented with glucose and pyruvate, and with 10% FCS. Both cells were grown in 5% CO2 at 37°C.HFF11 cells, originally constructed from HeLa cells , were kindly provided by K. Kotarsky . HFF11 cThe specificity of the polyclonal goat GPR30 antibody  was analyzed in MCF7 human breast cancer cells (American Type Culture Collection), a cell line used extensively to study native GPR30 . ImmunobTOT;PM; TOT was scored as overall intensity at 5 levels . Percentage of stained tumor cells was scored, but as the vast majority of tumors had either 0% or >50% stained cells, the intensity was used for further analysis. The staining was visually evaluated by two independent investigators , a well-established widely accepted method to evaluate IHC staining on TMA cores, and the mean score was used, rounding to nearest integer. Due to group sizes, GPR30TOT intensity was combined to two groups of weak (levels 0–2) vs. strong (levels 3–4). GPR30PM+ scores were combined to create a binary variable (levels 0 vs. 1–2).GPR30 expression was monitored by immunohistochemical (IHC) staining with GPR30 antibody (1:50) on 1.0 mm TMA cores. Receptor expression was evaluated as total cellular staining intensity , including BC death or death after documented distant metastasis, was used as primary end-point. Median follow-up time for patients alive at last follow-up was 9.1 years from BC2 diagnosis. Statistical analyses were carried out in RStudio 1.1.442  using R 3.5.1 . Associa package . Cox pro package . ProportAll procedures performed in studies involving human participants were approved by the Regional Ethical Review Board of Lund University (LU240-01) and in accordance with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.Since the study handled saved paraffin material, often several decades old, informed consent was not possible to retrieve from all patients. Nevertheless, all data was analyzed and presented anonymously, and a note was published in the local paper, informing previous BC patients to contact the research group if they did not want their tumor tissue to be used in scientific studies. This procedure was accepted by the Regional Ethical Review Board of Lund University (LU240-01).PM+; TOT; TOT was associated with a higher fraction of GPR30PM+ tumors in both BC1 and BC2  and the BC2/LGL2 pairs (p<0.0001).To assess GPR30 expression through tumor progression, GPR30ding LGL . A majorPM+ staining in both BC2 and LGL2 was associated with increased risk of BCD . A trend was also observed that higher GPR30TOT associated with increased BCD in BC2 and LGL2 . Similar trends were seen when looking at GPR30 expression in BC1.Previous studies by our group showed that characteristics of BC2 have the highest influence on prognosis after development of CBC, although the characteristics of BC1 continue to have some prognostic impact . GPR30PMTOT and GPR30PM+ staining in BC2 were graphically associated with increased BCD among patients with ER-positive tumors, but not ER-negative tumors . To address the combined prognostic information of GPR30 status in both BC1 and BC2, we created combination variables with information from paired BC1 and BC2 for GPR30TOT and GPR30PM respectively. This analysis showed that, although BC2 carried the higher prognostic value, BC1 also added prognostic information . Additionally, when studying the effect of tamoxifen on ER-positive BC2, there was a trend that patients with strong GPR30TOT or GPR30PM+ staining had a greater benefit from tamoxifen treatment than those with lower GPR30TOT or without GPR30PM staining . Finally, the prognostic effect of GPR30 in BC2 did not seem to be affected by whether tamoxifen had been given for BC1 or not .When stratified for tamoxifen treatment of ER-positive BC2, GPR30staining . HoweverGPR30 is a G protein-coupled receptor reported to mediate non-genomic estrogenic signaling and contribute to BC progression and tamoxifen resistance. However, the literature is inconsistent regarding the pathophysiological profile of GPR30 in BC, and the receptor function is still poorly understood. In this study, we sought to clarify the role of GPR30 during BC development and progression with or without tamoxifen exposure. To this end, we used a unique retrospective cohort of CBC, serving as a model of tamoxifen resistance. We show in this material that GPR30 staining is a strong prognostic factor for increased risk of BCD, particularly when expressed in the PM. We also show that the total GPR30 staining generally decreases during tumor progression. Interestingly, we find that total GPR30 staining was unrelated to tamoxifen treatment during