Antisense oligonucleotides (ASOs) have the ability of binding to endogenous nucleic acid targets, thereby inhibiting the gene expression. Although ASOs have great potential in the treatment of many diseases, the search for favorable toxicity profiles and distribution has been challenging and consequently impeded the widespread use of ASOs as conventional medicine. One strategy that has been employed to optimize the delivery profile of ASOs, is the functionalization of ASOs with cationic amine groups, either by direct conjugation onto the sugar, nucleobase or internucleotide linkage. The introduction of these positively charged groups has improved properties like nuclease resistance, increased binding to the nucleic acid target and improved cell uptake for oligonucleotides (ONs) and ASOs. The modifications highlighted in this review are some of the most prevalent cationic amine groups which have been attached as single modifications onto ONs/ASOs. The review has been separated into three sections, nucleobase, sugar and backbone modifications, highlighting what impact the cationic amine groups have on the ONs/ASOs physiochemical and biological properties. Finally, a concluding section has been added, summarizing the important knowledge from the three chapters, and examining the future design for ASOs. Antisense oligonucleotides (ASOs) are single-stranded (ss) oligomers composed of typically 10–25 nucleotides linked by negatively charged phosphorus-based linkages. ASOs have the distinctive ability to bind endogenous nucleic acid targets in a sequence-specific manner, thereby inhibiting gene expression and offering opportunities for the treatment of a broad range of diseases. As ASOs interact with their RNA (or DNA) targets through complementary Watson–Crick base-pairing, the sequence options of ASO lead compounds can be rationalized based on a knowledge of the endogenetic gene sequence to be targeted, thus further offering a potential against targets which are considered undruggable using conventional small-molecule drugs. The key features of ASOs further enable them to be transformed into personalized medicines, eventually even targeting patient-specific sequences and very rare diseases ..23) whicO-dimethoxytrityl-2’-O-methyluridine could be converted into an N4-triazole-modified 2’-OMe-cytidine phosphoramidite 18) [62].ynthesis .6 position has been the most explored attachment point. For example, a 2’-deoxy-N6-triazole-substituted adenosine monomer (9-(5’-O-dimethoxytrityl-2’-deoxy-β-ᴅ-erythro-pentofuranosyl)-N6-adenine) was reacted with spermine to yield 5'-O-dimethoxytrityl-N6--2'-deoxyadenosine (28) and subsequently incorporated into an ON  . I. IN-alkyN,N-(dimethylaminopropyl)phosphoramidate linkage (monomers 73 and 74) (DMAP) [N,N-diethyl-ethylenediamine phosphoramidate linkage (75) (DEED) [Rp or Sp) containing the DMAP modification were synthesized via dinucleotide derivatives obtained by a phosphitylation reaction followed by oxidative amidate coupling to create an epimeric mixture of the dinucleotide phosphoramidate-linked derivatives with subsequent separation of the two stereoisomers. These were then incorporated into the desired ONs after O3’-desilylation and phosphitylation of the dimers [Subsequently, two variants of this phosphoramidate linker strategy were synthesized, one being the ) (DMAP) , and the) (DEED) . Stereo-e dimers . The aute dimers .Tm increase of 27.0 °C for the ON/DNA duplex and 10.4 °C for the ON/RNA duplex [Tm towards their complementary RNA and DNA targets, which was ascribed to increased steric hindrance [75).Vasseur, Debart and co-workers introduced the DMAP modification into α-configured ONs –110. Theindrance , althougXenopus oocytes [The cationic phosphoramidate variant termed DEED was originally tested for its triplex-forming capabilities. The authors found that ONs containing the DEED modification were more capable at forming triplexes under conditions that approximated the magnesium, pH, and potassium levels found in vivo . A later oocytes . The stu oocytes .76) and guanidinobutyl (77) modifications were introduced into α-ONs which elicited significant stabilization of the formed ON/DNA and ON/RNA duplexes. Interestingly, towards RNA complements, these modifications resulted in more pronounced increases in Tm, relative to the DMAP modification mentioned above, with the guanidino phosphoramidate modification providing a 14.0 °C increase in Tm relative to the DMAP modification for fully modified ONs [Tm was observed for all fully modified α-TFOs irrespectively of modification  relative to the unmodified β-TFO control [The library of phosphoramidate variants was expanded when the aminobutyl phosphoramidate and the guanidinobutyl phosphoramidate were synthesized . A facilfied ONs . Further control . A cell  control .69) and aminoethyl phosphonate [70) linkages (Rp or Sp) thymidine dinucleotide linked through the 3’-5’ oxygen atoms modified with the phthalimidomethyl phosphonate linkage [Rp-isomer formed more stable duplexes with DNA and RNA complements relative to the control ON. The ONs carrying the Sp-isomer had a destabilizing effect. When tested for their nuclease resistance, an increase in stability was observed relative to their unmodified ON [69) modification was readily hydrolysed at pH 7 whereas the aminoethyl (70) modification was completely stable under the same conditions [Utilizing the phosphorus atom as an attachment point for cationic aminoalkyl groups has been employed with slight variations. Fathi, Cook and co-workers successfully introduced aminomethyl phosphonate  (69) andsphonate  (70) linlinkages . The int linkage . Later,  linkage . The des linkage –114. Forified ON –114. Int78–81 was accomplished as shown in 78 showed good yield in both phosphoramidite synthesis and coupling efficiency on the synthesizer, significant loss of the hydrocarbon-linked group was observed during the alkaline deprotection conditions [79 did not show any significant increase in Tm. This trend was changed when the modification 81 was inserted into a 21-mer 2’-OMe RNA-ON and hybridized to a complementary 2’-OMe RNA complement. Relative to the unmodified 2’-OMe RNA sequence, the modified ON carrying six insertions of monomer 81 gave an increase in Tm of 11 °C. The serum stability of modification 81 was evaluated in PBS/bovine serum at 37 °C for 8 h. Here, a significant improvement over the unmodified 2’-OMe RNA was observed for modification 81 [79–81 ethoxy)-2-propyl and bis(2-(dimethylamino)ethoxy)-2-propyl) were tested for their cell uptake properties. The BCNS carrying the dimethylamino groups had a higher effect on the cellular uptake for 5’-FAM-labelled ASOs relative to the oligomers carrying BCNS being primary amines. However, both modifications demonstrated an improved cellular uptake relative to the unmodified 5’-end FAM-labelled 2’-OMe ASO [A new class of internucleotide linkages has recently been introduced, termed branched charge-neutralizing sleeves (BCNSs). These cationic internucleotide linkages were synthesized through conventional phosphoramidate chemistry with a slight variation. In contrast to the standard method, bis(diisopropylamino)chlorophosphine was first reacted with either of the three diaminoalcohol groups, before subsequent phosphitylation with the DMT-protected nucleosides . After inditions . After iation 81 . The two-OMe ASO .2O 1:9) at 45 °C [82). However, cleavage products formed by guanine alkylation prompted a switch to a 12-mer sequence containing only cytosine and thymine [Another strategy to introduce cationic aminoalkylated moieties onto phosphorus atoms of the ON backbone involves aminoalkylated phosphorothioate linkages. In general, modifications of the backbone have been used in the context of the phosphodiester linkage, while only a few examples can be found for the PS linkage –121. Rahat 45 °C . Initialat 45 °C  (modific thymine .Rp or Sp) gave the aminoalkyl–PS linkages shown in Rp stereoisomer of the aminoalkylated PS linkage improved the stability of the formed duplexes, while the Sp stereoisomer destabilized the formed duplexes, all relative to their PS-ON controls. Against the complementary RNA target, the Rp stereoisomer either had a similar or slightly lower Tm than the control ON, while the Sp stereoisomeric linkages destabilized the formed aminoalkylated PS-ON/RNA duplex significantly. When tested for their triplex-forming properties, a similar pattern was observed. The Rp stereoisomeric linkages gave an improved stabilization of up to +6 °C, with the non-cyclic diamine (monomers 87 and 88) stabilizing duplex formation the most, while all Sp stereoisomeric linkages gave lower Tm than their control [82) and aminohexyl (86) PS linkages was tested relative to the PS-ON control. Interestingly, both modifications showed an improved resistance relative to the control ON; however, the Sp-aminoethyl (82) PS-linkage was slightly better than the Sp-aminohexyl (86) linkage while the opposite was true for the Rp stereoisomeric linkages [88) PS linkage was too heat-labile for nuclease tests [Conjugation of the desired aminoalkyl moieties with the stereo-pure PS-ON . Additionally, the dependency of the position of the modification had a large effect, since changing the position, for a single modification from the 5’-end to the middle and then to the 3’-end, resulted in different Tm increases of 7 °C, 1 °C, and 12 °C, respectively, for the ON/RNA duplex, relative to the DNA control [91 [Filichev and co-workers have recently synthesized a variant of the phosphoramidate internucleotide linkage  and have control . At pH 5 control . At pH 6trol [91 .The concept of introducing cationic backbone linkages extends beyond conjugating cationic