Influenza B viruses (IBV) drive a significant proportion of influenza-related hospitalisations yet are understudied compared to influenza A. Current vaccines target the head of the viral hemagglutinin (HA) which undergoes rapid mutation, significantly reducing vaccine effectiveness. Improved vaccines to control IBV are needed. Here we developed novel IBV HA probes to interrogate humoral responses to IBV in humans. A significant proportion of IBV HA-specific B cells recognise both B/Victoria/2/87-like and B/Yamagata/16/88-like lineages in a distinct pattern of cross-reactivity. Monoclonal antibodies (mAbs) were reconstituted from IBV HA-specific B cells, including mAbs providing broad protection in murine models of lethal IBV infection. Protection was mediated by neutralising antibodies targeting the receptor binding domain, or via Fc-mediated functions of non-neutralising antibodies binding alternative epitopes including the IBV HA stem. This work defines antigenic cross-recognition between IBV lineages and provides guidance for the rational design of improved IBV vaccines for broad and durable protection. Immune recognition of Influenza B virus (IBV) is poorly understood. Here, Liu et al. use flow cytometry to characterize IBV-specific memory B cell responses following seasonal vaccination and show that elicited cross-reactive antibodies can protect against infection, providing a platform for vaccine design. The clinical severity of IBV is equivalent to that of IAV3, with children and young adults most susceptible to IBV. Unlike IAV, which naturally resides within aquatic bird populations, IBV infection is almost exclusively restricted to humans, with only rare reports of infection in seals4. Mice and ferrets can however be experimentally infected with IBV, providing useful small animal models of human IBV infection and disease5. Owing to the limited host range and a markedly slower mutation rate than IAV6, circulating IBV strains exhibit more limited antigenic diversity than H1N1 and H3N2 IAV7. Nevertheless, since the first reports in 1940s IBV has gradually diverged into two distinct lineages—B/Victoria/2/87-like and B/Yamagata/16/88-like8 (referred to as B/Victoria and B/Yamagata lineages from here on), which are further divided into antigenic clades9.Influenza viruses are highly infectious respiratory pathogens that inflict annual epidemics and periodic global pandemics, a significant cause of mortality and morbidity. Two types of influenza viruses co-circulate in human populations, influenza A viruses (IAV) and influenza B viruses (IBV). Both IAV and IBV are formulated into current seasonal influenza vaccines. IBV historically accounts for ~20% of influenza-related hospitalisations in any given year10. Nonetheless, vaccine effectiveness of seasonal influenza vaccines against IBV can often remain low even when antigenic match with circulating strains is good12. Furthermore, protective serological titres elicited by current seasonal vaccines wane in immunised adults13. There remains a need for improved next-generation IBV vaccines to provide resilient and durable protection against a broad array of IBV strains.An increased awareness of the burden of IBV, coupled with growing antigenic complexity, prompted the recent deployment of quadrivalent influenza vaccines, incorporating components derived from both circulating IBV lineages in order to broaden coverage. Quadrivalent vaccines limit the likelihood of reduced vaccine effectiveness due to IBV lineage mismatch compared to superseded trivalent vaccines, which is thought to have significantly contributed to preventable deaths14, with more recent analysis of antibody repertoires after influenza vaccination suggesting a large fraction of antibodies elicited by primary infection15 or seasonal immunisation16 are cross-reactive against both lineages of IBV. Selected human monoclonal antibodies (mAbs) have been isolated and can bind and neutralise diverse IBV isolates from both lineages20, highlighting that cross-protective humoral immunity is feasible. These mAbs bind conserved epitopes localised within either the globular head, or the stem domain of hemagglutinin (HA), and can protect mice from challenge with diverse strains of IBV. Protection likely depends, at least in part, upon the elicitation of antibody-dependent cellular cytotoxicity (ADCC)17. In addition to HA-specific antibodies, a recent study suggests antibody binding the viral neuraminidase might similarly allow cross-protection against both IBV lineages21. Unlike the large number of mAbs characterised for IAV22, human immune recognition of IBV remains understudied. A greater understanding where and how human B cells and antibodies bind to IBV, the capacity of each site to mediate protection, and the extent of antigenic cross-recognition between IBV