Conjugation of variousreagents to antibodies has long been anelegant way to combine the superior binding features of the antibodywith other desired but non-natural functions. Applications range fromlabels for detection in different analytical assays to the creationof new drugs by conjugation to molecules which improves the pharmaceuticaleffect. In many of these applications, it has been proven advantageousto control both the site and the stoichiometry of the conjugationto achieve a homogeneous product with predictable, and often alsoimproved, characteristics. For this purpose, many research groupshave, during the latest decade, reported novel methods and techniques,based on small molecules, peptides, and proteins with inherent affinityfor the antibody, for site-specific conjugation of antibodies. Thisreview provides a comprehensive overview of these methods and theirapplications and also describes a historical perspective of the field. Therefore, many novel methods for conjugation have been developedduring recent decades,3 some of which willbe the focus of this review.Antibodieshave a long-standing reputation as excellent tools inmany medical and biological applications because of their capacityto selectively bind to specific target molecules with high affinity.Both in the diagnostic and in the therapeutic fields, antibodies arecommonly decorated with specific active groups, either to make themdetectable or to equip them with a specific characteristic or activity.The added characteristic could be a group that would be useful ina diagnostic setup, such as a fluorescent or radioactive label oran enzyme that can be used for detection. By adding such labels toan antibody, it can be used in a variety of analytical or diagnosticmethods such as immunohistochemistry (IHC), enzyme-linked immunosorbentassay (ELISA), or fluorescence-activated cell sorting (FACS). Furthermore,the number of therapeutically used monoclonal antibodies conjugatedto small therapeutic molecules, so-called antibody–drug conjugates(ADC), is continuously increasing and with that comes a demand forefficient, stable, and selective conjugation strategies.4 These conjugation methods can pose problems since it isdifficult to tune the labeling, as neither the position nor the exactnumber of labels per antibody can be controlled. Meanwhile, severalstudies have established the importance of homogeneous conjugatesfor increased therapeutic potential of ADCs.6 Fortechnical and diagnostic antibodies, unspecific labeling with fluorescentdyes might become problematic due to clustering and quenching of thefluorescence if several labeling molecules end up in close proximity.7 Furthermore, lysines are often located in proteinbinding surfaces due to their positive charge. If regions close to,or within, the paratope of the antibody contain lysines, the conjugatedmoiety may interfere with the antibody’s capability to interactwith its antigen. This might impair the efficacy of the antibody andthus give a less efficient therapeutic or diagnostic tool. To increasethe control of the labeling, conjugates based on maleimides, a commonthiol-reactive reagent, are utilized. However, these have been shownto undergo premature cleavage due to exchange reactions with otherfree thiol groups, such as those present in serum albumin.5 In addition, cysteines are most commonly naturallypaired, forming stabilizing disulfide bridges within the antibody,where the amount and location of the disulfides differ for differentImmunoglobulin G (IgG) subclasses. Consequently, these different subclassesmay be affected differently with regard to solubility and aggregationwhen these disulfides are broken.8 Furthermore,while one may achieve a higher control over the level of conjugationwhen utilizing cysteines compared to primary amine labeling, the exactlevel and site of conjugation can still not be decided in advance.There is also a recently developed method that utilizes glycans forattachment of the labeling group. This gives higher selectivity thantargeting side chains of common amino acids, although not as highas the methods discussed below. To enable this, a partial deglycosylationwith Endo S is performed where an artificial azide containing a galactoseresidue is bound to the remainder of the glycan group and can subsequentlybe utilized for labeling.9The traditional, and also mostwidespread, methods developed forprotein conjugation are nonspecific and based on the utilization ofside chains of frequently appearing amino acids, such as thiols, carboxylgroups and, most commonly, primary amines found in the N-terminusof the protein and on the side chain of lysines.To overcomethe limitations of unspecific antibody conjugation,several methods have been developed with the aim of directing theconjugation to a specific location on the antibody, to avoid interferencewith the antibody target binding, as well as to gain control of thenumber of labels per antibody.Although the main focus of thisreview will be on conjugation methodsthat are based on molecules with inherent affinity for the antibodyscaffold, some of the directed conjugation methods that require modificationof the actual antibody scaffold will be briefly discussed in the comingsection.10 and Kline et al.11 among others.While this engineering approach might sound cost- and time-consuming,it may very well be worth it in otherwise costly processes such asADC development, if it increases the therapeutic efficiency of thesaid antibody. Among these methods are, for example, those based onthe engineering of cysteines as conjugation handles, which has beena successful strategy even though it might lead to stability issuesdue to the breaking of pre-existing disulfides.12 Othermethods take advantage of nature’s way of carrying out site-specificconjugation by different enzymes, and these enzymatic reactions canbe based either on recombinantly introduced recognition sites in theantibody sequence or on natural moieties such as glycans.To avoid the heterogeneous labeling that is the result of traditionallyused conjugation methods, a plethora of different methods have beendeveloped with the aim of steering the conjugation to specific siteson the antibody. Many of these site-specific methods rely on engineeringof the actual antibody itself prior to conjugation and is excellentlyreviewed elsewhere by, for example, Zhou et al.15 Beyond the aforementioned natural,but rare, amino acids, there are also synthetically produced aminoacids equipped with functional groups, known as noncanonical aminoacids (ncAAs). These chemically orthogonal amino acids can be introducedrecombinantly directly in the antibody sequence. Hundreds of theseamino acids, with non-natural side chains, have been incorporatedin proteins using both prokaryotic and eukaryotic production systems,and some of these ncAAs possess the unique chemical properties thatmake them suitable for conjugation.16 Themost widely used strategy to introduce these amino acids invivo is by amber suppression, a method that was first introducedby the lab of Peter Schultz almost two decades ago.17 Commonly used unnatural amino acids for conjugation are p-acetylphenylalanine (pAcF), p-azidophenylalanine(pAzF), p-azidomethylphenylalanine (pAMF), and anazide derivative of lysine (AzK).18Inaddition to the 20 standard amino acids, there are also twoadditional amino acids, selenocysteine and pyrrolysine, that occurrarely, but naturally, in proteins that have been utilized for controlledconjugation of antibodies.19 spycatcher,20 and split inteins.21 The focus of this review, however, is methodsthat do not rely on antibody modification, and these will be reviewedin more depth in the coming section.Many more methods exist that rely on antibody modification forconjugation, such as engineered tags,The methods described above arein many situations excellent forantibody conjugation and can be very useful when developing new ADCs.However, many antibodies already exist on the shelves and there isa need to be able to efficiently and site-specifically label these.Fortunately, there are many naturally existing, as well as synthetic,affinity ligands that already specifically recognize certain siteson antibodies, that are not part of the antibody’s paratope.An overview of these affinity ligands, and their binding sites, canbe found in Engineering of these affinity ligands to achieve covalent antibodymodification present possibilities to site-specifically conjugatealso off-the-shelf antibodies. Furthermore, these binders providethe means to label antibodies in complex surroundings such as cellculture supernatants or solutions containing stabilizing protein additivessuch as bovine serum albumin, that would interfere with antibody labelingwhen using traditional methods based on side chain conjugation. Byutilizing a small molecule, peptide, or protein domain with affinityspecifically for the antibody for conjugation, these types of antibodyproducts can also be readily labeled in the solution in which theyare provided. Staphylococcusaureus and consists of five highly homologous, small helicalantibody binding domains with affinity mainly to the fragment crystallizable(Fc) part of IgG, but also to the VH domain of human VH3 family antibodies.40 Protein G stems fromgroup C and G Streptococci and is a multidomain protein that containsup to three highly homologous small β-sheet domains with anα-helix stacked on top, with affinity for both the Fc and theconstant part of the fragment antigen binding (Fab) on the heavy chainCH1 domain.42 With their inherent affinitytoward the conserved parts of IgG from many species and subclasses,43 these small bacterial domains are particularlysuitable for directed antibody conjugation and labeling at specificsites that do not interfere with the antibody binding region. Protein A and Protein G are naturally occurringbacterial surface proteins with inherent affinity toward the conservedparts of IgG antibodies. Protein A comes from 44 Normally, the benzophenoneis introduced in or in proximity to the protein–antibody bindingsite, and covalently cross-links the antibody at the interaction interface.In the following examples, this results in cross-linking to the IgGheavy chain, at either the Fc or the Fab fragment.In order to use thesesmall protein domains for antibody labeling, one common strategy hasbeen to introduce a benzophenone molecule to the Protein A or G domain.Benzophenones are UV-inducible at long-wavelength 365 nm light, whichis generally considered to be harmless to proteins, and can covalentlycross-link to nearby amino acids after activation or relax back totheir initial state 3A.44 Nor23First to publish this concept for antibody conjugation were Junget al., who coupled the benzophenone to a Protein G C3 domain at positions21 and 29, by mutating these to cysteine and conjugating the benzophenone,coupled to a flexible linker, to the protein domain using maleimidechemistry. Moreover, a N37Y mutation was introduced to abolish thedomain’s natural affinity for Fab, to ensure that the conjugationtook place only at the Fc fragment. The study showed that >50%ofthe antibodies had one or two Protein G domains covalently linked.Further, the cross-linking domain was equipped with an N-terminalbiotinylation peptide that could be used for attachment to streptavidin-coatedsurfaces. Site-specifically conjugated antibodies were used for immobilizationon glass surfaces and small particles, and it was shown that thisdirected immobilization provided a more efficient antibody–targetbinding compared to randomly biotinylated immobilized antibodies.p-benzoyl-phenylalanine(BPA) and incorporated it at position 5 of the Protein A-derived Zdomain45 using solid-phase peptide synthesis(SPPS) and added a C-terminal biotin to the domain for labeling purposes.This molecule was shown to