aminoalkyl groups to the phosphorus atom which, however, is beyond the scope of this summary. For a more in-depth account of cationic backbone modifications, we direct the reader to a recent review by Meng and Ducho .Rp stereoisomer is generally the preferred isomer for improved ASO/RNA stabilization. These considerations relate to the overall design of the ASOs, since the gapmer design allows for sugar-modified nucleotides to be used on the flanks, while for a mixmer- or a fully modified ASO a more diverse composition might be used.Cationic amine- and polyamine-conjugates/derivatives have the potential to improve the properties of ASOs for defined applications. Many different aspects need to be considered when optimizing an ASO for a defined application. This includes the position of a modification as well as the chemical composition of the cationic group, but also what scaffold the cationic group is to be attached to, i.e., is it a 2’-amino-LNA nucleotide or the common DNA/RNA nucleotides. This difference is reflected when evaluating groups attached to the base and sugar moieties. When the cationic group is connected via the nucleobase, thus allowing the group to be positioned in the major groove of the duplex, an increased target binding affinity is usually seen relative to modifications on the furanose ring (minor groove). However, when the 2’-amino-LNA scaffolds is used, a very high duplex stability is seen irrespectively of the target (DNA or RNA), as a synergistic effect between the locked sugar conformation and the cationic moiety emerges. Additionally, sugar modifications tend to bring higher nuclease resistance compared with nucleobase modifications. However, a drawback is the lack of RNase H activation for most of the sugar-modified derivatives. This is contrary to the nucleobase-modified variants that are in general well tolerated RNase H substrates. Another important aspect is the overall net-charge and the charge density of the ON. As the sugar and nucleobase substitutions compensate for the anionic charge carried by the PO and PS-backbone, a more densely charged ON is created which can result in synthetic difficulties. This can be circumvented by using internucleotide linkage modifications which generate net neutral or net positive ASOs, which usually have a high resistance towards nuclease degradation. Additionally, the internucleotide modifications result in ASOs that can stabilize the ASO/RNA duplex to a relatively high degree, although the Another consideration is the use of more densely modified nucleotides, i.e., carrying both modified base and sugar moieties. Although not as extensively explored, this strategy was employed by Fox and co-workers who elaborated on a 2’-deoxyuridine variant carrying a 1-propargylamino group  that had


ADAMTS13  cleaves von Willebrand Factor (VWF) multimers to limit the prothrombotic function of VWF. The deficiency of ADAMTS13 causes a lethal thrombotic microvascular disease, thrombotic thrombocytopenic purpura (TTP). ADAMTS13 circulates in a “closed” conformation with the distal domain associating the Spacer domain to avoid off-target proteolysis or recognition by auto-antibodies. However, the interactions of the distal TSP8 domain and the Spacer domain remain elusive. Here, we constructed the TSP8-Spacer complex by a combination of homology modelling and flexible docking. Molecular dynamics simulation was applied to map the binding sites on the TSP8 or Spacer domain. The results predicted that R1075, D1090, R1095, and C1130 on the TSP8 domain were key residues that interacted with the Spacer domain. R1075 and R1095 bound exosite-4 tightly, D1090 formed multiple hydrogen bonds and salt bridges with exosite-3, and C1130 interacted with both exosite-3 and exosite-4. Specific mutations of exosite-3 (R568K/F592Y/R660K/Y661F/Y665F) or the four key residues (R1075A/D1090A/R1095A/C1130A) impaired the binding of the TSP8 domain to the Spacer domain. These results shed new light on the understanding of the auto-inhibition of ADAMTS13. ADAMTS13 or auto-antibodies against ADAMTS13, impairs its proteolytic activity and eventually leads to TTP [ADAMTS13 is a metalloprotease that comprises a metalloproteinase (MP), a disintegrin-like (Dis), a thrombospondin-1 type 1 repeat (TSP1), a cysteine-rich region (Cys), a Spacer, seven additional type 1 repeats TSP2–TSP8), and two CUB  domains [–TSP8, ans to TTP .It is believed that wild-type (WT) ADAMTS13 circulates in a “closed” conformation in which the distal TSP8-CUB2 domains interact with the Spacer domain, resulting in impaired proteolytic activity ,6,7. ThiThe Spacer domain is a key player in mediating the auto-inhibition of ADAMTS13. Exosite-3 , the key epitope recognized by the auto-antibodies produced by TTP patients, is located on the Spacer domain ,13,14 anIn 2017, Kieron South and co-workers demonstrated that although both CUB1 and CUB2 domains bound to exosite-3, only the former was capable of inhibiting the proteolytic activity of MDTCS, a proximal truncated construct of ADAMTS13 with high activity . RecentlTo characterize the interaction of TSP8-Spacer and predict the key residues on the TSP8 domain, we used computational approaches to construct the TSP8-Spacer complex and analyzed the interaction of the TSP8 domain and the Spacer domain at the atomic level with MD simulation. Arg1075, Asp1090, Arg1095, and Cys1130 on the TSP8 domain were predicted to be critical in stabilizing the TSP8-Spacer complex. GoF mutation on exosite-3 weakened the interactions of Asp1090 with exosite-3, but enhanced the bindings of Arg1075, Arg1095, and Cys1130 to exosite-4. Mutation of these four predicted residues on the TSP8 domain to Ala impaired the binding of the TSP8 domain to the Spacer domain.Ninety-two percent of TSP8 residues were modeled with 99% confidence with its template, thrombospondin-1 TSR domains 2 and 3 (PDB: 3R6B). After docking by SwarmDock Server, 127 TSP8-Spacer complexes were obtained. According to the two criteria (Methods), two potential TSP8-Spacer complexes, 67d and 47d, were selected. The democratic scoring  displayeNHB) on the interface of Model A was ~6 with a Gaussian distribution, ~3-fold higher than that of Model B, suggesting that Model A was more stable than Model B  of heavy atoms showed that both models reached their equilibrium state at 20 ns A. Furthe Model B B. The st Model B C. The in Model B D. We als Model B . During NHB across the interface of three complexes were fitted to the Gaussian distribution . The time courses of the heavy-atoms RMSD ascended rapidly, fluctuated, and finally reached a stable plateau after 30 ns around 4.0 Å, 5.0 Å, and 7.5 Å for WT1, WT2, and WT3 , indicatribution , implyinNHB on the interface of three complexes were fitted to a Gaussian distribution  simulations . Thereforibution B, and thConsidering that each FMD simulation obtained a possible conformational state of the TSP8-Spacer complex, we analyzed the H-bonds and salt bridges across the interface of all three complexes. It was reported that exosite-3 was essential in mediating the auto-inhibition of ADAMTS13 ,12,20. OInterestingly, Glu634, Asp635, and Arg636 on the Spacer domain formed H-bonds and salt bridges with residues on the TSP8 domain with high survival ratios. Glu634, Asp635, and Arg636 are the residues of exosite-4 that play important roles in the proteolysis of VWF73 and multimeric VWF . Asp635 Arg1075, Asp1090, Arg1095, and Cys1130 on the TSP8 domain formed 11 out of 16 H-bonds and all four salt bridges with residues on the Spacer domain . More imTaken together, both exosite-3 and exosite-4, particularly Glu634, Asp635, and Arg636 in the latter, were critical for the TSP8-Spacer interactions. Arg1075, Asp1090, Arg1095, and Cys1130 were predicted to be the key residues on the TSP8 domain.NHB distributions of GoF1, GoF2, and GoF3 were fitted to the Gaussian distribution, with a mean NHB of 6.856 ± 1.564, 6.099 ± 1.641, and 4.628 ± 1.336, respectively (It was reported that after mutating the five key residues (R568K/F592Y/R660K/Y661F/Y665F) on the Spacer domain, the conformation of ADAMTS13 converted to an “open” state and its catalytic efficiency increased , indicatectively .NHB was reduced. The mean NHB of the GoF complex was less than that of the WT complex. Additionally, the key binding sites on the Spacer domain that stabilized the complex were changed from two to one. Interactions of exosite-3 with the TSP8 domain were dramatically reduced. Only a newly formed H-bond  and two weak salt bridges (Lys568-Asp1090 and Lys660-Asp1090) existed between the GoF exosite-3 and the TSP8 domain. Their average survival ratios were 0.129 ± 0.105 , 0.156 ± 0.127 (Lys568-Asp1090), and 0.167 ± 0.136 (Lys660-Asp1090), respectively  on the TSP8 domain are critical, these four residues were all mutated to uncharged Ala to obtain the Mut complex. The Mut complex was subject to 40 ns equilibrium simulation thrice, and three Mut complexes  were obtained. The RMSD of heavy atoms for the Mut1, Mut2, and Mut3 fluctuated around 4.0 Å, 5.5 Å, and 5.0 Å, respectively . The meamulation . Of noteNHB on the interface of these Mut complexes decreased dramatically  was significantly higher than those of the GoF complex (5.276 ± 1.803) and the Mut complex (2.064 ± 2.381). In addition, the mean NHB of the GoF complex was significantly higher than that of the Mut complex. The corresponding interaction energy for the WT, GoF, and Mut complexes were −356.8 ± 1.143, −330.1 ± 0.889, and −134.1 ± 0.884 kcal/mol, and the differences among these three complexes were statistically significant (p < 0.0001)  and Mut complex (647.6 ± 2.415 Å2), suggesting that mutations either in the Spacer or TSP8 domain partially exposed the hydrophobic