lineages is critical to guide efforts to develop improved influenza vaccines.While the extensive characterisation of human immune recognition of IAV spans many decades, the principal antigenic targets of IBV, including the degree of cross-recognition of the different lineages by B cells and antibodies, remain poorly defined. Cross-reactive humoral responses between IBV lineages were described as early as 199124, we interrogated the IBV-specific memory B cell response following seasonal tri- and quadrivalent vaccination of adult human volunteers. Both classical antibodies inhibiting hemagglutination, as well as novel broadly cross-reactive antibodies binding both IBV lineages were isolated from vaccinated subjects and demonstrated antiviral activity in vitro and in vivo. Furthermore, we demonstrate that protection mediated by non-neutralising antibodies was dependent upon interaction with cellular Fc receptors. The accurate definition of cross-reactive human anti-IBV antibody specificities will guide the design of novel IBV vaccines for broader protection.Using a flow cytometry-based approach previously described for IAV24 have proven utility for characterising B cells responses to influenza A vaccination using flow cytometry27. We designed analogous constructs to enable expression of soluble trimeric IBV HA ectodomains from B/Phuket/3073/2013 (B-PH13). We generated a wild-type (WT) and a T139G variant, selected to potentially disrupt hydrogen bonds within the receptor binding pocket28. With the HA proteins expressed and using flow cytometry  receiving 2015 trivalent Southern Hemisphere inactivated influenza vaccine (IIV3), including a B-PH13 component29. Vaccine-elicited expansion of B-PH13+ class-switched memory B cells  was clearly evident between baseline (d0) and d28 post-immunisation  and H1N1 A/California/07/2009 (H1-CA09)  was observed between the magnitude of IIV3-induced expansion in B-PH13+ memory B cell frequencies and the increase in strain-matched serum hemagglutination inhibition assay (HIA) titres  Fig. . We also09) Fig. . Moreoveres Fig. , confirm30. However, the dynamics of B/Yamagata and B/Victoria-lineage cross-reactivity at the level of the B cell remains unclear. We therefore compared lineage-specific and cross-reactive B cell responses by flow cytometry following either IIV3 or IIV4 vaccines containing B-PH13 (B/Yamagata component only) or B-PH13 and B-BR08 (combined B/Yamagata and B/Victoria components), respectively. Class-switched memory B cells from subjects receiving IIV3 or IIV4 were co-stained with B-PH13 and B-BR08 probes as before. Four populations of HA-binding memory B cells could be delineated  over trivalent (IIV3) for seasonal immunisation programmes. The ability of IIV4 to simultaneously promote serological responses to both B/Victoria/2/87-like and B/Yamagata/16/88-like components has been confirmed over triThe responsiveness of each memory B cell population following IIV3 and IIV4 immunisation was compared. B-PH13+ memory B cells were present at a median level of 0.17% (IQR: 0.13–0.22) at baseline in both cohorts and underwent significant expansion Fig.  in respo31. Out of 672 sorted B cells, 519 productive heavy sequences were recovered, 303 of which also had productive light chain sequences. On the basis of similarities in variable gene usage, complementarity-determining region 3 (CDR-H3) sequence length and CDR-H3 amino acid sequence, we identified 284 unique clonal lineages with a mix of larger clonal expansions and singletons in each population  or B-BR08-specific (N = 10) B cell populations displayed HA recognition largely restricted to the sample of three IBV lineage-specific HAs, with the exception of mAb W85–3F06, which was broadly cross-reactive , we found anti-IBV mAbs elicited by seasonal vaccines generally recognised all strains tested within a respective IBV antigenic lineage spanning over 20 years of antigenic drift.We next examined the specificity and breadth of monoclonal antibodies recovered from each of the four identified IBV HA-specific B cell populations. As with our previous IAV work, PBMCs from three IIV4 vaccine recipients with marked expansion of IBV-specific B cells post-immunisation were stained as before, followed by sorting of single B cells and BCR-sequencing as described33. These preliminary recombinant IBV stem proteins appeared misfolded and failed to bind the prototypic stem mAb CR9114, indicating antigenic changes compared to full-length HA  generally displayed much broader IBV reactivity across both lineages, with greatest breadth observed within the CR-E-derived mAbs. A subset of cross-reactive mAbs could mediate HIA and neutralisation in vitro, suggesting binding of epitopes proximal from the receptor binding domain. Eleven broadly cross-reactive antibodies were identified which bound