tumor development, and we found no clear relationship between the prognostic value of GPR30 and tamoxifen treatment.PM+) [Studies have indicated that GPR30 is influenced by the ER modulator tamoxifen, and this has been suggested to contribute to tamoxifen resistance –24. UprePM+) .To address if GPR30 expression decreases the benefit of tamoxifen, we performed a survival analysis of patients treated with tamoxifen after the diagnosis of an ER-positive BC2, stratified by GPR30 expression. In this material, GPR30 did not associate with worse prognosis. Instead, a trend was noted that among patients not treated with tamoxifen for their ER-positive BC2, PM-localized GPR30 has a higher hazard. Additionally, there was a trend that patients with PM-localized GPR30 staining in an ER-positive BC2 have a greater benefit from tamoxifen treatment. In summary, our data provides no clear evidence that tamoxifen exposure affects the prognostic value of GPR30 in BC2, or that the benefit of tamoxifen depends on GPR30 expression. However, results are not unanimous and the relationship between PM-localized GPR30 and tamoxifen needs to be further addressed in future studies.We also explored GPR30 through disease progression. The relationship between GPR30 staining in paired BC1 and BC2 appeared to be stochastic, in line with CBC being most often considered an independent primary tumor . On the www.proteinatlas.org [GPER1 differ between BC tissue and healthy controls [GPER1 gene are present in less than 2% [GPER1 mutation causes PM-GPR30 expression, which according to data from this study and earlier is present in around 7–23% of BCs [A limited amount of data exists regarding the expression of GPR30 in healthy breast tissue. However, one study assessed GPR30 in breast tissue from 12 healthy donors in which all were defined as GPR30 positive, with strong cytoplasmic GPR30 expression in ductal and lobular epithelium, myoepithelium, and stromal fibroblasts, but no expression in smooth muscle or vascular endothelium . In additlas.org ). Based tlas.org . Howevercontrols , 41. Amo than 2% –45. Henc% of BCs . HoweverAs seen here and previously , strong 2+ [2+-pump plasma membrane Ca2+-ATPase 4b [in vitro studies showing that receptor activity occurs both in the PM [Although associated with the same ligand, ER and GPR30 manifest considerable differences in terms of cellular function. ER is a nuclear receptor; a ligand-activated transcription factor that upon binding estrogen dimerizes and translocates to the nucleus, where it alters expression of target genes . On the 2+ , 19, cAM2+ , and ERK2+ , 48–50, 2+ , 48, and2+ . Further2+  and the TPase 4b , and stiTPase 4b . InteresTPase 4b . The subn the PM , 34, 35,n the PM , and nucn the PM . Today, n the PM , whereasin vitro data have demonstrated that GPR30-mediated ERK1/2 signaling depends on an amino acid sequence at the receptor intracellular C-terminal end, through which the receptor interacts with scaffold and adaptor proteins localized at the PM [Recent t the PM , and thit the PM , 35, 52.In conclusion, this study evaluated the estrogen-responsive receptor GPR30 in a unique and large cohort of CBC with long-term follow-up, serving as a model for tamoxifen exposure and resistance. We conclude that GPR30 has prognostic value in CBC. On the other hand, we find no clear evidence that GPR30 is involved in tamoxifen resistance. GPR30 staining correlates with BC subtype, with the highest total and PM-specific GPR30 observed in triple-negative BC. Additionally, GPR30 is most active in CBC when located in the PM. Thus, PM-localized GPR30 is an interesting candidate for future therapeutic exploitation.S1 Raw images(PDF)Click here for additional data file.S1 FigA, MCF7 cell lysates were immunoblotted with goat GPR30 antibody as previously described [B, HeLa TET-On/Off cells were incubated without (-TET) or with 100 ng/ml tetracycline (+TET) for 12 h and then lysed and immunoblotted with GPR30 antibody. C, MCF7 cells were stained live with GPR30 antibody for 30 min and then fixed and stained with Alexa488-labeled anti-goat antibodies (Life Technologies) as previously described [D, MCF7 cells were transiently transfected with a plasmid containing the cDNA of human GPR30 tagged in the N-terminus with the FLAG tag (FLAG-hGPR30), stained live with mouse M1 FLAG antibodies (Sigma-Aldrich) for 30 min, and then fixed and stained with Alexa488-labeled mouse IgG2b antibodies (Life Technologies) as previously described [C and D, 4',6-Diamidino-2-phenylindole (DAPI) was used for nuclear staining, and fluorescence images were collected using a Nikon Eclipse confocal microscope. The results are representative of experiments performed at least three times. Bar, 10 μm.escribed , 29, 34.escribed , 29, 34.