binding core  B. The ining core C. These 3 MCAo model, and reduced tissue hypoperfusion in a transient MCAo model of ischemia/reperfusion (I/R) injury. In the present study, we constructed the TSP8-Spacer complex by homology modelling and performed MD simulations. The key residues predicted in the present study, Arg1075, Asp1090, Arg1095, and Cys1130 on the TSP8 domain, might provide potential targets for new drug development.Plasma exchange, the main therapy for acquired TTP (aTTP), still has a mortality rate of 10–20% in aTTP treatment . The maiIt is well established that exosite-3 is crucial in mediating ADAMTS13 auto-inhibition ,6,8. GoFIn the present study, the GoF complex did not dissociate during 40 ns FMD, which is not consistent with previous reports. Under electron microscopy or small-angle X-ray scattering, the GoF ADAMTS13 adopts an “open” conformation ,7. The lOverall, we investigated the interaction of the TSP8 domain with the Spacer domain. MD simulations were used to depict the key residues on the TSP8 domain. The results revealed that Arg1075, Asp1090, Arg1095, and Cys1130 played important roles in maintaining the stability of the WT complex. Moreover, mutation of these four residues impaired the TSP8-Spacer binding, leading to an unstable complex.The 3D structure of the Spacer domain (residues: Ser556-Pro682) was derived from the crystal structure of ADAMTS13-DTCS resolved by Akiyama et al. in 2009   to perfoA region of the Cys domain, the adjacent N-terminal domain of the Spacer domain [The SwarmDock Server was utilized to do the flexible docking , withoutr domain .Subsequently, these two complexes were subject to the energy minimization and 20 ns equilibration simulation with NAMD  to analyze and select the most stable model for subsequent experiments. The optimal TSP8-Spacer docking complex was then subjected to energy minimization, equilibrium, and FMD simulations thrice to re-analyze the stability of the WT TSP8-Spacer complex .Based on the FMD simulation of the WT TSP8-Spacer complex, we mutated the predicted key residues on the TSP8 domain to Ala to obtain the TSP8-mutated complex (named Mut complex). We also mutated exosite-3 (R568K/F592Y/R660K/Y661F/Y665F) of the Spacer domain to obtain the GoF Spacer-TSP8 complex (named GoF complex). These two complexes were then respectively subjected to three independent energy minimization, equilibrium, and FMD simulations to further explore the effects of these mutations on the interaction of the Spacer domain and the TSP8 domain.+ and Cl− were added to the system at a concentration of 150 mM to mimic the physiological environment. Particle mesh Ewald (PME) [By utilizing VMD, the WT, GoF, and Mut complexes were immersed into a rectangular box composed of TIP3P water molecules. Protein atoms were 25 Å away from the wall. Nald (PME)  was usedNHB on the interface were analyzed to determine whether the system had been equilibrated. When three different equilibrated poses for a complex were obtained, they were used as the initial conformation for the following FMD simulations in parallel. The FMD simulation was performed for 40 ns without controlling the temperature and pressure, which is similar to the NVE ensemble [The system was subjected to the energy minimization in the CHARMM27 all-atom force field  with NAMensemble . Figure An H-bond was defined as a bond with an angle greater than 150° and a distance less than 3.5 Å between the donor and acceptor atoms. A salt bridge was defined as a bond formed by -O on the side chain of acidic amino acid and -N on the side chain of basic amino acid, with the bond length less than 4 Å. The survival ratio of an H-bond or a salt bridge was defined as the bond dwell time over the simulation duration, which was used to characterize the thermal stability of a complex. We used heat maps to illustrate H-bonds and salt bridges with an average survival ratio greater than 10%. The key residues were defined if those residues formed H-bonds or salt bridges with an average survival ratio greater than 30%.E) of a complex with a combination of the van der Waals energy and electrostatic energy.We used VMD tools to analyze the trajectories of all simulations. Cα-RMSD was applied to compute the deviation of a conformation from the initial state and describe the conformational stability . The SASNHB was fitted with Gaussian distribution. For a two-group comparison, the unpaired Student’s t-test (parametric) was used. For a multiple-group comparison, one-way ANOVA was used with a Bonferroni post hoc test. The value of p < 0.05 was considered statistically significant. GraphPad Prism 8.0.1 software was used to analyze the data.The


Listeria monocytogenes, is a multi-domain protein that binds and activates the human receptor tyrosine kinase MET. Three new InlB crystal structures reveal the relative orientation of the first two domains  for the first time. A recurrent packing contact formed by all five crystallographically independent molecules highlights functionally important binding sites in the internalin domain and the B repeat.InlB, an invasion protein from the facultative intracellular pathogen  392), there was no interpretable electron density for the B repeat. Here, three InlB392 crystal structures in which the B repeat is resolved are described. These are the first structures to reveal the relative orientation of the internalin domain and the B repeat. A wild-type structure and two structures of the T332E variant together contain five crystallographically independent molecules. Surprisingly, the threonine-to-glutamate substitution in the B repeat substantially improved the crystallization propensity and crystal quality of the T332E variant. The internalin domain and B repeat are quite rigid internally, but are flexibly linked to each other. The new structures show that inter-domain flexibility is the most likely cause of the missing electron density for the B repeat in previous InlB structures. A potential binding groove between B-repeat strand β2 and an adjacent loop forms an important crystal contact in all five crystallographically independent chains. This region may represent a hydrophobic ‘sticky patch’ that supports protein–protein interactions. This assumption agrees with the previous finding that all known inactivating point mutations in the B repeat lie within strand β2. The groove formed by strand β2 and the adjacent loop may thus represent a functionally important protein–protein interaction site in the B repeat.InlB, a bacterial agonist of the human receptor tyrosine kinase MET, consists of an N-terminal internalin domain, a central B repeat and three C-terminal GW domains. In all previous structures of full-length InlB or an InlB construct lacking the GW domains (InlB The N-terminal internalin domain  is characterized by a central leucine-rich repeat (LRR) region that binds to the first Ig-like IPT domain of MET with high affinity  higher potency in inducing MET phosphorylation than InlB321 . Interestingly, the InlB392 variants T332E and I334D/T336L , while the internalin domain alone stimulated cell scatter at 10 nM. Thus, InlB392 variants with a single substitution in the B repeat are substantially less active than a construct that completely lacks the B repeat. One possible mechanistic explanation for this perplexing result is that the wild-type B repeat contributes to MET dimerization, whereas the T332E or I334D mutation hinders dimerization of the internalin domains  and the inactive variant InlB392_T332E. While the wild-type protein was difficult to crystallize and the rare crystals generally showed no or rather low-resolution diffraction, we obtained crystals of InlB392_T332E under several conditions and were able to determine two structures at resolutions of 2.05 and 1.8 Å. Both domains are resolved in all three structures, revealing the arrangement of the internalin domain and the B repeat in InlB for the first time.Here, we describe the crystallization and structure determination of wild-type InlB2.2.1.et al. . All proteins were crystallized in MRC 2-well plates with drops consisting of 100 nl protein (10 mg ml−1) and 100 nl reservoir solution set up using a Crystal Gryphon robot (Art Robbins Instruments). All crystals were harvested directly from commercial or homemade standard screens without further optimization. InlB392_wt crystallized at 20°C in the MORPHEUS screen  pH 7.5, 20%(v/v) ethylene glycol, 10%(w/v) PEG 8000, 30 mM diethylene glycol, 30 mM triethylene glycol, 30 mM tetraethylene glycol, 30 mM pentaethylene glycol]. For harvesting, crystals were cryoprotected in reservoir solution to which an additional 20%(v/v) ethylene glycol was added before flash-cooling the crystal in liquid nitrogen. Crystal form I of InlB392_T332E was obtained at 4°C in the MORPHEUS screen condition G2 . These crystals were harvested directly from the crystallization drop and flash-cooled in liquid nitrogen without the addition of a cryoprotectant. Crystal form II of InlB392_T332E was obtained at 4°C in condition D3 of a homemade PEG smear screen with low- and broad-molecular-weight PEGs as described in Chaikuad et al. . These crystals were cryoprotected in reservoir solution additionally containing 15% glycerol before flash-cooling in liquid nitrogen.All three proteins were expressed and purified as described in Bleymüller  al. 2016. For cry al. 2015. The res2.2.392_wt were collected on beamline P13 operated by EMBL Hamburg at the PETRA III storage ring . Between 240 and 360° of fine-sliced data (0.1° per frame) were collected. The data were indexed and integrated with XDS  restraints and restraints to InlB392_T332E crystal form II (PDB entry 7nms) as a reference model were applied. Refinement statistics