covalently label human IgG1, mouse IgG2a,and polyclonal rabbit IgG at the Fc fragment.24 Yu et al. introduced a F5I mutation to the Z domain to increaseits affinity to mouse IgG1 and used a maleimide benzophenone (MBP)to introduce the cross-linking molecule at position 32 in the Z domain,after mutating it to cysteine. This domain showed a 64% coupling efficiencyto mouse IgG1 heavy chains, where 50% corresponds to one cross-linkedmoiety per antibody. An antibody biotinylated using this domain showedbetter binding to its antigen in a surface plasmon resonance (SPR)interaction compared to its randomly biotinylated counterpart whenimmobilized on a streptavidin surface.25 In a later publication, the ZF5IQ32MBP molecule was fusedboth N- and C-terminally to split enzyme halves of β-lactamase,that were then site-specifically conjugated to two different antibodiesbinding to separate epitopes on the same protein. This enabled analytedetection via split-enzyme complementation in a dual-antibody assay.46 Perols et al. used SPPS to introduce BPA orbenzoylbenzoic acid at positions 5 and 32 in Z, where Z32BPA could cross-link 41% of humanIgG1 heavy chains and Z5BBA cross-linked 66% of mouse IgG1 heavy chains.47 Further improving the cross-linking abilitiesof the Z domain, Hui et al. reported up to 80% cross-linking of mouseIgG3 heavy chains by introducing BPA at position 17 in the proteindomain and 47% conjugation efficiency for human IgG1 with BPA at position35.27 For this particular labeling domain,BPA was introduced in the backbone of the Z domain using the techniquedescribed in 48 The proteinwas equipped with a C-terminal sortase tag that could be used forconjugation of functional handles such as biotin or fluorophores tothe labeling domain, and subsequently to antibodies.27 The Z domain with BPA at position 17 has later been usedto conjugate DNA to antibodies that were used in an emulsion PCR setupto enable very sensitive protein detection.49 A Z domain with MBP coupled to a cysteine introduced at position35 has been used for pretargeting in radionuclide imaging by conjugationof a peptide nucleic acid probe to said Z domain using a C-terminalsortase tag, and then conjugating the domain to the human epidermalgrowth factor receptor 2 (HER2)-binding antibody trastuzumab.50 Further, Hui et al. showed covalent attachmentof antibodies to nanoparticles using a Z domain with BPA at position13 where an azide containing peptide was fused to the Z domain viaintein-mediated expressed protein ligation, which enabled cross-linkedantibodies to be conjugated to nanoparticles via click chemistry.51Similar approaches have subsequently beenused in multiple publicationsfor both Protein A and Protein G domains to further increase cross-linkingefficiencies and expand the applicability to other antibody subclassesand fragments, using various methods to introduce the benzophenonegroup. Konrad et al. used the ncAA 28 From the sameresearch group, a C2 domain with abolished Fc binding, through theN35W and D40T mutations, was used for labeling of antibodies site-specificallyat the Fab fragment. Introducing BPA at position 18 enabled 48–64%cross-linking to mouse IgG heavy chains depending on subclass, whileBPA at position 29 provided 43–58% cross-linking to human IgGheavy chains depending on subclass. It was further shown that thisFab labeling agent could be combined with the Fc labeling domain toincrease the signal in the ELISA setup used in the prior publication,and also that human antibodies labeled with these domains, eitherat Fc or at Fab, could still induce antibody-dependent cellular cytotoxicity.29 The Fab labeling domain was later also usedin a multiplex imaging Western Blot setup using laser ablation inductivelycoupled plasma mass spectrometry (LA-ICP-MS) by site-specificallylabeling mouse monoclonal antibodies with lanthanide metals.52 Another research group, led by professor Tsourkas,published a Protein G domain for Fc cross-linking with BPA at position24 for mouse, rat, and rabbit IgG labeling or at position 28 for humanIgG conjugation. That domain could also covalently label up to 90%of human IgG heavy chains. Similarly to their previously publishedZ labeling domain, discussed above, their Protein G labeling domaincontains a C-terminal sortase tag allowing for labeling with differentreactive groups.30 This labeling proteinhas been used for oligonucleotide conjugation to antibodies for incorporationto DNA nanostructures53 and for multiplexedcellular targeting.54 Further, site-specificconjugation of luciferase and fluorescent proteins to antibodies havebeen accomplished.55As for Protein G, apart from the initialstudy described by Junget al., Kanje and Hober developed a domain for Fc conjugation containingBPA at positions 25 and 42 of the C2 domain, which showed cross-linkingto 90% of polyclonal human IgG heavy chains and 57% of rabbit polyclonalIgG heavy chains. Human antibodies biotinylated with this domain showeda higher binding response when immobilized on a streptavidin surfacein an SPR setup, compared to the same antibody that was randomly labeledusing NHS-chemistry, and could also successfully be used for detectionin an ELISA experiment.in vivo as the protein domains, although small compared to the antibody,are still around 60 amino acids in size and foreign to the body. Furthermore,the labeled antibody pool would still be heterogeneous, normally consistingof a mix of antibodies with either zero, one, or two labels. Additionally,as the antibody domains used for labeling have a high natural affinityfor the antibody, it can be a tedious and chemically harsh task toremove those domains that have not covalently reacted with the antibody.Benzophenoneshave thus successfully been used in multiple variantsof Protein A and G domains for site-specific conjugation to antibodies,at both the Fc and Fab fragment. The antibodies have been labeledwith a plethora of different reporter molecules, and the conjugationefficiencies have constantly increased, leaving the scientific communitywith a vast variety of domains to modify their antibodies at specificsites with the desired number of labels, i.e. one, two, or three,depending on conjugation efficiency and labeling molecule combination.These methods provide a straightforward and mild way to site specificallylabel off the shelf antibodies in virtually any lab with access toa 365 nm UV light. With the improved labeling efficiencies, most ofthe published methods now make sure that on average at least one siteon each antibody is labeled. Drawbacks include risk for immunogenicityin cases where the antibody is to be used 31 pMet has the advantage of being smaller thanthe bulky benzophenone molecule, which could possibly lower the impactof exchanging an amino acid in or near the protein domain’sbinding site.Other endeavors have utilizedProtein A and G domains for antibody conjugation but exploited differentstrategies for cross-linking than the benzophenone approach. Lee etal. used the photoactivable ncAA photomethionine (pMet) to cross-linkantibodies using 365 nm UV light 3B. The C56 as its base protein, Mori et al. constructeda chemical conjugation affinity peptide (CCAP) with the introducedcross-linker disuccinimidyl glutarate (DSG)  3C that c57Also usingconventional cross-linkers for antibody conjugation,Shroeder et al. preactivated Protein A and Protein G coated microtiterplates with the cross-linkers succinimidyl (4-iodoacetyl) aminobenzoate(SIAB) and sulfosuccinimidyl (4-iodoacetyl) aminobenzoate (sulfo-SIAB)bifunctional reagents before adding antibody that could site-specificallycross-link at the Fc to the immobilized proteins on the plate, afterwhich non-cross-linked antibodies could be washed away under acidicconditions. The specific and directed immobilization gave higher signalscompared to undirected immobilization. Moreover, it enabled the possibilityto immobilize antibodies directly from a crude supernatant.34Yu et al. presented another proximityinduced site-specific conjugationof the B domain, of Protein A, to Fc, requiring no UV or chemicaltreatment, which was denoted pClick. The ncAA 4-fluorophenyl carbamatelysine that can react with nearby lysines 3D was in9 different disulfide-inducedcyclic peptides of the form XiCXjCXk . These peptides were then further matured by constructionof new libraries that resulted in the 13-mer peptide FcIII (DCAWHLGELVWCT),binding to the Fc region with a strength of approximately half ofProtein A and G, which both have equilibrium binding constants (KD) around 10 nM. The same publication also includesan X-ray structure of the complex between the FcIII peptide and theIgG Fc fragment (PDB ID: 1DN2). The protein structure shows that, despite the peptidehaving a completely different structure than other natural Fc-bindingproteins such as Protein A, Protein G, the neonatal Fc receptor, andthe rheumatoid factor, it does indeed overlap the same Fc bindingsite, namely, the interface between the CH2 and CH3 domains. The formation of a β-bulge conformation allows 8of the amino acids to form interactions with the Fc, covering a bindingarea of 650 Å, almost the same size as for the much larger naturalbinding proteins. Many of the interactions of the individual residuesalso share common features between the peptide and the naturally occurringFc-binding proteins.58 An 80-fold improvementof the affinity was later achieved by Dias et al. by transplantingthe FcIII peptide onto a stabilizing d-Pro-l-Protemplate creating a backbone cyclic peptidomimetic which stabilizedthe fold, previously only held together by the disulfide.59Althoughmany affinity reagents used for conjugation are protein domains likethose mentioned above, or even larger macromolecules, there are examplesdemonstrating that only a short stretch of amino acids can be enoughto ensure a large enough contact area for highly selective binding.As a smaller alternative to the naturally occurring Fc-binding proteinsA and G, a short cyclic peptide binding to the same region of IgGhas also been reported. DeLano et al. isolated two peptides, FcI andFcII, from a phage library of 4 × 1060 as well as immobilization on a sepharose resinfor high-affinity purification of antibodies.61 However, it took almost two decades since the first discovery ofthe peptide, before it was used for antibody conjugation. Park etal.35 and, shortly thereafter, Vance etal.36 both demonstrated conjugation ofa payload to antibodies through the introduction of the photoactivablencAA BPA into different positions of the peptide. Both groups drewthe conclusion that optimal conjugation results were achieved whenintroducing BPA in position 10. While Park et al. focused on monoconjugatedantibodies, Vance et al. achieved a drug-to-antibody-ratio of 1.9which corresponds to approximately 95% conjugation efficiency.The FcIII peptide has found several differentareas of applications, including that of fusion to a fluorescent modelprotein to demonstrate improvement in circulatory half-life,62 This same peptide, together with other previouslyknown peptides such as the original FcIII and the minimized Z domaindescribed above, also forms the basis of the AJICAP technology, describedby Yamada et al. This technology is based on the functionalizationof native antibodies with thiol groups through the use of these affinityreagents, which then allows for further conjugation of cytotoxic payloads.63 The same group later also describes the useof the AJICAP technology for gram-scale synthesis of stable and homogeneousADCs.64A closely related peptide, the IgG-BP, differing from FcIII onlyby the W6Y and L8R substitutions, have also been utilized for site-specificattachment. By introducing a lysine residue in position 8 of thispeptide and thereafter cross-linking it to the nearby Lys248 of anIgG-Fc through the cross-linker