(EPS)Click here for additional data file.S2 Figp were calculated using Wald test. A-B, cumulative incidence of BCD in relation to GPR30TOT in CBC patients with ER-positive BC2 (A) or ER-negative BC2 (B). C-D, cumulative incidence of BCD in relation to GPR30PM in CBC patients with ER-positive BC2 (C) or ER-negative BC2 (D).Cumulative incidence of competing event (death from other cause than BC) is shown for comparison. HR values were estimated using a cause-specific Cox proportional hazards model, and values of (PDF)Click here for additional data file.S3 FigTOT and PM-/PM- GPR30PM status were used as reference groups. A, cumulative incidence of BCD in relation to GPR30TOT in the tumor pair. B, cumulative incidence of BCD in relation to GPR30PM in the tumor pair.Presented HR values were estimated using Cox proportional hazards model and p values were calculated using Wald test, where the groups with weak/weak GPR30(PDF)Click here for additional data file.S4 Figp were calculated using Wald test. A-D, cumulative incidence of BCD in relation to tamoxifen treatment of BC2 in CBC patients with weak GPR30 staining (A) or strong GPR30 staining (B), and in patients without PM-specific GPR30 staining (C) and in patients with PM-specific staining (GPR30PM+) (D).Cumulative incidence of competing event (death from other cause than BC) is shown for comparison. HR values were estimated using a cause-specific Cox proportional hazards model, and values of (PDF)Click here for additional data file.S1 TableA, multivariate analyses of strong total GPR30 (GPR30TOT) adjusted for each variable separately. B, multivariate analyses of plasma membrane-specific GPR30 (GPR30PM+) adjusted for each variable separately.(PDF)Click here for additional data file.S2 TablePrognostic effect was calculated by cox proportional hazards model with Wald test. Interaction between GPR30 and the stratifying variable (ER or tamoxifen) was assessed using an interaction test. Relationship between GPR30 and risk of death from BC were assessed by Cox regressions.(PDF)Click here for additional data file.S3 TableTOT and PM-/PM- GPR30PM status were used as reference groups for survival analyses. Relationship between GPR30 and risk of death from BC were assessed by Cox regressions.Prognostic effect was calculated by cox proportional hazards model with Wald test. The groups with weak/weak GPR30(PDF)Click here for additional data file.


The aim of this study is to investigate the effects of cryogenic and room temperature pre-deformation on subsequent artificial ageing of Al–Mg–Si alloys. Naturally aged and pre-aged samples were strained to 5%, 10% and 20% at RT (25 °C) and under liquid nitrogen, and artificially aged at 185 °C. Pre-deformation generally increases ageing kinetics for both the naturally aged and pre-aged alloys, which increase in proportion to the degree of pre-deformation, and which are slightly more pronounced for the cryogenic condition. The peak strength is constant, except for when a low degree of pre-deformation is used, in which case it is slightly reduced. Cryogenically deformed samples show an increased strength and hardness, compared to samples pre-deformed at RT, when subjected to an equal magnitude of strain. This difference is reduced during artificial ageing. Synchrotron measurements reveal that this behaviour can be linked to the greater dislocation density, which is not completely recovered even after prolonged ageing at 185 °C. In the automotive industry, Al–Mg–Si alloys are preferably used as lightweight material due to their advantageous mechanical properties, such as good corrosion resistance, weldability, and great strength ,2. HowevIn the automotive industry, during the manufacturing of outer sheet panels, an additional heat treatment is applied. This heat treatment is applied after the last deformation step (deep drawing) and simultaneous to the coat of paint drying and hardening and is therefore called paint bake hardening . In receA modification of artificial ageing kinetics can not only be achieved with heat treatment prior to RT storage, but also through pre-deformation ,12. DefoIn general, deformation always occurs between the delivery of the material and the bake hardening process, because of forming operations like deep drawing. The degree of deformation varies between different processes, and also within a manufactured part itself. These variations can lead to different ageing responses and different remaining strain hardening