all 7 IBV HAs tested across the two lineages and the historical B/Lee/1940 strain. As many broadly reactive IAV-specific mAbs bind the HA stem, we generated recombinant IBV stem constructs  protected mice from mortality or weight loss following infection with the B/Yamagata-lineage B/Florida/4/2006 virus Fig. . In contIn mice challenged with B/Victoria-lineage B/Malaysia/2506/2004 virus, we observed three distinct patterns of protection by the panel of mAbs studied. Passive infusion of B/Victoria-lineage-specific HIA+ mAbs provided near complete protection from weight loss Fig. . Again, 35, with the ability to engage FcR being highly dependent on epitope location on the viral HA36. We therefore cloned and re-expressed a selection of IBV-specific mAbs using an Fc backbone with LALA mutations (L234A/L235A) shown to prevent engagement with murine FcRs37. Passive infusion of either B/Yamagata-lineage-specific mAbs or the cross-reactive mAb R95–1E07, all of which mediated HIA activity in vitro could still fully protect animals from weight loss in the context of a LALA Fc Pre-dominant antigenic sites of IBV cluster around the receptor binding domain (RBD), while previously resolved epitopes from broadly reactive mAbs bind epitopes located across both the globular head and stem domains of HA Fig. . When lo38. To confirm the selection of viral escape mutants, monoclonal isolates were recovered by plaque purification and similarly sequenced. We first determined the sensitivity of WT and mutant viruses to mAb-directed neutralisation  were identified, with recovered mAbs confirming broad IBV recognition by ELISA. Pan-IBV recognition was concentrated within mAbs derived from the CR-E population, with 64% (9/14) of mAbs binding all 7 IBV HA tested compared to 16% (2/12) of the CR-Y-derived mAbs. Nevertheless, it remains possible that the CR-E and CR-Y populations might derive from continuous spectrum of cross-reactivity and additional mAb isolation and characterisation is warranted. While administration of IIV3 was sufficient to drive expansion of both CR-Y and CR-E memory B cell populations, expansion of CR-Y was significantly enhanced following IIV4 immunisation. It remains to be seen if a differential ability to drive expansion of CR-Y B cells contributes to any potential increased serological protection from IIV4 vaccines. A limitation of our study is the restricted number of mAbs and the heterogenous patterns of IBV HA binding and neutralisation. However, in general we find seasonal influenza vaccines drive expansion of two pre-dominant populations of memory B cells; one binding neutralising epitopes proximal to the receptor binding site shared within each respective IBV lineage, and a second population of highly cross-reactive B cells binding both lineages and expressing antibodies that were generally non-neutralising. Although many human mAbs displayed a degree of protection in mice, the protective benefit of cross-reactive non-neutralising antibodies in human populations remains an open question. Nevertheless, IIV4 drove expanded serological reactivity against a broad range of IBV strains and simultaneously expands B cells expressing HIA+ antibody with pan-B/Yamagata or pan-B/Victoria reactivity, suggesting IIV4 has an improved capacity to combat antigenic drift in either lineage while removing the risk of lineage mismatch relative to IIV3.The elicitation of IBV cross-reactive serological responses to seasonal vaccines has been previously reported39 and in murine models42 found that prior priming with B/Yamagata antigens followed by immunisation with B/Victoria induces strong serological recall of B/Yamagata, but only low B/Victoria antibody responses. The converse is not the case, suggesting a degree of immunologic dominance of B/Yamagata HA. Elements of our study are consistent with these observations, such as (a) lower baseline frequencies of B/Victoria-specific memory B cells in adults compared with B/Yamagata-specific memory B cells, (b) a cross-reactive B cell population (CR-Y) that shows preferential binding to B/Yamagata HA by flow cytometry and (c) the absence of cross-reactive B cells that show the converse preferential binding to B/Victoria HA. Further resolution of the precise epitopes recognised by IBV cross-reactive B cells and antibodies may help elucidate mechanisms that underpin any differential capacity of antigens from the two IBV lineages to recall cross-reactive immunity.Interestingly, reports from immunised children20. Subsequently, the humanised murine antibody 12G6 was shown to potently neutralise and elicit HIA activity against both IBV lineages19, and binds a similar epitope to CR8033 overlapping the receptor binding domain. We identified at least four mAbs  with analogous breadth and HIA activity in vitro. Viral escape mutants generated with