DSG, this peptide has been used toprepare both an ADC by conjugation of the maytansine derivative DM1and a bispecific antibody by conjugation to a nanobody.Taken together, these examplesshow how nonmodified antibodiescan be efficiently site-specifically conjugated by utilizing a peptideas small as 13 amino acids. It should be noted that the function ofthe FcIII peptide relies on formation of a disulfide and that caremust be taken to ensure the desired redox state. This fact also precludesthe introduction of additional thiol groups to be used as functionalhandles. However, since the peptide is so small, it can be easilyproduced by a chemical synthesis approach, which in turn entails asimple way of introducing many other non-natural functional groups.KD of 75 μM.65 In 1997, the same researchgroup was able to show that they could utilize that binding site fordirected photolabeling with a biotin. The functional group used forbiotinylation was an azidoadenosine decorated with a biotin, wherethe azido group, photolyzed with 254 nm, a wavelength which couldpotentially affect the antibody more than the 365 nm used for benzophenonephotolabeling, was responsible for the covalent attachment.66 Later, in 2013, this concept was further developedby the use of a new ligand with higher affinity for the NBS, namely,indole-3-butyric acid (IBA).38 The labelingprocedure regarding buffer composition, ligand concentration, andUV energy was optimized to reach a high degree of specific labeling.By utilizing modified IBA, the authors were able to conjugate antibodieswith a number of different groups including biotin, fluorescein isothiocyanate(FITC), a peptide, and the chemotherapeutic paclitaxel. Furthermore,the same labeling method has also been utilized for directed solid-phaseattachment of antibodies to streptavidin-coated surfaces.38 In order to develop a method for antigen detection,the group of Ueda utilized the NBS to conjugate an antibody with afluorophore. Here, the proximity with the antigen binding site couldbe utilized, since the fluorescence of the conjugated molecule wasshown to be quenched by the free paratope, meaning that the fluorophorewas shown to fluoresce only when the antigen was bound.67 This proximity has also been utilized to inhibitIgE clustering on mast cells by blocking the paratope through covalentattachment of an optimized molecule to the NBS that simultaneouslybinds to the paratope of the IgE.68 Inconclusion, NBS provides an interesting site for antibody conjugationon the Fab fragment, which can be utilized for conjugation using smallmolecules rather than entire peptides or proteins. Drawbacks for themethod include the use of 254 nm UV light, which can be detrimentalto the antibody, and the need for a very pure antibody sample, whichrequires removal of preservatives and stabilizers in the sample priorto labeling.A new, unconventional binding site in the variable part of antibodieswas described by Rajagopalan et al. in 1996. The new binding site,later denoted NBS (Nucleotide Binding Site), was shown to bind adenosinetriphosphate (ATP) with an apparent The conjugation methodsdescribed above all rely on the covalent attachment of an affinityligand along with the probe of interest onto the antibody. However,other methods exist where the affinity ligand functions merely tobring a catalyst in close proximity to the conjugation site for acompletely traceless conjugation of the probe alone onto the antibody.This might be particularly interesting for many therapeutic applicationswhere, for example, immunogenicity of the affinity ligand could bea potential concern.69 presented an elegant approach for traceless labeling ofhuman IgG1, where a mutated Sortase A combined with an antibody bindingdomain, a Protein G domain, or a calcium-dependent Z domain,70 could facilitate labeling of five specific lysineresidues on the antibody heavy chain and one on the light chain witha labeling agent attached to a LPETG motif. The average number oflabels per antibody was determined to 2.3 or 2.9 depending on theanalysis method used, and the labeling did not interfere with theantibody’s ability to bind its antigen, the neonatal Fc receptor(FcRn), or the Fc gamma receptor I (FcγRI). After labeling,the Sortase A-antibody binding domain could be removed from the antibodywith an acidic wash or calcium removal by EDTA. An obvious upsideto the traceless strategies is that it does not leave a bulky proteindomain attached as the bridge between the antibody as its label. However,while the method described here was shown to attach the label to fivespecific lysines on the antibody, it appears to be less homogeneousin regard to the labeling site compared to the methods based on affinitydomains with one well-known binding site on the antibody.Recently, Yu et al.56 Ohata et al. further developed this domain by introducingthree di-rhodium centers at residues E3, E11, and E20 of the domain.The rhodium-containing domain was then used for catalysis of alkyne-functionalizeddiazo modification of IgG, which was not possible in the presenceof rhodium complexes on their own. The molecule was produced usingSPPS and could catalyze modification of antibodies by attachment ofthe chemical handle to position N79 in the Fc fragment. An alkynefunctionalized human IgG1 antibody was then shown to conjugate fluorophores,and could also conjugate drugs in order to make an ADC.32Usingthe previously mentioned minimized Z domain,71Another example utilizes the already describedFcIII peptide toform stable antibody–DNA conjugates. Here, the FcIII peptidefunctions to guide a template DNA to the binding site, while a complementaryDNA strand is conjugated to the Fc through a benzaldehyde moiety reactingwith a nearby lysine side chain, without the involvement of the actualpeptide. Authors of this study used these DNA-conjugated antibodiesto assemble IgG pentamers around a core DNA motif, mimicking the naturalIgM structure.72A somewhat analogousstrategy with the same aim of conjugatingDNA to the antibody utilizes a class of affinity ligands known asAptamers, which are single-stranded oligonucleotides that can bindspecifically to a target molecule. To achieve a traceless conjugation,the Aptamer is decorated with an oligonucleotide linker that, followingbinding of the target molecule, can hybridize to a partially complementaryreacting oligonucleotide containing an activated carboxyl residuethat reacts with a nearby amine residue at the specific site. Thetemplate Aptamer can then be removed by addition of a fully complementarycDNA. Cui et al. have demonstrated this method for several biologicallyrelevant proteins and used a particular Aptamer specific for the humanIgG Fc domain to create a traceless antibody–DNA conjugatewith high specificity.The methods describedabove, traceless or not, are all based on covalent conjugation ofan antibody payload to ensure stable conjugates in complex environments.In contrast to these methods, a few examples exist that instead relysolely on a noncovalent, high-affinity, selective interaction.73The first example of noncovalent antibody conjugation is based onthe same Z domain as the covalent methods mentioned above. To achievea high enough affinity for the stable interaction, Zhou et al. coupledtwo Z domains via a long enough flexible linker so that each domainof the protein could bind the two binding sites for Z on one Fc fragmentsimultaneously. This improved the affinity for Fc compared to thesingle Z domain and rendered a molecule with up to 84-fold sloweroff-rate compared to the Z domain on its own. The Z-linker-Z domainwas then circularized using a split intein approach and termed lasso.It was also equipped with an N-terminal biotinylation tag as wellas a cysteine for additional reporter conjugation. The lasso was usedas a capturing agent in an ELISA and shown to lower the limit of detectioncompared to an immobilized secondary Fab fragment by 12-fold and also,when conjugated to a fluorophore, used as a detecting agent in a confocalmicroscopy cell detection assay.in vivo xenograft model of human lung adenocarcinoma.74Another method that does not rely on covalent conjugation is themethod known as multivalent and affinity-guided antibody empowermenttechnology (MAGNET). Here, Gupta et al. report on a computationalapproach used to simulate and design affinity ligands that bind stronglyto certain conserved residues of an IgG1 antibody. As a template forthe simulations, the well-known affinity molecule 4-mercaptoethylpyridine(MEP) coupled to two different payloads was used. The resulting MAGNETlinkers were shown to rapidly associate to IgG by simply mixing thetwo molecules together. The authors also demonstrate that, despitethe interaction being noncovalent, the conjugated antibodies werestable in both mouse and human plasma for 14 days. In addition, theefficacy of the conjugate was demonstrated using an While very promising and obviously stable, some applicationsmaystill require covalent attachment of the label to ensure little tono label loss, in particular, when using detection antibodies in harsherreaction conditions for which these methods may not be optimal.in vivo. Thus, these antibody conjugation molecules arevery important tools available for the many research fields utilizingantibodies. We believe that the use of these affinity ligands willcontinue to contribute to the field of antibody conjugation and ADCdevelopment in the future as they will continue to develop. Futureendeavors should focus on improving the labeling efficiency as wellas stability and to evaluate different combinations of ligands toincrease the number of labels per antibody. This will be importantboth for development of ADCs with a higher drug-to-antibody-ratio(DAR) and for signal amplification during detection, to achieve morerobust tools and more potent drugs. The traceless approaches mentionedabove, where the affinity ligand works only as a catalyst holds greatpotential for simple and nonimmunogenic conjugates.Presented in this revieware methods for conjugation of reportermolecules and therapeutics to antibodies in a site-specific and controlledmanner. These methods are based on molecules with inherent affinityfor parts of the antibody that are outside of the paratope and providethe means to label any off-the-shelf antibody without modifying itfirst. These methods are important new tools, for making both detectionantibodies and ADCs. Many of the aforementioned examples have shownthe benefits of site-specific labeling compared to the traditionalrandom conjugation methods that rely on side chain modifications,which may impair both the antibody structure and binding site. Thepossibility of using these methods to label antibodies even when presentin complex mixtures makes them broadly applicable to many differenttypes of samples, possibly even including cells and tissues