are shown in Table 1The structures were solved by molecular replacement with 3.3.1.392. Crystals only grew in condition E6 of a MORPHEUS screen . Likewise, there are no major differences between the B repeats in the InlB392 structures and the published high-resolution structure of the isolated B repeat, with the exception of chain C in the InlB392_wt structure, which shows no clear density for residues 354–372 corresponding to strand β3 and the following loop in the other B-repeat structures . These residues may be flexible or adopt multiple conformations which cannot be resolved at this rather low resolution of 3.3 Å.As expected, there are no major differences between the internalin domains in the InlBon Fig. 1b. Likewes Fig. 1c. Thesea–2c). There are no polar contacts between the internalin domain and the B repeat to stabilize their arrangement. Many internalins have at least one domain C-terminal to the LRR or IR region .The orientation of the B repeat relative to the internalin domain varies substantially Figs. 2a–2 ▸c.et al., 2001392 chains reported here there are hydrogen bonds between the backbone carbonyl O atom of Glu321 and the backbone NH of Ala340 and between the side-chain carboxylate of Glu321 and the phenolic OH of Tyr323 . The only interaction of Glu321 and the internalin domain is a CH–π interaction of the aliphatic part of the glutamate side chain with the aromatic ring of Phe290.Glu321 was previously regarded as part of the internalin domain . The packing of InlB392_T332E crystal form II is different.None of the structures contained a twofold-symmetric arrangement of InlBis Fig. 3. Accordi392_wt and InlB392_T332E crystal form I is formed between the concave LRR side of one molecule and the cap region and the convex LRR side of another molecule , while the previously described LRR–cap contact has a positive ΔiG ranging from −0.4 to 16.7 kJ mol−1 (mean 5.4 kJ mol−1). The overall arrangement of the contact between the B repeat and the IR region is similar for all five instances of InlB392 described in this paper. The precise geometry varies somewhat and two groups can be distinguished. One group has three members, namely both crystal forms of InlB392_T332E and the B repeat of chain C packing against the IR region of chain A in InlB392_wt . The second group comprises two crystal contacts of InlB392_wt, namely the B repeat of chain B packing against the IR region of a symmetry-related chain B and the B repeat of chain A packing against the IR region of chain C . The different geometry of the two contact groups is illustrated in Fig. 4c). In the T332E variant, the mutated residue Glu332 substantially contributes to formation of this crystal contact and is well defined in the electron density .However, all five crystallographically independent InlBwt Fig. 4a. The s C Fig. 4b. The dty Fig. 4d.iG and is formed by all molecules. Potential functional implications will be considered in Section 4Taking into account the packing of all three crystal forms described in this paper, the binding of the IR region to the groove between B-repeat strand β2 and the loop connecting β2 and β3 is clearly the dominant packing interaction. This crystal contact has the largest mean interface area, the most negative Δ4.4.1.et al., 2016et al., 2016The T332E substitution is one of two point mutations that we had previously found to have a negative effect on the biological function of the B repeat  but an almost neutral effect of Glu332 (ΔiG ≃ 0 kJ mol−1). One explanation for this unexpected effect of the T332E substitution would be that the B repeat evolved to prevent fortuitous binding to the IR domain and that the T332E mutation counteracts this anti-aggregation property.Intriguingly, wild-type InlB4.3.et al., 2002α atoms of residues 319 and 393 in the Protein Data Bank. The distance to residue 393 of a symmetry-related copy of the GW domains . Given the highly flexible linkage between the internalin domain and the B repeat, this suggests that crystal-packing forces govern the observed orientations of the B repeat.Upon overlaying all five copies of InlBns Fig. 5. For cha4.4.et al., 2008et al., 2008et al., 2007392 structures (Fig. 6Strikingly, the contact between B-repeat strand β2 and the IR region of a neighbouring molecule is similar in all five chains. This is unusual as monomeric proteins rarely have common interfaces in more than one-third of their crystal forms and generally much less (Xu es Fig. 6. Upon bii.e. specific surface patches with properties that are thermodynamically favourable for cohesive interactions (Derewenda & Godzik, 2017iGP-value reported by the PISA server. The ΔiGP-value is a measure of interface specificity. A P-value larger than 0.5 means that the interface is less hydrophobic than it could be and therefore the interface is likely to be an artefact of crystal packing. A P-value smaller than 0.5 indicates an interface with a hydrophobicity that is higher than the average for the given structures would be, implying that the interface surface can be interaction-specific (Krissinel & Henrick, 2007iGP-value of 0.62, indicating a pure crystal-packing contact. In contrast, the interface between the B repeat and the IR region that is present in all structures has a ΔiGP-value of 0.28, indicating higher hydrophobicity than would be expected on average (Fig. 7Instead, we assume that this is a fortuitous interaction between two binding sites lacking their native binding partner. The MET binding site in the IR region and strand β2 of the B repeat may thus represent ‘sticky patches’, ge Fig. 7.Y. enterocolitica type III secretion protein SycD. Recombinantly produced SycD showed weak, concentration-dependent homodimerization in solution. Two crystal forms showed putative homodimers; however, the arrangement of protomers in these dimers differed (Büttner et al., 2008Aeromonas hydrophila homolog AscH with AopB suggested that SycD probably employs this region of TPR1 for interaction with its binding partner YopB, when present (Nguyen et al., 2015We previously observed a similar phenomenon for the et al., 2016392 structures presented here are compatible with the role of the B repeat in MET activation that we proposed previously. However, additional experiments will be required to corroborate or disprove this model, as we did not observe homodimerization of the B repeat even in crystals of wild-type InlB392.Our assumption that the hydrophobic groove formed by strand β2 and the loop connecting strands β2 and β3 forms the primary ligand-binding site in the B repeat Fig. 7 is suppo5.392, as the B repeat needs to adopt yet another position to fit into the structure of full-length InlB. Therefore, additional crystal forms and further structures are likely to show even larger inter-domain movements.While crystal-packing contacts are often suspected to lead to structural artefacts, they can also help to reveal functional information when comparing different packing environments. Here, five crystallographically independent molecules reveal protein dynamics, highlighting the high inter-domain flexibility between the internalin domain and the B repeat of InlB. However, the five structures presented here apparently do not completely map out the conformational space of InlB392 involves the known binding site for the MET Sema domain in the IR region and a surface patch in the B repeat that has previously been shown to be functionally important through mutagenesis and cellular assays. The structures analysed in this paper thus represent a good example showing that recurrent crystal contacts can highlight physiologically relevant binding sites. Due to the extremely high protein concentration in crystals, crystallography allows the visualization of protein–protein interactions that are too weak to be studied in solution. The biological relevance of such contacts needs to be addressed by complementary functional assays (Kobe et al., 2008et al., 2007et al., 2021et al., 2021The largest packing contact that is similarly formed by all five crystallographically independent instances of InlB392, wild type, 7pv9InlBPDB reference: 392_T332E, crystal form I, 7pv8InlBPDB reference: crystal form II, 7nmsPDB reference: 10.1107/S2059798322000432/jb5037sup1.pdf. DOI: crystal form II, 7nms


Despite the ubiquity of physical systems evolving on time-dependent spatial domains, understanding their regular and chaotic dynamics is still in a rudimentary state. While chaos implies that the system’s behavior can be altered by small perturbations, this sensitivity proves to be useful for control purposes. Here we report on the experimental discovery of a novel mechanism to control chaos by time-variation of the system  size: depending upon the rate of the latter, the chaotic state may be completely prevented. Our experimental observations are disentangled with theoretical insights and numerical modeling, which also reveals the ability to control spatio-temporal chaos, thus making the findings relevant to a wide range of natural phenomena. To illustrate how one can traverse the edge of chaos by varying the domain size, as a testbed we have chosen the Faraday waves phenomenon2, which is a paradigmatic example in pattern-forming systems, known to exhibit temporal chaos as well. When a container is filled with a liquid and vibrated with sufficient acceleration A in the direction of gravity, standing surface waves, historically named after Faraday2, are formed and oscillate at a frequency f. Such waves exhibit patterns with a large variety of shapes and symmetries  takes place in the overlap region of the two stability curves commonly known as the pattern competition regime5. In this regime slow amplitudes 7. According to -space surrounding the