R95–1D05 and CR8033 both entailed glycan removal at N212, suggesting binding to highly similar epitopes. Interestingly, conserved pathways of escape were observed for both lineage-specific and cross-reactive mAbs that mediated HIA. For B/Yamagata, this was focused at two regions: substitutions in the 150-loop at the glycine residue 156 , and substitutions at residues 212 and 214 within the 190 helix. Analogous G156R and G156E mutations were reported for cross-reactive mAb C12G619 and double substitutions at D212 and T214 previously reported for B/Yamagata-specific human mAbs 3A2 and 10C418. For B/Victoria, we commonly observed removal of glycan at N212 via N212S (R95–1D05) or T214P  mutations, generally coupled with deletion of K177 to D179  in the 160-loop. These HA modifications lie immediately proximal to residues identified in B/Brisbane/60/2008 essential for binding by cross-reactive mAb C12G619. Common pathways of escape for both lineage-specific and cross-reactive mAbs may complicate the design of IBV HA immunogens for preferential induction of pan-IBV immunity. In addition, HIA+ cross-reactive antibody specificities like CR8033 might be susceptible to a loss of efficacy in the face of antigenic drift, potentially driven by strain-specific or lineage-specific humoral responses at the population level.Several human antibodies with broad recognition of both IBV lineages have been isolated and are of interest as potential diagnostic, therapeutic and prophylactic agents for IBV infection. Broad IBV recognition and HIA activity was first demonstrated by the isolation of CR8033, which binds and neutralises both IBV lineages and has HIA activity against B/Yamagata strains20 and 46B817, or localised in the IBV stem domain, as seen with CR911420. While we have identified antibodies with properties suggestive of both CR8071-like  and HA stem-reactive specificities , the absence of neutralisation activity in vitro means accurate epitope localisation via crystallographic or electron microscopic approaches is required.Broad IBV recognition and broad prophylactic and/or therapeutic protection against IBV infection in vivo can be mediated at alternative epitopes. In particular, within the vestigial esterase domain at the base of the HA, as seen with CR807143. However, the degree to which B cells targeting the different epitope clusters are recruited into protective serological responses remains unclear, although some initial studies suggest cross-reactive serum antibody may be significant16. In terms of protective capacity, we observed a clear hierarchy following passive infusion studies whereby cross-reactive antibodies capable of mediating HIA showed the greatest protective effect in vivo. This is consistent with the reported activity of mAb 12G6, which demonstrated greater prophylactic and therapeutic protection against experimental IBV challenge than antibodies binding distal to the receptor-binding domain44. Next in that order were mAbs that conferred intermediate protection, characterised by broad IBV recognition but no HIA activity, in a manner akin to CR8071. Consistent with reports for IAV35, protection mediated by such mAbs was highly dependent upon engagement with cellular Fc receptors. Finally, we found mAbs binding the IBV stem domain failed to neutralise in vitro and provided the weakest protection against experimental challenge. The fact that mAb CR9114 can neutralise IAV but not IBV strains20 suggests the IBV stem may exist in a conformation comparatively resistant to antibody-mediated neutralisation. This is supported by the poor epitope accessibility and low relative neutralising potency of the IBV stem-proximal mAb 5A718. In contrast, a recent report detailed the isolation of multiple human IBV stem-binding antibodies that can both neutralise in vitro and provide potent cross-lineage protection in mice45. In human populations, serum antibodies binding the IBV stem are widely prevalent, with titres increasing with age46 or following IBV infection15. However, while the IBV HA stem is highly conserved and can be targeted in mice to protective effect47, the utility of the IBV stem as a human vaccine target remains to be clarified.The ready detection of broadly cross-reactive B cells in humans based on our study presented here is supportive of efforts to broaden vaccine protection. Moreover, a recent study using phage display to derive mAbs from a seasonal vaccine recipient also identified multiple cross-reactive antibody lineages including those with intra-Yamagata, inter-lineage and even CR9114-like specificitiesIn summary, we demonstrate broadly cross-reactive memory B cells are common in humans, express both neutralising and non-neutralising immunoglobulins and are efficiently recruited into the humoral response elicited by seasonal immunisation. Further, IBV