One key event in insect evolution was the development of mandibles with two joints, which allowed powerful biting but restricted their movement to a single degree of freedom. These mandibles define the Dicondylia, which constitute over 99% of all extant insect species. It was common doctrine that the dicondylic articulation of chewing mandibles remained unaltered for more than 400 million years. We report highly modified mandibles overcoming the restrictions of a single degree of freedom and hypothesize their major role in insect diversification. These mandibles are defining features of parasitoid chalcid wasps, one of the most species-rich lineages of insects. The shift from powerful chewing to precise cutting likely facilitated adaptations to parasitize hosts hidden in hard substrates, which pose challenges to the emerging wasps. We reveal a crucial step in insect evolution and highlight the importance of comprehensive studies even of putatively well-known systems. DurinThe evolution of parasitoidism in Hymenoptera has led to one of the largest species radiations within insects –19. SeveIn an undescribed species of parasitoid wasps, we discovered peculiar antler-like extensions on top of otherwise ordinary-looking chewing mandibles . These eTo test whether flexible mandibular movement represents a singular evolutionary event or might play a larger role in parasitoid evolution, we analysed the occurrence of this type of mandible throughout Chalcidoidea and all major lineages of Hymenoptera and correlated the morphological characters with the most recent molecular phylogenies of Hymenoptera  and Chal. 2 (a)a,b).The face of Colotrechninae sp. is excavated and bears a pair of lateral facial processes next to the inner eye margins, which are each flanked by a single elongate seta. These processes are pointed ventrally and are slightly curved inwards. They are situated slightly below the level of the antennal insertions. The antennae are inserted high on the face within deep antennal scrobes. The mandibles possess five teeth each. Their outer surfaces feature conspicuous, distally pointed extensions, reaching distally to the lateral facial processes. Further, each mandibular extension carries a distinct inner tooth a,b.c,d). Two separate pairs of muscle bundles are developed and connect the mandible to the cranium. One pair (M1) has its anterior bundle (M1a) originating from the frons and its posterior bundle (M1b) from the gena. Both bundles insert at the inner angle of the mandibular base. The second pair (M2) has its anterior (M2a) and posterior bundles (M2b) originating at the ventral part of the gena and inserting to flanges of the outer margin of the mandibular base .The mandible is loosely articulated to the head capsule by a single anterior condyle. A posterior condyle is completely absent. All mandibular muscles insert directly to the mandible and not via sclerotized tendons c,d. Two lar base c,d. (b)a,d, Austrotoxeuma, a posterior condyle is slightly indicated but not articulated to the head capsule. The mandibular musculature of all other Chalcidoidea is also characterized by two muscles (M1 and M2) with two bundles each, which individually insert on the mandible. As in Colotrechninae sp., M1a originates always from the frons and M1b from the gena. In most Chalcidoidea, M2a and M2b originate from the ventral gena. A notable exception is the flattened head of Ceratosolen (Agaonidae), where M2a originates from the frons.Despite huge variation in overall mandibular shape, all other Chalcidoidea examined (figure 2e and Belytus (Diaprioidea) and Exallonyx (Proctotrupoidea)), the posterior condyle is completely reduced (no posterior articulation with the head capsule), while it is indicated but without form closure around the condyle in the more distantly related lineages (Telenomus (Platygastroidea) and Andricus (Cynipoidea)).Regarding mandibular morphology, we also found a reduction of the posterior condyle in other Proctotrupomorpha, while it was distinct in all other groups figures e and 5. b and Belytus (Diaprioidea) and Exallonyx (Proctotrupoidea)). With the exception of Netelia (Ichneumonoidea), the tendons of M1 are split distally and attach to separate muscle bundles. Both muscles (M1 and M2) originate from the gena. This largely corresponds to the condition found in other mandibulate insects, such as beetles .The mandibles in all hymenopteran lineages except Chalcidoidea have single insertion points for M1 and M2 a and 2b,c and 2b,c and figure 1e,f). By contrast, flexible mandibular movement along multiple degrees of freedom is realized by the interplay of all four muscle bundles acting independently. For upward-directed movement of mandibles, the two posterior bundles M1b and M2b would act as protractors and their anterior counterparts M1a and M2a as retractors. In Colotrechninae sp., this movement allows for a closure between the tips of the mandibular antlers and the lateral facial processes .In Chalcidoidea, the mandible and its articulation as well as the associated musculature are highly modified compared to other pterygote insects, with fundamental functional consequences. The mandible is articulated to the head capsule by just a single anterior condyle, instead of two condyles as generally postulated for pterygote insects with biting mouthparts. This abolishes a functional restriction of mandibular movement to a single plane. Instead, in combination with highly modified mandibular musculature, a flexible movement of mandibles can be achieved, including adduction, abduction, protraction, retraction, rotation and any combination of these movements. In Chalcidoidea, M1 is therefore not restricted in its function as an adductor and M2 not as abductor as in other pterygote insects. By contrast to all other groups examined, M1a originates from the frons (figures 2 (b)Lariophagus distinguendus   is typical for the majority of chalcid wasp species. Flexible mandibular movements during its host eclosion are clearly visible : both mandibles can move independently at the same time. This allows precise cutting, as the mandibles can operate under different angles to the substrate and to each other. This flexible movement might be especially helpful in an environment with spatial constraints, where force has to be applied with minimal movements of the head itself. These constraints can either be caused by arthropod host eggs, as in the earliest chalcid lineage Mymaridae, or by the substrate surrounding parasitoids emerging from their enclosed hosts, as in the majority of Chalcidoidea. Therefore, we assume that flexible mandibular movement played an important role in the evolution of diverse host associations.Chalcidoidea are unique among parasitoids in targeting the largest diversity of host taxa and in exhibiting the largest number of feeding types defined for parasitoid wasps . HoweverThe flexible articulation of the mandibles represents a modification unique among insects. Close relatives of Chalcidoidea (Diaprioidea and Proctotrupoidea) already show at least a partial reduction of the posterior condyle , which me–h, electronic supplementary material, movie S1). A potential grasping mechanism could be used for clasping the hosts prior to oviposition. Currently, the host biology of Colotrechninae sp. is obscure and the new species is only known from a single female specimen. The face and mandibular morphology of Colotrechninae sp. is unparalleled among extant insects but shows a staggering similarity to the ‘hell ants’ (Formicidae: Haidomyrmecinae) described from Cretaceous amber deposits [The bizarre mandibles in Colotrechninae sp. represent a unique evolutionary step that was facilitated by their flexible articulation. The antler-like extensions of the mandibles can interlock with the lateral facial processes. In combination with the excavated face, this strongly hints to a grasping mechanism, while the standard biting function of the mandibles can be maintained (figure 1deposits . ‘Hell adeposits . The man (c)The mandible of Chalcidoidea represents an evolutionary novelty that likely played an important role during an extremely large insect radiation process leading to the estimated 500 000 species of this superfamily. It has long been known that the evolution of mandibles in the Mandibulata  and its modifications in the dicondylic insects have triggered large species radiations . Our res. 4 (a)Representative taxa of ethanol-preserved Hymenoptera and one species of Coleoptera  have been selected and studied and voucher specimens are deposited at the State Museum of Natural History, Stuttgart. (b)syris [Tomographic scans of ethanol-preserved insect heads were performed at the UFO-I station of the Imaging Cluster at the KIT light source using a parallel polychromatic X-ray beam produced by a 1.5T bending magnet. The beam was spectrally filtered by 0.5 mm aluminium and the resulting spectrum had a peak at about 15 keV, with a full-width at a half maximum bandwidth of about 10 keV. A fast indirect detector system was employed, consisting of a 12 µm LSO:Tb scintillator  and a disyris  and the syris . (c)https://biomedisa.org) [Tomographic slices were converted to 8 bit and cropped to the region of interest. In Amira 5.6. heads, mandibles and mandibular muscles were pre-segmented in the software's segmentation editor. The labels served as input for automated segmentation, which was performed using the online platform Biomedisa (isa.org) . Segment (d)L. distinguendus used in this study originate from the laboratory colonies of the Biologische Beratung GmbH (Berlin), where they were bred on larvae of Sitophilus oryzae  (Coleoptera: Curculionidae) that developed in grains of the common wheat Triticum aestivum L. The infested wheat grains were observed and the hatching wasps were recorded using a Nikon DSC D90 camera mounted on a Leica MZ 12.5 stereomicroscope.The specimens of


Initially, the Cochrane risk of bias (RoB) tool had a domain for “blinding of participants, personnel and outcome assessors”. In the 2011 tool, the assessment of blinding was split into two domains: blinding of participants and personnel (performance bias) and blinding of outcome assessors (detection bias). The aims of this study were twofold; first, to analyze the frequency of usage of the joint blinding domain (a single domain for performance and detection bias), and second, to assess the proportion of adequate assessments made in the joint versus single RoB domains for blinding by comparing whether authors’ RoB judgments were supported by explanatory comments in line with the Cochrane Handbook recommendations.We extracted information about the assessment of blinding from RoB tables  of 729 Cochrane reviews published in 2015-2016. In the Cochrane RoB tool, judgment  needs to be accompanied by a transparent comment, in which authors provide a summary justifying RoB judgment, to ensure transparency in how these judgments were reached. We reassessed RoB based on the supporting comments reported in Cochrane RoB tables, in line with instructions from the Cochrane Handbook. Then, we compared our new assessments to judgments made by Cochrane authors. We compared the frequency of adequate judgments in reviews with two separate domains for blinding versus those with a joint domain for blinding.The total number of assessments for performance bias was 6918, with 8656 for detection bias and 3169 for the joint domain. The frequency of adequate assessments was 74% for performance bias, 78% for detection bias, and 59% for the joint domain. The lowest frequency of adequate assessments was found when Cochrane authors judged low risk – 47% in performance bias, 62% in detection bias, and 31% in the joint domain. The joint domain and detection bias domain had a similar proportion of specified outcome types .Splitting joint RoB assessment about blinding into two domains was justified because the frequency of adequate judgments was higher in separate domains. Specification of outcome types in RoB domains should be further scrutinized.The online version contains supplementary material available at 10.1186/s12874-021-01339-1. Risk of bias (RoB) assessment is a crucial methodological aspect of systematic reviews and an obligatory part of Cochrane reviews . A 2008 We have shown previously that Cochrane reviews authors frequently made inadequate RoB judgments using the 2011 RoB tool –10. MoreThe aims of this study were twofold: first, to analyze the frequency of usage of the joint blinding domain, and second, to assess the proportion of adequate assessments made in the joint versus single RoB domains for blinding in Cochrane reviews by comparing whether authors’ RoB judgments were supported by explanatory comments in line with the Cochrane Handbook