pattern competition regime of the modes L(t). Four distinct regions are observed above the threshold (thick curve): (a) chaotic competition between the two modes; (b) periodic competition with a time-dependent envelope; (c) periodic competition with a time-independent envelope; and (d) the pure mode  oscillating periodically with a time-independent envelope. The shaded areas represent the hysteretic regions: if one starts from below the threshold curve and increases the amplitude, in the lowest shaded area a flat surface is observed, but if the starting point is in region (a), then by decreasing the amplitude and entering the shaded area, chaotic competition is exhibited. The surface patterns presented in Fig. To navigate the experiments on time-dependent domains, first we developed the roadmap on the time-fixed domain analogous to that in the literaturementclass2pt{minimmentclass2pt{minimkey observations can be made from Fig. First, the fractal dimension 10 in which Second, using the initial phase and envelope within the time-independent periodic pure mode regime as the reference, during the domain growth  over 11 and therefore requires a longer transition regime in order to regularize the system. Indeed, on the one hand, since a chaotic set, on which the trajectory of the chaotic process lives, has embedded within it a large number of unstable low-period orbits, sensitive dependence on small changes to the chaotic state implies that the system’s behavior can be altered by using smaller (domain) perturbations for larger We start by illustrating how domain growth can lead to regularization of the chaotic regime corresponding to entclass1pt{minimaentclass1pt{minimaentclass1pt{minimaentclass1pt{minimaentclass1pt{minimaentclass1pt{minimaC  in time, since these two methods to control dynamics may seem to be interrelated. Indeed, from the dispersion relation mentclass2pt{minim0mm Fig. . To be amentclass2pt{minimmentclass2pt{minimmentclass2pt{minimC of f crosses the border of the Arnold tongue leading to the loss of synchronization (zero detuning) between the driving and oscillator frequencies and thus to infinite growth of the phase difference13. This growth is not uniform: there are epochs when the phase difference is nearly constant, and other, much shorter, epochs where the phase difference changes relatively rapidly by 14 as in our case, cf. Figs. 15 and could be the result of some symmetries underlying the dynamical system16.Considering that in Fig. entclass1pt{minimaentclass1pt{minimaentclass1pt{minimaC in Fig. C of both modes. In general, while frequency chirping leads to a “domain flow” linear term in the corresponding amplitude equation17 similar to that for the actual domain flow18 in equation  and the total phase changes by 25.Spatially extended  systems are prone not only to temporal (amplitude) chaos, which is well studied in finite-dimensional contexts, but also to spatio-temporal chaos26. Although the Faraday wave phenomenon is two-dimensional and would require two coupled GLEs to describe the two cross-roll hydrodynamic system27, its qualitative understanding can still be achieved using a single 1D complex GLE (cGLE) evolving on the slow time 22:18 due to domain evolution v; here x. Chaotic behavior exhibited by the cGLE on time-fixed domains has been extensively studied28 and will be used as a reference. Depending on the values of 28. The chaotic regime occurs beyond the Benjamin-Feir-Newell curve 28. When U has no zeros, |U| remains saturated and only its phase will be dynamically active, leading to phase chaos. On the contrary, if U vanishes at some point x, then the complex phase is undefined there and a phase slip occurs leading to defect chaos. In the bichaotic regime, taking place for the values of 22.To gain further insights into controlling chaos by variation of the domain size, as a minimal model we study the Ginzburg-Landau equation (GLE) universally valid near critical points of pattern forming systemsWe first consider domain shrinkage and its reverse process in analogy with experiments in Fig. beta )=0.5,-1.4$$\1 in 1D and experimentally in 2D29, thereby leading to a faster change in the wavenumber structure of a pattern. Hence, if the route to chaos requires the change in the wavenumber structure of the original periodic mode, then the difference in the phase-slip occurrence characteristic times between domain shrinkage and growth is responsible for the observed asymmetry. If, on the other hand, the route to chaos from a given original periodic mode simply requires an excitation of another mode, the interaction of which with the original one leads to temporal chaos 6, then the difference lies in the subcritical nature of the transition to chaos, which can occur directly. The latter happens in other contexts, e.g. when a modulationally destabilized monochromatic wave in a fluid system undergoes a subcritical bifurcation directly into chaos, provided dissipation is weak enough30. It is known that finite amplitude perturbations are needed in order to trigger subcritical instabilities32. In the context of Faraday waves, such finite-amplitude perturbations are always present, e.g. in our system due to meniscus waves and wall motion when domain size is varied. The direct transition from periodic to chaotic via subcritical bifurcation is justified not only by the periodic and chaotic regions being adjacent33 in the amplitude-frequency -plane, but also by the fact that superposition of modes, where chaos is observed, belongs to the subcritical side of the resonant tongue8. Given that in the case of domain shrinkage modes from the subcritical side of the resonant tongue are excited via a finite-amplitude instability, while in the case of growth modes from the supercritical side emerge, one indeed expects irreversibility and therefore hysteretic behavior of Faraday waves29, which is fundamentally due to viscous dynamics of the bulk flow underlying the surface pattern34.An important observation from the results of integration in Figs. entclass1pt{minimaIn the course of the above numerical investigation, we also observed that domain shrinkage is more effective at controlling chaos; for instance, the domain growth fails to regularize the stronger chaotic state presented in Fig. 29 showing that pattern formation is naturally impeded during fast domain evolution as phase slips have no time to develop. Furthermore, these observations are consonant with the 1D theoretical findings35 indicating that more complex pattern sequences can be expected during slow domain evolution, which allows for more phase-slips to occur and therefore leads to changes sooner in the state of the system.Finally, a numerical study was performed to demonstrate the effect of domain evolution rate similar to experiments in Fig. ate Fig. a,b—reachbeta )=0.,-1.4$$\ebeta )=0.,-1.4$$\ebeta )=0.,-1.4$$\e36 and hence often balance on the edge of chaos as known from studies on cardiac and neuronal activity37, for example. The latter concept has also been encountered in many other areas38: in economy, creative destruction represents the driving force within a market economy; in social science, the dynamic interaction between individuals and macro-levels such as laws, religions, and governments imposing too much order and limiting individual development in the name of conformity, ultimately leading to stasis; in human cognition and creativity39, the states at the edge of chaos can be seen to be maximally novel while still connected to ones in the ordered regime—the hallmark of innovative thinking.In the presented work we demonstrated, both experimentally and theoretically, the ability to control chaos by the system size variation. These findings may shed some light on spatially evolving biological systems and life, that require ‘a healthy dose of chaos’ for proper operation29. Second, considering the sensitivity of Faraday waves to possible evaporation and thus to water layer depth changes40, in our setup the liquid level was also maintained constant at The designed experimental setup shown in Fig. 42. This method is based on an optical system composed of a video projector (ViewSonic PJD7820HD) casting a grating pattern on the water free surface and a camera (Nikon D5200) recording the reflection of this pattern from the free surface. The grating pattern distorted due to deformation of the surface is then recorded and compared to the reference image of the undistorted grating pattern on the flat free surface in order to produce a phase-shift map—the difference between phases of light intensity at each pixel in these two images—from which the height of the deformed surface is reconstructed using a relation between the phase shift and the object’s height. The highly accurate common-optical axis implementation of FTP for water surface waves introduced recently29 guarantees a vertical resolution of 29.Figure 29 was modified in order to investigate temporal chaos. To that end, measurement of the waves amplitude at a single point on the surface is sufficient7 as explained below; hence, we resorted to a laser displacement sensor (Optex FA CDX-30A) capable of measuring the surface deformation with the accuracy of 7.For the present study, the setup reported in Ref.An example of controlling chaos by domain deformation is provided in Fig. tre Fig. a, the Fotre Fig. b revealsCollecting data at the rate 150 per second and with a vertical resolution of entclass1pt{minimaThe real and imaginary parts of entclass1pt{minima(τ) Fig. e, wave f(τ) Fig. d are recReU Fig. e and \doequation .Blocks of data were recorded continuously over a 45, which with the measurement at a single location on the surface not only reveals the type of regularity of the regime (the dimension of the strange attractor) but also the number of modes involved. This technique assumes that all the important dynamical features are contained (embedded) in a single time series. A strange attractor is characterized as an aperiodic one, in which the surrounding trajectories diverge exponentially from each other in time, and, most importantly, is an object of fractal dimension D, i.e. the number of small cells of size m-dimensional embedding phase-space coordinates are constructed as 45 assures that the topological properties extracted from the above embedding, such as dimension and Lyapunov exponent, are equivalent to that of the attractor provided that D from an experimental data set47; however, there exist equivalent estimates of D, the most common of which is based on the correlation dimension:N denoting the total number of data points. The numerical implementation of (48. Once m, the limiting slope in the scaling region (e.g. in Fig. m beyond four and is saturated at the value of To determine the dynamic type of the Faraday wave regime we resorted to the analysis of the total amplitude