cross-reactive mAbs demonstrate protective potential in vivo and may constitute a substrate amenable to selective targeting by improved vaccine designs. Accurate definition of conserved sites of vulnerability on the IBV HA, in particular, clarifying the molecular basis of narrow versus broad IBV HA recognition, will greatly inform rational vaccine design efforts for eliciting broad and durable protective immunity against IBV.The study protocols were approved by both the Alfred Hospital Ethics Committee (# 432/14), and the University of Melbourne Human Research Ethics Committee (# 1443420) and all associated procedures were carried out in accordance with the approved guidelines. All participants provided written informed consent in accordance with the Declaration of Helsinki. Animal studies and related experimental procedures were approved by the University of Melbourne Animal Ethics Committee (#1714193).29 and registered as NCT02632578. Briefly, 30 healthy Australian adults (mean age 40.4 years) were vaccinated with the 2015 IIV3 (bioCSL Fluvax®) containing 15 μg of hemagglutinin from A/California/7/2009-like (pdmH1N1), A/Switzerland/9715293/2013 (H3N2)-like and B/Phuket/3073/2013-like strains. Blood samples were taken at baseline (d0) and d28 post-immunisation and sera, plasma and PBMCs were cryopreserved prior to use. For the 2016 IIV4 trial, 20 healthy adults were administered Sanofi FluQuadri® vaccine containing A/California/07/2009-like virus (pdmH1N1), A/Hong Kong/4801/2014-like virus (H3N2), B/Phuket/3073/2013-like virus and B/Brisbane/60/2008-like virus components. Sera, plasma and PBMCs were collected and cryopreserved from d0 and day 28. Participant information is summarised in Supplementary Figure The 2015 IIV3 immunisation trial is fully described elsewhere24. Analogous probes were prepared for influenza B encompassing the HA ectodomain C-terminally fused to the trimeric foldon of T4 fibritin, a biotinylatable AviTag sequence GLNDIFEAQKIEWHE, and a hexa-histidine affinity tag  suspension cultures and purified by polyhistidine-tag affinity chromatography and gel filtration. For use as flow cytometric probes, HA proteins were biotinylated using BirA (Avidity) and stored at −80 °C. Prior to use, biotinylated HA proteins were labelled by the sequential addition of streptavidin (SA) conjugated to PE, APC, Alexa488 (ThermoFisher) or BV421 (BD) and stored at 4 °C.Recombinant HA proteins for use as flow cytometry probes derived for IAV A/California/7/2009 and A/Switzerland/9715293/2013 strains were previously describedHA-specific B cells were identified within cryopreserved human PBMC by co-staining with HA probes conjugated to SA-PE, SA-APC, SA-BV421 or SA-Ax488. Monoclonal antibodies for surface staining included: CD19-ECD , CD20 Alexa700 , IgM-BUV395 , CD21-BUV737 , IgD-Cy7PE , IgG-BV786 , CD14-BV510 , CD3-BV510 , CD8a-BV510 , CD16-BV510 , CD10-BV510  and CD27-BV605 . Background B cells interacting with SA were excluded by staining with SA-BV510 (BD 563261). Cell viability was assessed using Aqua Live/Dead amine-reactive dye (ThermoFisher). For mouse samples, splenocytes or lymph node suspensions were stained with the following panel: B220-BUV737 , IgD-BUV395 , CD45-Cy7APC , GL7-Alexa488 , CD38-Cy7PE , SA-BV786 , CD3-BV785  and F4/80-BV785 . Samples were collected using a BD Fortessa configured to detect 18 fluorochromes and analysis was performed using FlowJo software version 9.5.2 (TreeStar).48. Productive, recombined heavy chain (V-D-J) and light chain (V-J) immunoglobulin sequences were synthesised (Genscript), cloned into expression plasmids and transfected into Expi293F cells using ExpiFectamine (Invitrogen). Recombinant monoclonal antibodies were purified from culture supernatants using sepharose Protein-A or G (Pierce). BCR sequences have been deposited in Genbank accession MK311355 - MK312159.The sequencing and cloning of BCRs from single B cells and the expression of recombinant mAbs was performed as previously described50) using a fitted curve (4 parameter log regression). For serum samples, endpoint titres were using a fitted curve (4 parameter log regression) and a cutoff of two times background.Antibody binding to IBV HA, HA1 and stabilised stem proteins was tested by ELISA. 