contributions, due to different dislocation densities in the final products.The influence of RT and cryogenic pre-deformation for different degrees and delivery states  on the paint bake process have not been fully explored in the relevant literature. Moreover, a clarification on the effect that the decay of the dislocation density has upon the thermal load of a paint bake process for the above-mentioned cases is lacking. In this article, we illustrate the effect of RT and cryogenic pre-deformation on the artificial ageing response and on the alteration of the dislocation density.All specimens were produced from sheet material with 1.5 mm thickness of alloy EN AW 6016, with a chemical composition of Mg (1.1 wt.-%), Si (0.35 wt.-%), Mn (0.08 wt.-%), Fe (0.15 wt.-%) and Al . The alloy was tested in two different conditions, naturally aged (T4) and pre-aged (PA). Both conditions were solution annealed at 540 °C in a convection furnace  and water quenched. Pre-ageing was executed at 100 °C for five hours. The specimens exhibit a recrystallised microstructure with a grain size of 36 ± 12 µm. All specimen were stored in a Memmert IPP 400 incubator  at 25 °C for two weeks.After the intermediate storage, the actual measurements were carried out in the following sequence: Initially, the samples were pre-deformed via straining at either RT or −196 °C and then again stored for two weeks at 25 °C. Subsequently, a heat treatment at 185 °C for different times was applied. Directly afterwards, hardness measurements and tensile tests were carried out at RT.2), the reduced section of the tensile testing sample, as well as the extensometer, were kept in a Dewar filled with liquid nitrogen. Previous measurements with this setup are explained in detail by Gruber et al. [−1 and a gauge length of 50 mm in accordance with ISO 6892-1 [For the pre-deformation tensile test, samples were cut from the sheet material perpendicular to the rolling direction, with a width of 12.5 mm and a reduced section of 57 mm in accordance with ISO 6892-1 . Pre-defr et al. . After pr et al. . AdditioO 6892-1 . Due to 2  standards, respectively. The samples were also cut from the pre-deformed samples and stored at 25 °C for two weeks prior to the measurements. To evaluate the dislocation density independence of the heat treatment, the samples were measured in situ during heating from 25 to 185 °C, and during the 20 min treatment at 185 °C. Afterwards the diffraction patterns with respect to the azimuthal range were integrated using pyFAI software  [Additionally, synchrotron measurements were conducted using a fixed photon energy of 60 keV on the Petra III beamline P02.1 at DESY . Diffraction patterns with a beam size of 200 × 200 µm were recorded by a 2-dimensional Perkin-Elmer XRD1621 detector . The wavelength and sample to detector distance was calibrated using LaB6  and CeO France) . The cha France) , with a  France) ,28.2 pre-strained samples. In contrast, an increase in hardness at the beginning of the artificial ageing treatment can be achieved in the PA condition in In With greater pre-straining, the initial hardness increases for both conditions. Simultaneously, the ageing kinetics accelerate and the peak hardness appears earlier. Pre-straining at RT promotes the nucleation of β’’, and also leads to a faster coarsening of the precipitates and to a decrease in hardness ,17. The 2-deformed samples are greater than those of the RT deformed samples. This results from the higher strain hardening rate at lower temperatures in aluminium and its alloys [2 pre-straining. Two potentially overlapping effects can be assumed: (i) The dislocation densities are greater at the beginning of the heat treatment and remain so, to a certain extent, for up to 30 min at 185 °C, despite an increased recovery rate [Moreover, prior to the heat treatment, all hardness values of the LNs alloys . Subsequs alloys ,30. Figuery rate ; (ii) Thery rate ,18,19,202 resulted in reduced maximum hardness, while still accelerating kinetics.It can be observed from In order to gain an overview of the strength level during the paint bake hardening, tensile tests were conducted after 10, 20 and 30 min at 185 °C, and also at 0 min for the condition without heat treatment. In 2 leads to an overlap of the tensile testing curves. No significant difference in strength with the pre-deformation temperature can be seen directly after straining and at heat treatment times from 10 to 30 min. Obviously, the pre-deformation is too low to result in noticeable differences between RT and LN2. Please note that the tensile samples were stored at RT for two