Fagopyrum tataricum Gaertn.). Normal rutinosidase seeds can be distinguished from trace-rutinosidase mature seeds and ripening young seeds using a rutin solution by comparing the muddiness, which corresponds to quercetin generation. In dough, we detected a threefold relative increase in rutinosidase activity corresponding to 1% contamination of normal rutinosidase flour with trace-rutinosidase flour within 65 min. These methods do not require expensive apparatuses and toxic chemicals and are therefore promising for detecting and preventing contamination by rutinosidase, e.g., in food processing factories.Contamination of rutinosidase is a major problem for rutin-rich food due to the hydrolysis of the functional compound rutin and generation of strong bitterness caused by the hydrolyzed moiety quercetin. This problem sometimes occurs between the trace and normal rutinosidase Tartary buckwheat varieties. Here, we developed a simple and rapid method for rutinosidase detection in ‘ripening seeds using UV light’ and in ‘dough using alum-flavonoid complexation’ from Tartary buckwheat ( Therefore, Tartary buckwheat is generally used as a functional food and/or subsidiary material [Tartary buckwheat is cultivated in mainly in China, Nepal, eastern Europe, and Japan. One important trait of Tartary buckwheat is the high rutin concentration in its seeds ,2, whichmaterial ,4,5,6. Fmaterial ,8,9 and material , blood cmaterial ,12, and material . In addimaterial , headachmaterial  were fouHowever, Tartary buckwheat seeds also have high rutinosidase activity , which iRutin is a functional compound. Therefore, the hydrolysis of rutin is a disadvantage of Tartary buckwheat. In addition, the hydrolysis of rutin triggers the generation of a strong bitter taste in subsequent food products ,17,18,19Food-processing technology, such as the heat treatment of flour, can prevent rutin hydrolysis. However, strong and long-term heat treatment is needed to inactivate rutinosidase, and this treatment leads to serious deterioration of taste, texture , color, These methods do not use HPLC but rather employ common family cups, reasonable plastic tubes, or microtiter plates with nontoxic solutions. In this paper, we described the detailed procedure and effectiveness of the methods.In the control, which contained only the rutinosidase reaction mixture, precipitation did not occur , which iFirst, we confirmed that steam-heat treatment to flour completely inactivates rutinosidase using HPLC. After 16 h of storage of intact MK dough, rutin was completely hydrolyzed B, whereaThe effects of alum against rutin hydrolysis are shown in 3 with low-temperature dough storage can prevent rutin hydrolysis with up to 1% contamination of MK flour by T8 flour. This method, with the simple and rapid detection described in this paper, produces rutin-rich foods with reduced bitterness efficiently.The results of the application of the method to evaluate contamination of MK flour by normal rutinosidase flour are shown in Two Tartary buckwheat varieties, trace-rutinosidase variety MK and normal rutinosidase variety T8, were sown in early June in the experimental field of Memuro Hokkaido Japan . In late August, we harvested mature plants and dried them at 35 °C for approximately one week. Harvested plants were threshed, and seeds were purified using a grain fan, then subjected to stone removal. After that, seeds were stored at 10 °C.To evaluate rutinosidase activity during ripening in Tartary buckwheat, we observed the seed set every day at maturation and marked flower buds when we found seed set, which occurred at 3, 6, 12, 18, and 24 days after pollination (DAP). We used T8, MK, and perennial buckwheat seeds for the experiment. Harvested seeds were immediately applied for the simple detection of rutinosidase activity.® Junior, Brabender® GmbH & Co., Duisburg, Germany). Flours were passed through a 2-mm mesh, and the flour milling percentage was adjusted to 63%. We have confirmed that flour milling percentage, rutin concentration, and flour milling between T8 and MK were almost the same [Tartary buckwheat seeds harvested at maturation were milled using a test role mill (Quadrumatthe same .v/v) of 50 mM acetic acid pH 5.5: 40% (v/v) of 2-methoxy-ethanol). We used plastic tubes or 96-well microplates for evaluation. For one seed, 150 μL/well rutinosidase detection solution was added. When the seeds have normal rutinosidase activity, such as T8 seeds, generated quercetin is precipitated in the well because the solubility of quercetin is much lower than that of rutin in the solution. In the seeds with trace-rutinosidase activity such as MK, rutin hydrolysis was very slow compared to that of T8. Therefore, quercetin was not precipitated. Fifteen minutes after the addition of rutinosidase detection solution, we observed the precipitation of quercetin in the well. Compared to the T8 and MK seeds, we can clearly identify whether the tested seeds have normal rutinosidase or trace rutinosidase. For more rapid detection of rutinosidase, 0.5 μL of reaction solution was spotted on paper 5 min after the addition of rutinosidase reaction solution. The paper was irradiated with UV light from a commercially available black light , and the spot color was observed. Quercetin had luminescence upon irradiation with UV light and a yellow color was observed at the spot, whereas rutin did not have luminescence (black spot). To identify the substance of the precipitate in the T8 well, the rutinosidase reaction mixture containing the precipitant was centrifuged and washed with water 3 times. Then, the precipitate was dissolved in methanol containing 20% (v/v) 0.1% (v/v) phosphoric acid. After filtration, the solution was analyzed using HPLC [3CN/H2O/Trifluoroacetic Acid, 7.5:92.5:0.1) to solvent B .Harvested seeds during ripening were cut in half from the top of the seeds in the horizon direction. The cut seeds were suspended in rutinosidase detection solution that contained 80 mg rutin/100 mL solution (60% (ing HPLC . HPLC waw/v) alum was poured into 10 g of subject flour. After mixing well, the dough was allowed to stand at room temperature (approximately 25 °C). We observed and compared the color of the dough and supernatant at 0, 1, 15, and 65 min after the addition of the alum solution. To evaluate differences of color in the supernatant, l*, a*, and b* at a contamination rate of 0–1 at 65 min were measured from the picture in open black box in https://www.gimp.org/downloads/ accessed on 11 January 2022). The measurement was performed 10 times and mean ± SD was expressed. Means were statistically compared by Bonferroni’s multiple comparison test [To evaluate rutinosidase activity, we employed the alum solution to boost the yellow color of Tartary buckwheat dough. The alum forms a complex with flavonoids, such as rutin and quercetin. The alum–quercetin complex has a more concentrated yellow color than the alum-rutin complex. Therefore, we can evaluate rutinosidase activity in dough by assessing the intensity of the yellow color in dough or the supernatant of flour-alum mixed solution and comparing it with that of the standard flour, whose rutinosidase activity (contamination rate) is known. To evaluate the detection ability of rutinosidase by this method, we prepared standard flours as follows: ‘zero-rutinosidase flour’ and ‘artificial contaminated flours’, in which a known amount of T8 flour was mixed with MK flour. The relative rutinosidase activities of the flours were 0 (zero-rutinosidase flour) and 1, 3, 7, 19, 55, and 163 . To prepare ‘zero rutinosidase’ flour, 10 g of MK flour packed in a nonwoven bag with 1 cm thickness was steamed using a steamer for 15 min. After steaming, partly caked flour was crushed into powder using a motor. To evaluate the rutinosidase activity in the flour, 100 mL of the alum solution containing 0.1% (son test . We emplThe developed methods can distinguish normal rutinosidase seeds from trace-rutinosidase mature seeds and ripening young seeds using a rutin solution by comparing the muddiness, which corresponds to quercetin generation. These are reasonable and effective for detecting the contamination of rutinosidase compared to PCR-based variety discrimination methods.Detection methods based on differences in the solubility of substances, such as rutin and quercetin, are effective as simple detection methods based on visual muddiness. In addition, these methods are promising for detecting and preventing contamination by rutinosidase, e.g., in food processing factories, because they do not require expensive apparatuses and toxic chemicals.