96-well Immunosorp plates (ThermoFisher) were coated overnight at 4 °C with 2 μg/mL recombinant IBV HA expressed in Expi293 cells or sourced commercially . After blocking with 1% fetal calf serum (FCS) in PBS, duplicate wells of IBV-specific monoclonal antibodies at different dilutions  or human sera  were added and incubated for one hour at room temperature. Plates were washed prior to incubation with 1:30,000 dilution of HRP-conjugated anti-human IgG (KPL) for 1 h at room temperature. Plates were washed and developed using 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (Sigma) and read at 450 nm. HA-binding intensity was calculated as the antibody concentration giving half-maximal signal (EC50) viral assays. Challenge stocks were titrated in mice and assessed for pathogenicity  cells (ATCC CCL-34) per well to form confluent monolayers. The next day, serial dilutions of recombinant mAbs were incubated with B/Phuket/3073/2013 and B/Brisbane/60/2008 for one hour at 37 °C in Flu-media (Dulbecco’s Modified Eagle’s Medium (DMEM) with 0.8% bovine serum albumin (BSA), 1% penicillin/streptomycin and 0.1% l-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin), before adding the virus-antibody mixture to MDCK cells. After 2 to 3 days culture media supernatants from wells with visible cytopathic effect were collected and used to infect a fresh monolayer of MDCK cells in the presence of increasing concentrations of mAb. After serial passaging, culture supernatants were harvested, viral RNA was extracted and the IBV HA gene sequenced. Putative mutant viruses were identified based upon sequence comparison to similarly passaged media-only or irrelevant mAb (anti-HIV VRC01) controls. Where mutants were identified, single virus isolates were recovered by plaque purification using standard techniques. Single plaques were rescued, expanded in MDCK cells, and mutant viruses within culture supernatants sequenced and TCID50 determined as before.The generation of IBV escape mutants was based upon previously described protocols52. MDCK cells were seeded in 96-well plates at 1.5 × 105 per well. The next day, serial dilutions of recombinant mAbs were incubated in Flu-media with 100TCID50 of WT or mutant B/Phuket/3073/2013 and B/Brisbane/60/2008 viruses for one hour at 37oC, before addition to MDCK cells. After 18–24 h, supernatants were removed, cells were fixed and cellular cytopathicity was visualised by ELISA using mouse anti-influenza B nucleoprotein  primary and goat anti-mouse HRP-conjugated secondary antibodies. Plates were developed using TMB substrate and read at 450 nm. The concentration of mAb preventing 50% infectivity (IC50) was calculated.The neutralisation activity of recombinant mAbs was examined using a modified microneutralisation assay50 WT or mutant B/Phuket/3073/2013 and B/Brisbane/60/2008 viruses and incubated at 37 °C for 18–24 h. Cells were resuspended by manual scraping and infectivity confirmed by staining with mouse anti-influenza B nucleoprotein  and goat anti-mouse Alexa647 . The binding of human anti-IBV mAbs (5 μg/mL) or an anti-HIV negative control (VRC01) to surface expressed HA was detected using goat anti-human Alexa647 .The ability of recombinant mAbs to bind cell-surface HA on infected cells was examined by flow cytometry. MDCK cells were seeded into 6-well plates and infected with ~10,000 TCIDTo characterise conservation of the IBV HA, a cross-section of 2000 B/Yamagata and B/Victoria-lineage viral sequences spanning 1988–2018 were exported from the EpiFlu database [gisaid.org], HA protein sequences aligned using Geneious 11.1.3 (Biomatters) and weighted conservation scores accounting for year of isolation determined at each residue position. Amino acid conservation was visualised using Pymol.U tests. Correlations were analysed using Spearman’s rank-order tests. Curve fitting was performed using 4 parameter logistic regression. All statistical analyses were performed using Prism (GraphPad).Data is generally presented as median ±  interquartile range. Statistical significance was assessed by Mann–Whitney Further information on experimental design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Malassezia globosa, MgCA. These MTCs incorporate various scaffolds, among which aliphatic amine with 1–4 carbons atom in their molecule, morpholine, piperazine, as well as phenethylamine and benzylamine derivatives. All the reported MTCs displayed a better efficacy in inhibiting MgCA compared to the clinically used sulphonamide drug acetazolamide (KI of 74 μM), with KIs spanning between 1.85 and 18.9 μM. The homology model of the enzyme previously reported by us was used to rationalize the results by docking some of these MTCs within the fungal CA active site. This study might be useful to enrich the knowledge of the MgCA inhibition profile, eliciting novel ideas pertaining the design of modulators with potential efficacy in combatting dandruff or other fungal infections.A series of monothiocarbamates (MTCs) was investigated for the inhibition of the β-class carbonic anhydrase  from the fungal parasite  Such results showed for the first time relevant antidandruff effects by targeting MgCA, which were equivalent to those of the standard azole drug ketoconazole,M. globosa, one of the main fungi belonging to this genus which infects humansRecently, we cloned and characterized a β-CA in the pathogenic