recommendations.https://osf.io/fmjxz/.This was a primary methodological study in which we analyzed the methodology of Cochrane reviews published in the Cochrane Database of Systematic Reviews (CDSR). The study protocol was prepared a priori, but the protocol was not published. Raw data generated in this study are available on the Open Science Framework project page on the link CDSR was searched for all reviews of randomized controlled trials (RCTs) of interventions  published from July 2015 to June 2016. This was a large, one-year convenient sample based on our previous studies , 11, 12,Titles and abstracts of Cochrane reviews were screened for eligibility by the first author (OB) and verified by another author (SD). The second author was verifying that no reviews were erroneously included/excluded. A list of analyzed Cochrane systematic reviews and studies included is presented in Supplementary file https://osf.io/fmjxz/) [The first author (OB) wrote series of macro-instructions in Visual Basic for Applications  to automate data scraping of all the CSRs included in the study from The Cochrane Library webpage to Microsoft Excel 2010  workbook. The automatic extraction of RoB tables for every eligible Cochrane review was then done with a new set of coded instructions, as in our previous studies (/fmjxz/) . Errors During error checkup and manual search for missing data, in two separate analyses of the domain for blinding of participants and personnel  and for In our previous study , the firPilot tests (adjustments of the tool) were done in the studies mentioned above by most experienced authors  on samples of 500 RCTs each. These authors used the same tool in this study, with no changes in appearance or coding.The support for judgement provides a succinct summary from which judgements of risk of bias can be made and aims to ensure transparency in how these judgements are reached.’. These supporting comments should be sufficiently informative for making a judgment. Thus, we assessed whether Cochrane authors’ RoB judgments were supported by the comments provided by authors in RoB tables.The Cochrane Handbook explicitly instructs authors: ‘In the first step of assessing judgments’ adequacy we made a new assessment of RoB based on supporting comments from Cochrane reviews, based on instructions from the Cochrane Handbook. In the second step, we compared these de novo judgments with judgments published in Cochrane reviews.The new assessment of RoB for the joint domain was made for RCTs in which Cochrane authors provided both a judgment  and accompanying comment. The only source for these assessments was the accompanying comment from the RoB table and the description of the intervention provided by the Cochrane authors, not the full texts of the original studies. The mentioned user interface was used just to enhance the visualization of the mentioned data and to ease the fulfilling of the MS Excel table. No full texts of the primary studies were analyzed. We followed instructions for rating detection bias from the Cochrane Handbook (Sects. 8.11.2 and 8.12.2)  and defiTwo authors  reassessed the RoB for their respective half of the sample. Due to the redundancy of the questions (Q#1 vs Q#3 and Q#2 vs Q#4) the lead author checked for the discrepancies and eventually corrected the assessment in about 20% of the cases. Lastly, we compared our new RoB assessments with the assessments made by the Cochrane authors. The proportion of RoB assessments by Cochrane authors matching the reassessment adhering to the Cochrane Handbook was termed – adequacy. The opposite term, inadequacy, does not necessarily mean the original judgment is incorrect but simply not justified by the supporting comment , 15.RoB judgments, for the joint blinding domain, assigned by Cochrane authors were analyzed by number and adequacy . The definition standard in our assessment was the Cochrane Handbook, as specified in Table 8.4.d . We consWe analyzed the distribution of different types of outcomes  in the performance bias domain, detection bias domain, and the joint domain. Primarily, our user interface offered a variety of pre-specified outcomes: all outcomes, not specified, objective , outcomes rated/related/reported (RRR) by the clinician , patient-rated/related/reported or patient RRR  and subjective in general. Due to overlap of characteristics of some types of outcomes and relatively small numbers, we used and analyzed a reduced list of outcomes: (i) all outcomes or not specified, (ii) objective outcomes (or subject independent), and (iii) subjective outcomes (including both clinician RRR and patient RRR). “Not specified” outcomes were the ones with a cell left blank in the RoB table  and thus were grouped. We also compared the distribution of severity of reassessed judgments  for all three domains .Apart from the analysis of the whole joint blinding domain and comparison between two separate standard blinding domains, we performed analyses of subsamples when the joint domain for performance and detection bias was split into multiple subdomains according to the various outcomes. Here, we compared distribution  and adequacy of judgments to the whole sample.We presented descriptive data as frequencies and percentages. We used type I error α = 0.05 and type II error β = 0.2 for all statistical tests. Statistical analyses were performed using MedCalc for Windows, version 12.5.0.0 . Kolmogorov–Smirnov test was used to assess normality for all the datasets. For comparison of independent samples of non-parametric data, the Mann–Whitney test was used, and the Wilcoxon test was used for paired samples. A chi-squared test was utilized to asset the difference in proportions. Tukey fences were used for suspected outliers. Hypotheses, outcome measures, statistical tests used, and results are logged in Supplementary file The analysis was conducted on 729 Cochrane reviews, with 10,527 included trials. There were a total of 6918 assessments for performance bias, 8656 for detection bias, and 3169 for the joint domain  and versus 78% (6747/8656) for detection bias domain  was the lowest 59%; 1860/3169) in the joint domain  that authors specified was found for detection bias domain (13% / 5% / 82%) and joint blinding domain  of those trials , and ‘better’ RoB judgments in 273 (8.5%) trials , as shown in Table .4% of thp < 0.05; detection bias domain 86.9%, p < 0.05); see Table N = 111) out of 187 inadequate judgments in the subjective outcomes group (Table Assessment of subjective outcomes demonstrated significantly lower adequacy in the joint blinding domain 57.3%) than in the two separate domains  was significantly different  than the whole sample. On the other hand, judgments in the rest of the trials which judged the risk of detection bias differently were as (in)adequate as in the whole sample .Distribution of categories of outcomes in the whole joint blinding domain  and its subsample of trials with domain split according to the type of outcome , compared to one joint domain for all those three groups of individuals.Separate domains force the review authors to provide a separate judgment for different groups of individuals; thus, assessments become more precise with split domains. This separation is relevant because there are specific difficulties for blinding different groups . There mSometimes Cochrane reviewers specified the type of outcome for which the domain was judged, i.e., whether they considered an outcome objective or subjective. The distribution of assessments according to different types of outcomes demonstrated certain similarities between the joint domain and the detection bias domain. Both domains had a very low rate of specified outcomes , but in contrast to the performance bias domain (with less than 5%), this might be seen as a success. We might conclude that much more effort has to be introduced to identify outcomes susceptible to bias related to blinding in trials, as well as taking care of proper blinding of the subjects.In our previous study , assessmClinician-related outcomes that were judged with low risk by Cochrane authors contributed the most to inadequate judgments. Among these, the majority defined such an outcome as objective, even though it was not , a problem linked to the detection bias domain. Some RoB tables did not have enough detail in supporting comment, e.g., they used a vague “double-blind” comment without specifying who exactly was blinded, which is a frequent explanation that reviewers use when describing their rationale for assessing the performance bias domain. This likely stems from the primary studies, where the usage of the term “double-blind” without any further details about blinding of key individuals is widespread; however, it has been shown repeatedly that the term is ambiguous and that it means different things to different researchers –19. ThusAdditionally, we found that Cochrane authors sometimes split the joint domain into multiple subdomains for different outcomes. While this approach may be considered more transparent regarding different types of outcomes , such reviews had much worse results in terms of RoB adequacy. Therefore, we have demonstrated that splitting a joint blinding domain only according to the outcomes is not a preferable solution. Splitting  should be used based on the different groups of individuals  and the susceptibility of an outcome to be influenced by knowledge of intervention received, such as in RoB 2.Cochrane methods are continuously evolving. Our findings indicate that the decision to split the domain about blinding into two separate domains was justified, as the adequacy of judgment was better in separate domains. This is easily understood, as the joint domain refers to multiple groups of participants, and therefore it may be unclear how Cochrane authors are judging RoB related to blinding in domains covering more than one group of participants. For this reason, we hypothesize that the decision to split further the assessment of RoB related to blinding to three assessments in the RoB tool 2 will prove to be even more advantageous for accurate assessments . HoweverOur study's strength is that we have analyzed a large number of Cochrane reviews with more than ten thousand trials included. We have focused on Cochrane reviews because the use of Cochrane methods, i.e., Cochrane RoB tool, is mandatory in Cochrane reviews, but our results are also relevant for non-Cochrane systematic reviews. Although the majority of non-Cochrane reviews do not report on RoB , 21, wheThere are also some potential limitations to our work. Firstly, even though we prepared a study protocol before commencing this study, we did not publish the study protocol, as there is still no requirement in the international community for publishing protocols of studies other than clinical trials. However, we are aware that publishing the study protocol prospectively could be important for readers for appraising the risk of selective reporting and any other biases that may have occurred due to changes to the protocol during the study.Additionally, there may be differences between assessments made in the original Cochrane reviews in data availability, as the Cochrane authors have appraised reports of included RCTs, and they might have contacted trial authors for clarifications. For this reassessment, we relied on comments provided by Cochrane authors in RoB tables. Cochrane authors should provide informative comments to explain the rationale for their judgments as instructed by the Cochrane Handbook. If the authors did not report all the key information transparently in the supporting comment, the judgment might not be sufficiently justified. The concept of adequacy, used in this study, might still be subjective because it was ultimately determined by the authors