Viruses have evolved numerous strategies to maximize the use of their limited genetic material, including proteolytic cleavage of polyproteins to yield products with different functions. The poliovirus polyprotein 3CD is involved in important protein-protein, protein-RNA and protein-lipid interactions in viral replication and infection. It is a precursor to the 3C protease and 3D RNA-dependent RNA polymerase, but has different protease specificity, is not an active polymerase, and participates in other interactions differently than its processed products. These functional differences are poorly explained by the known X-ray crystal structures. It has been proposed that functional differences might be due to differences in conformational dynamics between 3C, 3D and 3CD. To address this possibility, we conducted nuclear magnetic resonance spectroscopy experiments, including multiple quantum relaxation dispersion, chemical exchange saturation transfer and methyl spin-spin relaxation, to probe conformational dynamics across multiple timescales. Indeed, these studies identified differences in conformational dynamics in functionally important regions, including enzyme active sites, and RNA and lipid binding sites. Expansion of the conformational ensemble available to 3CD may allow it to perform additional functions not observed in 3C and 3D alone despite having nearly identical lowest-energy structures. RNA viruses need to produce proteins that help evade the host immune defense, allow the virus to replicate, and establish new infections ,2,3. A tPoliovirus 3C, 3D and 3CD are all important proteins for the viral replication cycle. Here we will briefly discuss the structure and function of each. Structurally, 3C is cysteine protease with a chymotrypsin-like fold with the active site between two six-stranded β-barrels, with four helices . The cat3D, the RNA-dependent RNA polymerase (RdRp), has the “cupped right hand” topology common to most nucleic acid polymerases , consistPoliovirus 3CD is largely a structural composite of the 3C and 3D proteins, with a short linker connecting these domains . Unlike The distinct and emergent functions of 3CD compared to the fully processed 3C and 3D proteins cannot be explained by structural considerations alone, given the nearly identical X-ray crystal structures of 3C/3D compared to 3CD . It is nHere, we have used nuclear magnetic resonance (NMR) based methods to explore the conformational dynamic differences between 3C, 3D and 3CD as a potential explanation for their different functions. These experiments capture conformational dynamics across many timescales, including chemical exchange saturation transfer (CEST)  that monEscherichia coli BL21(DE3) pRARE cells. In 3D and in the 3D domain of 3CD, the following amino acid substitutions (relative to type 1 Mahoney strain) were present to prevent aggregation: L446D and R455D. In 3C and in the 3C domain of 3CD, the following amino acid substitutions were present to prevent aggregation in 3CD: E55A, D58A, E63A, and C147A to prevent intermolecular cleavage of 3CD  labeling, 10–100 μL from the 10 mL M9 culture were plated onto M9 agar plates and incubated for 18–22 h at 37 °C. M9 minimal media (10 mL) with 50% D2O  was inoculated with colonies from the M9 plate for 16–20 h until OD600 > 0.6, then 1 mL was transferred to 50 mL M9 minimal media with 100% D2O for 12–24 h until OD600 > 0.6. Cells (20 mL) were then added to 950 mL of 100% D2O M9 media. In the 100% D2O M9 media, glucose was replaced with deuterated glucose  and the concentration of potassium phosphate buffer and magnesium sulfate were doubled  samples and uniformly labeled 15N 3C were prepared as described above. Data was processed using NMRpipe software [1H-13C heteronuclear multiple quantum coherence (HMQC) spectra are from the first plane of the triple quantum relaxation violated coherence transfer allowed experiment. For 1H-13C spectra, 64 points were used in the indirect dimension (t1) and 768 points were used in the direct dimension (t2).To prepare for methyl NMR spectroscopy, samples were buffer exchanged to 100 mM potassium phosphate, 100 mM sodium chloride, 60 μM zinc chloride, 5 mM β-mercaptoethanol, 20% d-8 glycerol  in Dsoftware  on NMRbosoftware . The 1H-13CH3 groups of 3C were determined using three-dimensional correlation experiments described in ref [Resonance assignments for the δ1-d in ref  using prd in ref . ResonanTriple quantum relaxation violated coherence transfer experiments were performed at 600 MHz at 298 K and analyzed as previously described . The alla/Ib is the ratio of intensities of the forbidden and allowed experiments, η is the intramethyl 1H-1H cross-correlated relaxation rate, δ is a parameter that accounts for relaxation due to external protons, and T is the relaxation delay period. η and δ for each residue were determined by nonlinear least squares fitting using the Trust Region Reflective algorithm. Uncertainty in fit parameters calculated by Monte Carlo bootstrapping resampling from a normal distribution using the standard deviation of repeating measurements was very small, so residual bootstrapping was used to determine uncertainty. The data were fit to Equation (1):2) were calculated according to Equation (2):c is the rotational correlation time for the protein molecule, μ0 is the vacuum permittivity constant, γH is the gyromagnetic ratio for a proton spin, rHH is the distance between methyl proton pairs, P2(x) is the second order Legendre polynomial 1/2(3x2−1), θaxis,HH is the angle between the methyl 3-fold axis and a vector connecting a pair of methyl protons [∥/D⊥ < 1.17 [c for each residue using an axially symmetric model results in a difference in order parameters of less than 3%. For 3CD, the rotational diffusion tensor calculated by HydroNMR [∥/D⊥ = 1.66), raising the possibility that the methyl 3-fold axis orientation relative to the principal axis of the diffusion tensor could affect the intramethyl cross- relaxation rate, with bond vectors aligned along the principal axis experiencing lower rotational correlation times than bond vectors aligned perpendicular [c and relative order parameters calculated using different values of τc for each residue calculated from the axially symmetric rotational diffusion tensor [Methyl axis order parameters (S protons . For 3C ⊥ < 1.17 ), so we HydroNMR  is approndicular . Howevern tensor ,68,69, s1H-13C multiple quantum CPMG relaxation dispersion experiments were performed at 850 MHz at 295 K and 298 K, as previously described [−1. Error bars were calculated from spectral noise. Rex was calculated by subtracting the value of R2,eff at the highest νcpmg from R2,eff at the lowest νcpmg for each residue. If both values of R2,eff were within error due to noise, Rex was considered to be 0. Curves for Ile151 in 3C and 3CD were fit using GUARDD in MATLAB [escribed , with a n MATLAB .1H chemical exchange saturation transfer (CEST) experiments were performed at 850 MHz at 298 K, as previously described [escribed . An exch13C CEST experiments were performed at 850 MHz at 298 K on 3C, as previously described [escribed . An exchAs the structural similarities between 3C and 3D with their corresponding domains in 3CD largely failed to explain functional differences, we sought to understand these differences by comparing conformational dynamics via NMR spectroscopy. Since our interest is primarily in comparing the conformational dynamics between the fully processed 3C and 3D proteins to their counterpart domains in the 3CD protein, we report first on NMR experiments with 3C and comparisons against the 3C domain of 3CD, before reporting on the NMR experiments of 3D and comparisons against the 3D domain of 3CD. At the end of this section, we then compare the rotational correlation times of 3C and 3D by themselves and in the context of 3CD to learn about the degree of independence of the domains in 3CD. To avoid confusion, residues belonging to the 3C and 3D proteins will be identified according to 3C and 3D superscripts, respectively. We will also refer to residues in the 3C domain of 3CD with a superscript 3C*D and residues in the 3D domain of 3CD with a superscript 3CD*. In this way, we can keep the numbering convention for 3D while referring to the corresponding domain in 3CD.1H-13C methyl HMQC NMR spectra were collected for poliovirus 3C labeled with 13C at the delta-1 carbon of all Ile residues , which report on ps-ns timescale dynamics [c) of 24.8 ns so that the highest order parameter was 1, the average value of the order parameters calculated for 3C was 0.69. This value for τc agrees very well with the value calculated by HydroNMR (25.1 ns). Order parameters calculated using the τc determined by TRACT NMR are given in parentheses . The side chains of Ile363C and Ile723C are contacting via hydrophobic interactions, and Ile723C is adjacent to the active site residue Glu71, which is part of the catalytic triad. Ile1513C has the lowest methyl axis order parameter in 3C, 0.46 (0.39), indicating that it is the most dynamic on the ps-ns timescale.While slower conformational dynamics include potentially functionally relevant larger movements such as domain motions, faster motions such as loop motions on the ps-ns timescale may also be important for substrate binding or allostery ,56. Methdynamics , were dedynamics ,80  for 3CD . Here, w3C*D in the RNA-binding loop and Ile1513C*D in the protease active site. Ile903C*D may be associated with a larger Rex value in 3CD suggesting changes in kinetics and/or thermodynamics associated with conformational exchange, and/or a change in the exchanging conformation. It is notable that there was also a small chemical shift difference for Ile90 between 3C and 3CD  was determined by assuming the highest order parameter was 1 because residues in the 3C and 3D domains could not be analyzed for τc separately due to 1H-15N resonance overlap. More specifically, measurement of the rotational correlation time by the TRACT NMR experiment resulted in a value lower than used here because without separation of the resonances in the 1H and 15N dimensions, slower-relaxing resonances overpower the faster-relaxing resonances that are needed to accurately determine τc [For analyzing methyl axis order parameters in 3CD, the rotational correlation time  appeared to have higher order parameters, while other residues  had lower order parameters for 3C compared to 3CD  of the independent domains to the domains in the context of 3CD. The τc value can be thought of as the average time it takes for a molecule to rotate 1 radian. If the 3C and 3D domains in 3CD tumble independently and do not interact, τc for each domain would be less than τc for the whole protein considered as a rigid body, with τc for each domain roughly proportional to its molecular weight [c values would be identical and larger than the τc of the individual domains [c, we will focus on the value of the intramethyl 1H-1H cross-correlated relaxation rates, η , they could contribute to each domain having a larger τc by limiting the rotational diffusion, which would result in a longer rotational correlation time. Interdomain contacts, i.e., 3CD adopting a more compact conformation in solution than in the X-ray crystal structure , especially in comparisons between 3CD and 3C/3D. To calculate order parameters from intramethyl cross-correlated relaxation rates (η), we used the simplest version of the Lipari-Szabo model-free formalism [2 and τc. Any comparison of order parameters between different proteins depends on the chosen values of τc. When discussing order parameters, we are comparing the distribution of order parameters within the protein rather than absolute values of S2 because the true value of τc is not known for 3CD due to its large molecular weight and separate domains of 3CD making traditional methods for determining τc unavailable. To assess the accuracy of our use of the highest order parameter to determine τc, we used the TRACT NMR experiment [c for 3C  are exchanging in 3C, they do not appear to be exchanging in 3CD. Ile153C is adjacent to the PIP binding site containing Lys12 and Arg13. Ile943C*D and Ile1033C*D also do not appear to undergo detectable exchange, although the resonances are of low intensity and the relaxation dispersion profiles were noisy, likely due to the slow tumbling time, and hence elevated transverse relaxation rate, of 3CD. Ile943C is adjacent to the RNA and lipid binding site in 3C [While Ile158te in 3C ,21,91. Tte in 3C ,15,16. 3D, Ile3003D and Ile3313D, which are in the palm domain in between the N terminus of 3D and the active site, have been proposed to stiffen the structure of the polymerase via a hydrogen bonding network [3D and Ile3243D, located in the palm domain. Ile3243D is on motif C, which contains the metal-coordinating Asp328. Ile3053D and Ile3163D, in the palm domain on motif B, are distant from the N terminus and 3C domain and appear to be in a very similar chemical environment in the x-ray crystal structures. However, they have different chemical shifts between 3D and 3CD. Ile3053D is on the same α-helix as Asn297, which contacts the 2′ OH of the incoming NTP [3D, also on this helix, does not appear in the 3CD spectrum indicating that it is likely broadened by conformational exchange. Ile2953D, also on motif B, has a much higher Rex in 3CD than in 3D (3CD* is likely exchanging to a state with a larger difference in chemical shift and/or a higher relative population. Alternatively, Ile2953D may be exchanging too fast for the contribution to relaxation from conformational exchange to be refocused as effectively by the CPMG pulse train. If the larger Rex in 3CD is due to a higher excited state population, this would be consistent with the idea that 3CD adopts additional conformations in solution relative to its isolated domains [3D is less ordered in 3CD than in 3D; this residue contacts Phe258, which has been shown to be necessary for recruitment of guanine nucleotide exchange factors required for membrane reorganization in 3CD but not 3D and 3C separately [Ile3 network ,84. Thesming NTP . Ile3003an in 3D . This di domains . Ile903Dparately . 3D and Ile583D, located near the N terminus of 3D in the fingers domain, have chemical shift differences between 3D and 3CD (3D and Ile1503D, near these two residues, also have chemical shift differences between 3D and 3CD (1H CEST experiments revealed that Ile1303D is exchanging with a conformation that has a large difference in 1H chemical shift on the slow timescale in both 3D and 3CD. Insertion of four amino acids after Asp146, which is distant from the 3C domain, has been shown to decrease proteolytic processing of P1 by 3CD [3D, which is on the same loop as Asp146, has a much higher order parameter in 3D than in 3CD, so the conformational dynamics observed for Ile1503CD* may be important for allosteric communication with the 3C domain.Ile17 and 3CD . These d and 3CD  and both and 3CD c. These 1 by 3CD . Ile1503We identified differences in conformational dynamics on the picosecond to second timescales between the poliovirus 3C, 3D and 3CD proteins via NMR spectroscopy that may be important for understanding the differences in protease specificity, RNA and lipid interactions and polymerase activity. Differences in conformational dynamics near the protease active site and RNA binding site in 3C may be important for differentiating functions between the 3C and 3CD proteases. Similarly, enhanced flexibility of the 3D domain of 3CD may help to explain its lack of polymerase activity. Addition of protease substrates, RNA and/or PIP lipids may further accentuate these dynamic differences. Our results indicate that allosteric coordination between the 3C and 3D domains of 3CD likely operates through changes in these conformational dynamics.