fungus provoking dandruff 1–17 used in the experiments were reported earlierCompounds 2 hydration activity4 for maintaining constant the ionic strength, following the initial rates of the CA-catalysed CO2 hydration reaction for a period of 10–100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor at least six traces of the initial 5–10% of the reaction have been used for determining the initial velocity. The uncatalysed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (10 mM) were prepared in distilled-deionized water and dilutions up to 0.01 µM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay, in order to allow for the formation of the E–I complex. The inhibition constants were obtained by non-linear least-squares methods using the Cheng–Prusoff equation whereas the kinetic parameters for the uninhibited enzymes from Lineweaver–Burk plots, as reported earlierAn applied photophysics stopped-flow instrument has been used for assaying the CA catalysed COThe dimeric form of the homology built model of MgCA3D ligand structures were prepared by MaestroGrids for docking analysis were centered in the centroid of the catalytic cavity residues. Docking studies were carried out with the program Glide2– type for trithiocarbonate and DTCs– type for MTCs– ZBG coordinating (through the sulphur atom) to the metal ion from the enzyme active site. Recently, we also developed a homology model for MgCA alone and in complex with DTCs– ZBG may also lead to effective MgCA inhibitors. The series of compounds 1–17 previously reported1–15, the trithiocarbonate 16 and the xanthate 17  hCA I and II, as well as MgCA, with the sulphonamide inhibitor acetazolamide (AAZ) as standardInhibition data with compounds 1–17 can be compiled from the data of The following SAR for the inhibition of MgCA with compounds AAZ (KI of 74 μM), with KIs ranging between 1.85 and 18.9 μM.(i) Independently from the nature of the substituents attached to the MTC nitrogen, all reported derivatives showed better MgCA inhibitory properties than the clinically used sulphonamide drug R and R1 groups demonstrated to possess a limited influence on the inhibitory effectiveness of compounds 1–17, which allegedly mainly lean on the ZBG interactions with the Zn ion and the residues nearby. On the other hand, it is necessary to stress the importance of lipophilic moieties (R and R1) present in the scaffold of the inhibitors, which may favour the overall interactions within the catalytic cleft, allowing by far a better inhibitory efficacy compared to simple inorganic anions (ii) A rather flat SAR can be extrapolated from the data reported in n-propyl derivative 4 demonstrated the best efficacy in inhibiting the enzyme among the reported MTCs (KI of 1.85 μM), sign that two short, unbranched aliphatic chains could better fit within the lipophilic pockets of the active site. The two secondary MTCs 1 and 2 were observed to generally possess slightly worse inhibitory potency (KI of 14.1 and 18.9 μM) compared to 4 and the remaining tertiary MTCs, showing comparable KIs (ranging between 4.22 and 9.16 μM). The only exception was represented by derivative 14, which incorporate a N-(3-Cl-phenyl)-piperazine moiety, this compound being in fact the less active tertiary MTC investigated here. In detail, the presence of a Cl atom in the meta towards the heterocyclic ring could elicit a steric hindrance within the MgCA binding pocket that reduced the KI to 15.9 μM. Finally, whereas the benzyl trithiocarbonate 16 possessed a KI comparable to those of most MTCs (KI of 8.12 μM), the phenethyl xanthate 17 showed a roughly two-fold reduced efficacy (KI of 16.2 μM).(iii) The di-(iv) The effectiveness of MTCs in inhibiting the MgCA was shown to be scarcely reduced, but anyhow comparable, to that of the DTCs investigated earlier by us2, 8, 9 and 10) endowed with acceptable inhibitory properties and a varied structure were selected as representatives of the synthesized MTCs. These derivatives were submitted to quantum mechanics optimization (B3LYP/6–31 G*+) in order to compute the charge distribution and optimal geometry, prior to dock the molecules into the recently developed homology-built model of MgCA,Docking simulations were performed to elucidate the binding mode of MTCs within the MgCA active site. Four inhibitors from 2a and 9a and the side chain of F88(A). The benzyl and phenethyl tails of these derivatives were further stabilized by the π-alkyl interactions established with the aliphatic side chain of V71(B) and L132(B)  and F88(A), respectively (The scaffold fragments of the four derivatives accommodate into a hydrophobic pocket defined by residues from both monomers. π–π interactions occur between the phenyl moieties of derivatives  L132(B) . These s32(B)  and Q38 (chain A) residues, the oxygen atom of the MTC was able to bind