of this manuscript  , 15.The categorization of outcomes as objective or subjective was made by our team. It needs to be emphasized that outcomes are often not fully objective or fully subjective but instead fall somewhere on the continuum between objective and subjective. It is possible that clinician input during the execution of the Cochrane reviews could have influenced the risk of bias judgments, at least partially explaining why the assessments in the reviews would be different from those undertaken in this study.https://osf.io/fmjxz/.Furthermore, some may consider that blinding is not well defined in the Cochrane Handbook and that neither Cochrane authors nor our team could categorically determine whether the Cochrane Handbook criteria have been met. For this reason, we have transparently reported our judgments and rationale behind our assessments: raw data generated in this and related manuscripts can be located on the Open Science Framework project page on the link It could also be argued that blinding is a poorly defined construct. For example, blinding could be a property of the trial methods (in which case assessment of blinding would involve assessing the presence/adequacy of the placebo or sham), but also it can be manifested in the knowledge or beliefs of key individuals about the allocation of interventions; in the latter case evaluation of blinding would involve assessing knowledge or beliefs of the key individuals about the allocation .In this study, we analyzed Cochrane reviews published within a limited date range from July 2015 to June 2016. However, we have no reason to believe that the results would be different if we have used a more extended period after June 2016. We did not choose an earlier period than July 2015 because the analyzed Cochrane RoB tool was published in 2011, and we considered it essential to leave out the first few years after its publication to allow Cochrane reviewers to adopt the new methodology. Regarding the inclusion of a higher number of more recently published Cochrane reviews, we have evidence from our recent methodological study that this is not needed . In thatOur results indicate that splitting the joint RoB domain about blinding key individuals into two separate domains was justified. Cochrane authors more frequently made adequate judgments in separate domains for blinding. We anticipate that this should result in an even higher adequacy of judgments in the Cochrane RoB 2 tool, but this will need to be confirmed after its full implementation in Cochrane reviews.Additional file 1. List of analyzed Cochrane systematic reviews and studies included.Additional file 2. Table overview of the hypotheses, outcome measures, statistical testsused and results.


The results showed that the rheological curve showed a typical work hardening stage, and there were three different stages: work hardening, transition and steady state. A strain compensation constitutive model was established to predict the flow stress of the Mg-2.5Nd-0.5Zn-0.5Zr alloy, and the results proved that it had high predictability. The main deformation mechanism of the Mg-2.5Nd-0.5Zn-0.5Zr alloy was dislocation climbing. The processing maps were established to distinguish the unstable region from the working region. The maps showed that the instability generally occurred at high strain rates and low temperatures, and the common forms of instability were cracking and flow localization. The optimum machining range of the alloy was determined to be 592–773 K and 0.001–0.217 s−1. With the increase in deformation temperature, the grain size of the alloy grew slowly at the 573–673 K temperature range and rapidly at the 673–773 K temperature range. Isothermal hot compression experiments were conducted on Mg-2.5Nd-0.5Zn-0.5Zr alloy to investigate hot deformation behavior at the temperature range of 573–773 K and the strain rate range of 0.001 s Mg alloys have the benefits of low density, high stiffness, high specific strength and machinability . They haMg-Nd-Zn-Zr alloys are favored by many researchers for their excellent mechanical properties and low RE content ,16,17,18It is well known that constitutive equation analysis has become an effective method to present the thermal deformation behavior of metal materials . The flo−1. Zheng et al. [−1, and established the hot working diagram. The optimum hot working condition temperature was 380 °C and the strain rate was 0.006 s−1.The dynamic material model (DMM) of Prasad based on processing maps is a powerful tool for optimizing thermal processing and controlling the microstructure of a variety of alloys ,25,26,27g et al.  studied In this paper, the deformation behavior of the extruded Mg-2.5Nd-0.5Zn-0.5Zr alloy was investigated using the Gleeble-3500D thermal simulator. A strain-compensated Arrhenius constitutive equation was established to predict the flow stress. A DMM-based processing chart was constructed, which provided important guidance for optimizing thermal deformation parameters. In addition, the evolution of the microstructure of the alloy was analyzed.The initial material  was an Mg-2.5Nd-0.5Zn-0.5Zr (wt%) extrusion bar with a diameter of 25 mm. The actual chemical composition  was examined by X-ray fluorescence spectrometer . The compression sample was processed along the extrusion direction (ED), and the cylindrical sample was Φ8 × 12 mm. Firstly, the surfaces were polished smooth with 400#, 800#, 1200# and 1500# sandpaper, and then the samples were cleaned by ultrasonic shock for 5 mins in acetone and alcohol solution, in that order. The isothermal compression experiment was conducted using the Gleeble-3500D thermal simulator. Experimental parameters are shown in 2H5OH and H3PO4 electrolyte, electropolishing for 2 min at 0.5 A and 5 min at 0.25 A).With the inhomogeneous deformation of the sample taken into consideration, the microstructure was observed in the center area of the longitudinal section, as shown in the brown area in −1 in −1 and the minimum peak stress was 16.6 MPa at 773 K 0.001 s−1.−1), An Arrhenius-type equation was used to characterize the relationship between the hot-deformation parameters ,29,30,31From Equations (6)–(9), the parameters The relationship between strain rate (essed as :(10)Z=ε˙Linearizing Equation (11) by logarithmic processing:1, Q, and lnA were 0.121014, 0.021944, 3.778, 5.5147, 171.929 kJ/mol and 27.65, respectively. In this study, the detailed steps to determine the material constants were illustrated by taking a strain of 0.4 as an example. In The flow stress can be expressed as:Hence, the flow stress of the Mg-2.5Nd-0.5Zn-0.5Zr alloy when the strain was 0.4 can be expressed as:It can be seen from −1, as shown in Equation (15).The constant equation with the strain factor (ɛ) was coupled to the constructed constitutive equation. Therefore, the hyperbolic sinusoidal Arrhenius constitutive equation of the Mg-2.5Nd-0.5Zn-0.5Zr alloy was obtained at 573–773 K and a strain rate of 10.0–0.001 sGB) of pure Mg was 82–105 kJ/mol, and the lattice self-diffusion activation energy (QL) of pure Mg was 135 kJ/mol [−1 and the deformation temperature was 723–773 K  and Q in the constitutive model. The change of n value was related to the movement of crystal defects during deformation. In 5 kJ/mol ,37. AccoTo further verify the applicability of the true strain-coupled high-temperature constitutive equation established in the above section, the correlation coefficient R, square root error (RMSE) and average relative error (AARE) were used for comprehensive evaluation ,26,28. AFrom Equations (16)–(18), E was the experimental flow stress, P was the predicted flow stress, and i stands for the ordinal. N was the number of data sets for statistics. As shown in Prasad et al. ,27 develFor arbitrary strain rate and deformation temperature, the power distribution between J and G was described as follows ,31:(20)∂equation :(21)η=JJThe variation of η with strain rate and deformation temperature constitutes the power dissipation diagram. This graph was a contour map of the variation of deformation temperature–strain rate field efficiency, with different regions representing specific deformation mechanisms. Furthermore, a criterion of flow instability was defined using the extremum principle of irreversible thermodynamics ,40:(22)ξ−1 followed the power equation of Equation (15). This showed that the DMM and Prasad criterion could be used to draw the processing maps of the Mg-2.5Nd-0.5Zn-0.5Zr alloy under different strain conditions.Flow instability occurred when the ξ, 1.717 × 10−4 nm−2 (623 K), 1.105 × 10−4 nm−2 (673 K), 1.758 × 10−4 nm−2 (723 K) and 1.387 × 10−4 nm−2 (773 K), respectively. However, no matter what the deformation temperature was, the local misorientation was not uniform during hot compression, and the misorientation at the grain boundary was generally higher than that in the grain [he grain . These rhe grain . As show−1 was studied using an isothermal compression experiment system. The main results were as follows:In this paper, the high-temperature deformation behavior of the Mg-2.5Nd-0.5Zn-0.5Zr alloy in the range of 573–773 K and 0.001–10 s−1 was very sensitive to the deformation temperature, strain rate and strain variables. The whole hot-working process was divided into three stages: work hardening, dynamic softening and dynamic balancing.(1) The flow stress of the Mg-2.5Nd-0.5Zn-0.5Zr alloy in the range of 573–773 K/0.001–10 s(2) A hyperbolic Arrhenius-type strain compensation constitutive model with high accuracy for the Mg-2.5Nd-0.5Zn-0.5Zr alloy was established. This constitutive model had high acceptable predictability. The dominant deformation mechanism of the Mg-2.5Nd-0.5Zn-0.5Zr alloy was dislocation climbing.−1.(3) The thermal processing maps of the Mg-2.5Nd-0.5Zn-0.5Zr alloy under different strains were established, and the optimal processing range was determined to be 592–773 K and 0.001–0.217 s(4) With the increase in deformation temperature, the grain size of the Mg-2.5Nd-0.5Zn-0.5Zr alloy grew slowly at the 573–673 K temperature range and grew rapidly at the 673–773 K temperature range.Hot deformation was performed in compressive modes.A manufacturability map of the Mg-2.5Nd-0.5Zn-0.5Zr alloy was established for the first time.A strain-compensated constitutive model for determining flow stress in this alloy was established with highly acceptable predictability.The dominant deformation mechanism of the alloy was dislocation climbing.Highlights:


Recent studies have demonstrated that details of verbal material are retained in memory. Further, converging evidence points to a memory-enhancing effect of emotion such that memory for emotional events is stronger than memory for neutral events. Building upon this work, it appears likely that verbatim sentence forms will be remembered better when tinged with emotional nuance. Most previous studies have focused on single words. The current study examines the role of emotional nuance in the verbatim retention of longer sentences in written material. In this study, participants silently read transcriptions of spontaneous narratives, half of which had been delivered within a context of emotional expression and the other half with neutral expression. Transcripts were taken from selected narratives that received the highest, most extreme ratings, neutral or emotional. Participants identified written excerpts in a yes/no recognition test. Results revealed that participants’ verbatim memory was significantly greater for excerpts from emotionally nuanced narratives than from neutral narratives. It is concluded that the narratives, pre-rated as emotional or neutral, drove this effect of emotion on verbatim retention. These findings expand a growing body of evidence for a role of emotion in memory, and lend support to episodic theories of language and the constructionist account. The role of memory in language has been the major focus of an extensive body of empirical research over the past half century. It has interested scholars across a wide range of disciplines ranging from linguists and psychologists to cognitive neuroscientists. Originating from a classic study by A burgeoning literature has demonstrated that emotion enhances memory . It has A primary goal of this study was to examine the role of emotional nuance in encoding verbatim information of written language in memory. Operationally, emotional nuance was defined as narratives or excerpts produced with high emotional expression and high engagement ratings. We assessed recognition memory of emotional and neutral written excerpts taken from transcribed spontaneous narratives. Based on the episodic and constructionist accounts, we pursued the hypothesis that episodic details of verbal materials would be specifically encoded in memory and further strengthened when laced with emotional nuance. That is, emotional nuances would boost the verbatim retention of written text, resulting in higher recognition memory of verbatim text in emotional contexts than in matched neutral contexts.The existence of verbatim memory for language has generated considerable support from the constructionist approach to language. Also known as construction-based grammar, the constructionist approach posits that language is composed of a network of constructions, learned pairings of form and function/meaning , 2013. WIt is interesting to note that “surface” forms of sentences are retained especially well when the materials imply pragmatic inferences such as sarcasm , politenSimilarly, in the current study, it was hypothesized that emotional nuance will encourage readers to personally engage with the text, thereby enhancing the reading experience of the text for the reader. In order to elicit emotional nuance in a spontaneous speech monolog, we used transcribed narratives that were spontaneously produced by performers with emotional or neutral expression. The narratives obtained from the performers were rated in terms of emotional expressiveness and engagement to select the texts that represent extremes on both continua for the recognition task. This procedure was done to ensure that differences in overall emotional or neutral expression between the two types of narratives are easily discerned. Using these materials, we constructed two types of written narratives: ones rich in emotional nuance, referred to as “emotional narratives” and ones devoid of nuance, referred to as “neutral narratives.” In the selected narratives, strong emotional expressiveness or neutral, indifferent expression was present throughout the respective discourse units. Unlike prior studies that examined verbatim memory for spoken language , this stAn extensive body of empirical research confirms that emotional experiences generate highly durable and vivid memories . The vasEmotional content in language has been commonly identified along the dimensions of valence and arousal . In the Based on previous work studying the impact of emotional significance on language processing, it seemed reasonable to assume that people retain episodic details of verbal materials when laced with emotional nuance. Yet existing literature does not provide an adequate understanding of the link between emotion and verbatim memory of written language beyond the lexical level. The general aim of this study was to explore the effect of emotional nuance on verbatim memory in written language. Specifically, are verbatim excerpts deriving from emotional narratives better remembered than those from neutral narratives? To this end, participants silently read transcriptions of spontaneous narratives, half of which had been produced with high emotional expressions and half with neutral, as originally spoken by performers. The text excerpts included excerpts taken from the narratives that participants had seen (target excerpts), and excerpts from narratives by the same performers that participants had not seen (foil excerpts). They subsequently completed a yes/no recognition test, in which they distinguished seen from unseen written excerpts. Within each condition, half of these test excerpts were emotional and the other half were neutral; half had been seen previously and half had not. It was anticipated that participants would recognize verbatim excerpts taken from previously seen emotionally nuanced narratives better than excerpts taken from the previously seen neutral narratives or the unseen narratives.The current study was approved by the Institutional Review Board (IRB) at New York University.M = 24.1, SD = 4.9). The age of one female participant was not recorded, but assumed to be in her 20’s based on her status as a Master’s student at the time of testing. All had learned English as a first language from birth. All participants were native speakers of American English except for one British and one Singaporean English speaker. All participants had normal hearing and signed informed consent form prior to the beginning of the study. One participant who failed to follow instructions was excluded, and thus the final sample consisted of fifteen participants .Sixteen healthy native speakers of English  participated as readers in the study. Participants ranged in age from 18 to 34 years  and to the “A priori: Compute required sample size” power analysis. The analysis indicated that a sample of 15 would be sufficient to detect a large sized effect with an alpha = 0.05 and power = 0.80.g GPower  to calcuThe stimuli were transcribed narratives from video recordings of spontaneous speech produced in emotional or neutral tones by performers. These performers  were recruited from an improvisational and comedy training center to provide emotionally intoned and neutral narratives. All acquired American English as a native language from birth and ranged in age from 20 and 40 years. Each performer was randomly assigned to an emotional or a neutral presentation of the narrative and instructed to talk freely about topics of their choice for 3 min using the intended tone of voice . A list of topics that are common to adult English speakers was provided as prompts , but narrators were encouraged to suggest any topics that they were interested to talk about. They could also create fictional scenarios and use emotions of different or mixed valences (positive and negative). For the emotional condition, they were asked to tell their stories in an expressive, emotively extreme manner; for the neutral condition, they were asked to tell their stories with neutral expression, using a calm, deliberate manner of delivery. Two or three narratives were elicited by each performer, each of which lasted for 3 min, resulting in 85 narratives in total. All narratives were audio- and video-recorded and carefully transcribed verbatim, including all verbal detail for presentation in the experiment.N = 85) and then rate the degree of (1) emotional expressiveness and (2) engagement, respectively, on two 7-point rating scales. Participant instructions for the video ratings were provided in very little to no emotional expression) to 7-expressive . The second rating, engagement, was to quantify the extent to which the performer is engaging in her narrative, ranging from 1  to 7 (very engaging). The ratings were obtained from spoken materials, instead of written transcripts. It was our intention to see if the overall emotional vs. neutral status of the spoken narratives is conveyed in written form. Our view was that each narrative, as a whole, conveyed an overall emotional or neutral orientation as a Gestalt; excerpts taken from transcripts of these narratives should carry enough of this material to influence participants.The obtained videotaped narratives were then submitted to five adult female raters, all of whom were native speakers of American English. The goal was to identify extremely emotional and neutral videotaped narratives, as listeners perceive them. The raters were instructed to watch each videotaped narrative  were devised, each containing 12 transcribed narratives. Half of the videos in each stimulus set consisted of emotional narratives and half contained neutral narratives see . The aveA list of 120 test excerpts was obtained from the two stimulus sets, half of which were taken from the emotional and half taken from neutral narratives. Five excerpts were selected from each narrative (5 excerpts 24 narratives = 120 excerpts). There was thus a total of 60  + 60  = 120 excerpts presented for each participant see . All excThe test stimuli consisted of written excerpts from transcriptions of narratives that participants have read (target excerpts), and excerpts from transcriptions of narratives by the same speakers that they have not read (foil excerpts). In two cases, proper nouns were removed from the test stimuli. The eliminated words were replaced with an ellipsis consisting of three dots, as in: “So last week I got cast in a new… show at the,” and “and I invited my friend from high school,…, with us.” In order to more directly evaluate the status of the written material with respect to the independent variable , detailed corpus analyses of the narratives and the excerpts were performed, using two methods: LIWC  and ANEW-ed, noun inflections indicating a plural -s), or derived words  whose stems are matched to the word list and contractions  were considered.The ANEW is a standardized material for quantitative analysis of emotional words in the English language. Specifically, based on the dimensional view of emotion in psychology  or low  in valence were categorized as valenced words, while words rated between these two poles (4.0–6.9) were categorized as neutral words. Similarly, words high in arousal (6.0–9.0) were considered arousing, whereas words rated lower than 6 in arousal were considered nonarousing. For the purpose of this study, “emotional words” include the words that are evaluated as either (1) valenced (positive or negative) on the valence dimension or (2) arousing on the arousal dimension.All participants were tested individually in a quiet room during a single session lasting approximately 1 h. Participants were seated in a comfortable chair and stimuli were presented on paper. Before starting the experimental session, participants filled out a list of background questions pertaining to their gender, educational background, and native language.The experiment consisted of two phases: an exposure phase and a recognition test phase. In the exposure phase, participants were instructed to silently read written stories originally narrated by female speakers. Participants were informed that they would subsequently be asked to recognize the excerpts taken out of the stories. Each participant read one of two sets of 12 transcribed narratives (set A or set B), self-paced. Accordingly, half of the participants read six emotional and six neutral narratives in set A, while the other half read six emotional and six neutral narratives in set B. The narratives were presented in a pseudo-random order so that no more than three narratives from the same category appeared sequentially. The narratives appeared in the same order in each stimulus set. The mean number of transcript words across narratives was 184 words. The total group of stories for each participant constituted between 2097 (set A) and 2,388 words (set B) depending on which set was assigned to the participant.In the subsequent recognition test phase, the participants were told that they would be presented with written excerpts, some of which were taken from the stories that they had read and some were not. For each excerpt presented, participants indicated whether