This study was undertaken to determine the effect of the partial replacement of wheat flour (WF) with barley brewer’s spent grain (BBSG) and barley-buckwheat brewer’s spent grain (BBSG + B) on dough quality and bread properties, including nutritional value. The contents of brewer’s spent grain (BSG) in the blend with wheat flour were 0, 10, and 20%. The quality of the flour blends was assessed with intermediate methods and based on laboratory baking. Analyses were also carried out to determine contents of basic nutrients and energy value. The replacement of part of wheat flour with BBSG and BBSG + B diminished gluten yield and deteriorated its quality . Changes were also observed in the starch-enzymatic system, resulting in a decreased falling number and maximum paste viscosity. Breads containing both BSG types featured higher yield and lower loaf volume. They had also higher contents of protein, dietary fibre, fat, and ash as well as a lower energy value compared to the wheat bread. Considering the organoleptic traits of breads, the 10% replacement of wheat flour with BSG is recommended in the blend. The BBSG + B was found to elicit more beneficial effects on bread properties than BBSG. Today, there is enormous political and social pressure around the world to reduce the amount of industrial waste. For this reason, many laboratories investigate the possibility of using various by-products to produce new, desired goods. Large amounts of by-products are generated by the food industry, many of which have a valuable composition and can still be used as raw material for further processing. The by-products generated by the brewing industry include waste yeast slurry, leavings, and brewer’s spent grain (BSG). Given the volume of global beer production (1.94 billion hectoliters in 2018) [Authors  reportedFagopyrum esculentum) deserves special attention owing to its exceptional nutritional value. Its seeds are rich in protein featuring a high biological value approximating 90%. These properties are due to the high content of most essential amino acids, especially lysine, tryptophan, threonine, and the sulfur-containing amino acids [Due to the valuable composition of BSG, attempts have been made to use it to produce many food products, such as bread ,5,7, coono acids . Buckwheno acids  report tThe available literature lacks reports concerning the influence of barley-buckwheat brewer’s spent grain on the properties of wheat flour blends and bread made of them. With this in mind, a study was undertaken to compare and evaluate the effect of replacing a part of wheat flour with barley brewer’s spent grain and barley-buckwheat brewer’s spent grain on the baking value of flour blends determined with direct and indirect methods and on the nutrient composition of the bread made of these blends.2 retention in the dough [3 for a flour with an average baking value and 41 cm3 for a strong flour. The value of Zeleny’s sedimentation index determined in the present study for wheat flour (37.5 cm3) indicates the good quality of its gluten proteins  a. It wasWhen assessing the correlations between the features describing the properties of the amylase-starch complex and the quality of bread based on the PCA analysis, it was found that the value of the first principal component was 68.62% (PC1) and that of the second principal component was 18.59% (PC2) b. There Based on the calculated values of Pearson’s correlation coefficients, a correlation was also demonstrated between the quality attributes of the bread. Yield of bread (YB) negatively correlated with bread volume , while the overall score in the organoleptic assessment negatively correlated with bread yield and positively with its volume.Commercial wheat flour type 650 (WF) was from mill company Dolnośląskie Młyny S.A. . Two types of brewer’s spent grains (BSG): barley (BBSG) and barley-buckwheat (BBSG + B) came from the brewery Browar Stu Mostów . BBSG and BBSG + B were preserved by drying at a temperature of 55 °C for 3 h in the UF110 Plus dryer . Before starting the research, both BSG were ground in a laboratory hammer mill WŻ1 . The granulation of milled BSG was below 250 μm. Wheat flour and ground BBSG and BBSG + B were used to prepare blends with BSG contents of 10 and 20%. Wheat flour served as the control. The other ingredients used in the production of bread were evaporated iodized salt produced by Cenos Sp. z o.o.  and pressed baker’s yeast produced by Lesaffre Polska S.A. , bought in the local market.Wheat flour and its blends with BBSG and BBSG + B were determined for: wet gluten content  and fall3), at mixing time 5 min. The doughs were put in the molds , greased with oil. The molds were placed in a fermentation chamber  for 90 min. During fermentation, the dough was degassed after 60 and 90 min and left for the final fermentation (45–50 min). The breads were baked in two replications in a GT800 electric furnace , at a temperature of 245 °C for 30 min, with steaming in the first 3 min of baking.Bread was baked under laboratory conditions with the single-stage method, according to the following recipe: wheat flour or its blend with BSG—250 g, salt—3.8 g, and compressed yeast—7.5 g. The dough was prepared in a farinograph mixer (bowl for 300 g of flour) by adding tap water having a temperature of 30 °C, in a volume ensuring dough consistency of 300 FU  filled with millet grain. Analyses were performed at least in duplicate. Bread weight was used to determine bread yield in respect of the weight of flour used for baking. Bread volume was converted as bread volume per 100 g of flour and specific volume (bread loaf volume divided by bread loaf weight). The crumb porosity was assessed according to the Dallmann scale .A team of 10 trained panelists conducted the organoleptic assessment of bread according to the Polish Standard . All stuBreads were determined for: total protein content—with the Kjeldahl method  using a p ≤ 0.05) between the mean values were determined using the Duncan’s Multiple Range Test. The Principal Component Analysis (PCA) was performed to determine correlations between the quality attributes of bread and parameters indicating the quality of protein and amylose-starch complexes. Statistical calculations were carried out at p = 0.05 using the Statistica 13.0 data analysis software system .).The results presented are mean values ± standard deviation (SD). Statistical analyses of results obtained for WF, BBSG, BBSG + B, blends thereof, and breads with 10 and 20% content of BBSG and BBSG + B were conducted with the one-way analysis of variance (ANOVA). Significant differences (The partial replacement of wheat flour with BSG resulted in a decreased content of wet gluten and its deteriorated quality assessed based on the Zeleny’s sedimentation index and farinographic analysis. The falling number and viscosity of the flour pastes decreased along with the increase in the BSG content in the flour blend. The BBSG + B contributed to unfavorable changes in the baking value of the flour blends to a lesser extent than BBSG. An increase in bread yield and a decrease in the volume of loaves were found in laboratory baking, under the influence of both BSG types. Considering the nutritional value, the breads containing both BSGs turned out to be superior over wheat bread. With the increase in the content of both BSG types in flour blends, the contents of protein, dietary fiber, fats and ash significantly increased and the energy value decreased, while BBSG + B turned out to be a better additive than BBSG. In the organoleptic assessment, the breads with 10% BBSG and BBSG + B were classified as of quality level I, similarly to the control bread. The breads with BBSG + B were scored higher than these with BBSG.