weeks prior to the paint bake treatment, which has been shown to already reduce increased dislocation density created via deformation in cryogenic conditions [2 yield strength before heat treatment, despite the increased strain hardening at low temperature pre-deformation. In addition to the gain in yield strength, the only indication of pre-deformation is the flow onset, especially in the not heat-treated samples. Unaffected by previous deformation temperature, this is expressed by a small plateau at the beginning of the flow onset, which disappears during the paint bake process. The stronger the applied pre-deformation, the more pronounced this plateau is and the longer the heat treatment is necessary to restore an original flow onset. This is the result of simultaneous processes during further deformation: the competition between strain hardening and softening due to dynamic recovery [In nditions . This carecovery . The florecovery ,17,18. Trecovery  which arrecovery ,20, or f2 pre-strained samples exhibit tensile test curves that have shifted to greater values. As mentioned above, the initial variation is due to a greater strain hardening rate at lower temperatures [At 10% pre-deformation in eratures  and the 2 deformed samples show a greater strength at the beginning, which deteriorates during the paint bake process but is still present after 30 min.As expected, all samples in the naturally aged T4 condition show, at the beginning of the heat treatment, a decrease in strength because of cluster dissolution and the negative effect of natural ageing see a. Howeve2 is characterized by a greater work hardening than at RT, and thus, greater strengths are achieved with the same degree of deformation, as explained previously. At 20% strain, a strength level has been already reached which exceeds the ultimate tensile strength achieved at RT. Therefore, in the case of direct subsequent deformation, early failure occurs , a material constant (α = 0.6), the shear modulus (μ = 2.5 × 104 MPa), the Taylor factor (M = 3.06) and the Burgers vector (b = 2.8 × 10−10 m) [To compare the synchrotron measurements with the tensile tests, a rough estimation of the dislocation density (ρ) with respect to stress (σ) can be obtained from the Taylor Equation (1) . It incl10−10 m) .(1)σ=σ0If only the impact of dislocation density is considered, Equation (2) leads to Δσ of ~7 MPa for alloy EN AW 6016 T4 and to Δσ of ~12 MPa for alloy EN AW 6016 PA after 20 min of artificial ageing.2 deformed samples is mainly due to a greater dislocation density of the samples deformed in cryogenic conditions, which is maintained during the paint bake process. Therefore, it is confirmed that mechanism (i) plays an important role, which assumes that the dislocation densities are greater at the beginning of the heat treatment and remain so, to a certain extent, for up to 30 min at 185 °C, which could be approved by the synchrotron measurements.This difference in stress matches very well with the results of ~8 MPa and 10–15 MPa from the tensile tests in 2 deformation during the first 30 min of artificial ageing. This could be explained by the clusters formed upon pre-ageing, which may stabilize the dislocations structure via coherency strengthening effects [In the naturally aged and pre-aged samples, the dislocation density as well as the ageing kinetics increases through cryogenic pre-deformation compared to pre-deformation. The relation of these two mechanisms is on the side of the maintenance of greater dislocation densities during heat treatment. Above all, alloy EN AW 6016 PA shows a significantly greater difference in hardness, strength and dislocation density between RT and LN effects , and thu effects .In this study, the effects of cryogenic deformation on subsequent artificial ageing were investigated. Naturally aged (T4) and pre-aged (PA) samples of alloy EN AW 6016 were strained to 5%, 10% and 20% at room temperature and under liquid nitrogen, and afterwards aged at 185 °C, for different durations. 2 pre-deformation after the full paint bake treatment. Synchrotron measurements revealed that this behaviour could be linked to the greater dislocation density, which is not completely recovered even after prolonged exposure to a temperature of 185 °C and stays at greater values in the case of cryogenic pre-deformation. The results led to a deeper understanding of the industrial paint bake process of cryogenically deformed samples.Hardness measurements and tensile tests display an acceleration of artificial ageing kinetics after pre-deformation at RT, as already known from various studies ,18,19,20