the excerpt had been previously seen during exposure. They were instructed to select “Yes” if they saw it before at encoding or “No” if they did not. The order of excerpts was randomized, with each participant receiving the same sequence.N = 60) and half neutral written excerpts (N = 60). A total of 60 excerpts taken from the subset of narratives that participants read (A or B) were presented along with additional 60 excerpts taken from the other subset of unexposed narratives (B or A). To ensure an unbiased selection, the test excerpts were randomly chosen by the experimenter blind to the emotional/neutral distinction of the narratives. Since participants were exposed only to narratives from either set A or set B and the test excerpts were taken from both sets (sets A and B), half of the excerpts had been witnessed before  and half of the excerpts were new  for each participant. Such a design, called a block design, was used to control for any possible effects that might arise from the salience of words or linguistic structures in narratives, which is not of primary interest in this study.The test phase consisted of a total of 120 trials, containing half emotional (d′) and response bias measured by c. The formula for d′ was d′ = z(hit rate) − z and the formula for c was c = −0.5[z(hit rate) + z] ] . Table 5d′) was 1.22 (SD = 0.34) across participants. A one-sample t-test on recognition accuracy (d′) revealed that the mean recognition accuracy (d′) was significantly greater than zero, t(14) = 13.71, p < 0.001, Cohen’s d = 3.54. These results indicate that participants reliably recognized the differences between excerpts that they had seen verbatim  and excerpts originally obtained from the same speakers but with different content . These results also suggest that written materials were encoded verbatim in memory and such verbatim memories were accessed on the subsequent test to endorse whether the written excerpts have been seen before.Overall, the mean recognition accuracy (d′) as a function of mode . In order to examine whether emotion affects the retention of verbatim representations, we conducted a paired-samples t-test on recognition accuracy (d′) for excerpts taken from emotional and neutral excerpts. Verbatim representation was significantly better recognized for excerpts taken from the emotional narratives  than excerpts taken from the neutral narratives , t(14) = 2.43, p = 0.029, Cohen’s d = 0.63. This finding confirms that participants were more likely to retain verbatim representations of excerpts from emotional narratives compared to the ones from neutral narratives. An overview of the results can be seen in c) was 0.03 (SD = 0.43) across participants. A one-sample t-test demonstrated that the overall response bias c was not significantly different from 0, t(14) = 0.31, p = 0.763, Cohen’s d = 0.08, indicating that there was neither a bias toward responding yes nor a bias toward responding no.Overall, the mean response bias  and neutral excerpts , t(14) = −0.67, p = 0.516, Cohen’s d = 0.17. That is, participants were not inclined to favor either the yes response or the no response for both emotional and neutral excerpts.A paired-samples t-test revealed that there are statistically significantly higher proportions of emotional words in emotional transcribed narratives compared to neutral narratives, t(22) = 2.36, p = 0.014, Cohen’s d = 0.96, Memotional = 6%, SDemotional = 1.92% vs. Mneutral = 4.15%, SDneutral = 1.89%. Given that each narrative averaged 184 words in length, there were, on average, about 11 emotional words (184 × 0.06 = 11) in emotional narratives, and about 7 emotional words (184 × 0.04 = 7) in neutral narratives.Using the LIWC data, an independent sample The emotional and neutral excerpts were analyzed using LIWC. The LIWC results showed that there were greater proportions of emotional words in emotional excerpts (6.2%) vs. neutral excerpts (3.51%). Despite the difference in proportions of emotional words in emotional excerpts vs. neutral excerpts, it is worth noting that the numerical differences between the two were fairly small. Considering that our data was expressed as a percentage, the average number of emotional words in each excerpt was less than a word (0.5 words) and many excerpts contain 0% of emotional words. Due to many data points that were zero, no statistical analyses were conducted on these data. These data reflect the experimental design that excerpts were randomly selected throughout the two sets of narratives. This result suggests that presence or absence of words classified as emotional was not the key determining factor in participants’ responses. This supports our hypothesis that the original narratives transmitted sufficient affective nuance to be discerned in excerpts taken from them.t-test showed that there was no significant difference in overall proportion of emotional words contained in emotional vs. neutral narratives, t(22) = 0.882, p = 0.387. When the overall analysis using the ANEW list failed to reach statistical significance, careful inspection of their ratings provided a clue that emotional valence  and arousal levels differed for the independent variable. That is, the results of independent samples t-test indicate that there are greater proportions of negative, t(22) = 2.879, p = 0.009, and arousing words, t(22) = 2.539, p = 0.019, in the emotional narratives, than in the neutral narratives. In contrast, there was no significant difference in the proportions of positive words in emotional and neutral narratives, t(22) = 0.888, p = 0.384. The means and standard deviations of these measures are reported in Using the ANEW identification procedure, an independent sample As was the case with LIWC, each excerpt contained only 0–3 words that match those in the ANEW list, limiting statistical analyses. The proportion of emotional words in emotional excerpts was 7.73% (62 words / 802 words × 100 = 7.73%) and 6.74% (50 words / 742 words × 100 = 6.74%) in neutral excerpts. Overall, there was a negligible effect of positivity in emotional and neutral excerpts: emotional excerpts contained 5.36% positive words, while neutral excerpts contained 5.66%. The proportions of negative and arousing words in the two types of excerpts mirrored those in the narratives. Emotional excerpts expressed more negative and arousing words than did the neutral excerpts . See Building on previous literature on the effect of language and emotion on verbal memory, the goal of the present study was to investigate the influence of emotionally nuanced verbal materials on verbatim memory. To address this, memory for emotional and neutral written excerpts, taken from written narratives seen or unseen by readers, was assessed through a recognition test. First, participants were able to successfully endorse whether or not they had seen excerpts presented to them, corroborating the existence of verbatim memory for written language originally spoken in a spontaneous manner. Second, a key finding of this study is a facilitative role for emotional nuance in verbatim retention of verbal materials; participants demonstrated better recognition memory for excerpts from transcribed emotional narratives compared to those from transcribed neutral narratives. This pattern of results establishes that emotional nuance enhances verbatim retention of written excerpts and further highlights the ability of written language to communicate emotional nuance in spontaneous discourse.The narratives used in the experiment were originally spoken by performers with emotional or neutral expression and then selected by raters according to which narratives received the highest and lowest ratings for emotional expressiveness and engagement to ensure that distinctions between emotional and neutral texts were significantly large. According to the result of the lexical analysis, measures of emotionally laden lexical items in the excerpts and transcripts reveal small differences between emotional and neutral stimuli. It is likely that the difference between the emotional and neutral narratives lies in the overall, pervasive emotional nuance or neutral context carried in each narrative as a whole. We conclude that the web of emotional nuance created by emotional connections throughout the discourse is accountable for the impact on the participants.Evidence of verbatim memory for language obtained in this study is consistent with the claim of current notable episodic (or exemplar) and constructionist theories that the verbatim form of text is specifically represented and maintained in memory . StructuA fundamental claim of the constructionist theory is that linguistic structures, combined with function, are stored as constructions and emerge from actual usage of these constructions . The preWith regard to stimuli, it is worth noting that the transcribed narratives used in the current study were long enough to ensure that participants were unlikely encode or rehearse the sentences intentionally. The entire narratives averaged 2,242.5 words in length  (compare this to ≈ 300 words in The distinctiveness and attention-grabbing nature of emotional stimuli has been shown to drive performance. These cognitive characteristics of emotional stimuli are closely related to each other because relatively distinctive items automatically capture attentional resources . The disIn a similar vein, an increased attention toward emotional stimuli can also account for the preferential encoding of verbatim representations for the emotional narratives. There has been a substantial amount of study demonstrating that emotional stimuli trigger attentional capture, leading to prioritized processing . ConsistIn our study, valence was not treated as an independent variable. It is worth noting that there is an extensive literature that examines how valence, as one dimension of emotionality, differentially affects memory performance, using positive and negative items . For insOne potential limitation of this study is the sample size. The study needs to be replicated with a larger number of participants to provide stronger support for the generalizability of results. The findings of this study have practical implications for educational settings. Incorporating an emotional text to educational materials is more likely to lead to better learning outcomes by facilitating retention of knowledge. Such materials could potentially promote effective learning as students tend to retain verbal material when tinged with emotional nuance. The current findings are also potentially useful for writers who want to engage and influence their readers. Writing strategies that integrate emotionally charged elements will improve reader’s retention, potentially influencing their opinions.To conclude, the findings of the present study argue in favor of the existence of verbatim memory for written language and provide further evidence that verbatim memory is boosted by emotional nuance. Taken together, current findings provide support for the episodic theory and the constructionist account of language and contribute to existing knowledge on memory by establishing a link between emotion and verbatim memory.The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by the NYU IRB/University Committee on Activities Involving Human Subjects. The patients/participants provided their written informed consent to participate in this study.All authors contributed to the design of the study, participated in the development of stimulus materials, and approved the final version of the manuscript before submission. YK performed data collection, data analysis, and interpretation under the supervision of DS and JS. YK drafted the manuscript. DS and JS provided critical revisions and comments.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.