only to the side chain OG atom of S48 or NE2 atom of Q38. Hence, it is reasonable to hypothesize that the shorter length of the CO bond (1.25 Å) compared to that of the CS (1.75 Å) oneM. globosa, a validated anti-dandruff drug target,I of 74 μM), with KIs spanning between 1.85 and 18.9 μM. Docking procedures were applied to the homology model of the enzyme we previously reported to shed light on the binding mode the MTCs exhibited within the fungal CA active site. This study might be of help to better decipher the MgCA inhibition profile, raising the discovery of novel modulators with potential efficacy in combatting dandruff or other fungal infections.Kinetic and computational approaches were applied to investigate a series of MTCs as novel inhibitors of the β-class carbonic anhydrase from the fungal parasite


As flavonoids are promising modulators of this enzyme’s activity, a panel of 44 flavonoids, organised in five groups, was screened for their inhibitory activity of α-glucosidase, based on in vitro structure–activity relationship studies. Inhibitory kinetic analysis and molecular docking calculations were also applied for selected compounds. A flavonoid with two catechol groups in A- and B-rings, together with a 3-OH group at C-ring, was the most active, presenting an IC50 much lower than the one found for the most widely prescribed α-glucosidase inhibitor, acarbose. The present work suggests that several of the studied flavonoids have the potential to be used as alternatives for the regulation of PPHG.α-Glucosidase inhibitors are described as the most effective in reducing post-prandial hyperglycaemia (PPHG) from all available anti-diabetic drugs used in the management of type 2 diabetes   According to the International Diabetes Federation, there are 415 million of adults with diabetes, probably reaching 642 million in 2040DM is characterised by absolute or relative deficiency in insulin secretion by pancreatic β-cells, increased insulin resistance and/or impairment of insulin action in target tissues,,An effective control of hyperglycaemia in type 2 DM includes the retarding, regulation and/or inhibition of carbohydrate hydrolysing enzymes,50 values,The use of α-glucosidase inhibitors started in the 1970s and the first related therapeutic agents for diabetes were approved in the 1990s,,,,,,,,,,,Some flavonoids, such as morinacarbose. Molecular docking calculations were also applied to complement the inhibitory activity studies and to predict the binding model of the selected flavonoids to the three-dimensional structure of α-glucosidase.The present study aims to establish a structure–activity relationship of a panel of flavonoids , dividedThe overall obtained results constitute an important step for the establishment of the adequate and ideal chemical structure of flavonoids to target α-glucosidase and consequently, to manage type 2 DM.Saccharomyces cerevisiae, p-nitrophenyl-α-D-glucopyranoside (pNPG), DMSO, NaHPO4, Na2HPO4, compounds D3 , D4 (apigenin), D6 (kaempferol), D7 (luteolin), D8 (quercetin), D9 (morin), D16 (acacetin), D18 (rutin), E1 (naringenin), E2 (eriodictyol), E3 (taxifolin) and acarbose. Compounds A1, A2, A3, A4, A5, A6, B1, B2, B3, B4, C1, C2, C3, C4, C5, C6, C8, C9, D1 (chrysin), D2  and D5 were obtained from Indofine Chemical Company, Inc. . The flavonoids C7, C10, C11, C12, C13, D10, D11, D12, D13, D14 and D17 were synthesised as previously describedThe following reagents were purchased from Sigma-Aldrich Co. LLC : α-glucosidase from pNPG into α-D-glucose and p-nitrophenol, at 405 nm. Briefly, in a 96-well plate, the enzyme (0.05 U/mL), dissolved in 100 mM phosphate buffer (pH 6.8), was pre-incubated at 37 °C for 5 min with the flavonoids (0–200 μM). In sequence, pNPG was added (600 μM) and incubated in the reaction mixture at 37 °C for 30 min. The enzymatic reaction was monitored spectrophotometrically, in a microplate reader , by measuring the absorbance at 405 nm. The obtained values correspond to the slope measured between 5 and 20 min. The amount of DMSO used had no interference with the assay. Acarbose (0–2000 μM) was used as positive control. The obtained results represent at least three independent experiments.The α-glucosidase activity was measured using a method of Tadera et al.A5 (0–62.5 μM), B3 (0–50 μM), C7 (0–5 μM), D8 (quercetin) (0–12.5 μM), D10 (0–18.75 μM), E3 (taxifolin) (0–100 μM) and for the positive control, acarbose (0–2000 μM).The inhibitory kinetic analyses were performed for the most active flavonoids of each group (A–E): pNPG was added  and incubated in the reaction mixture at 37 °C for 30 min.Briefly, the enzyme (0.05 U/mL), dissolved in 100 mM phosphate buffer (pH 6.8), was pre-incubated at 37 °C with the above mentioned flavonoids in a 96-well plate, for 5 min. In sequence, Vmax) (y axis) against the flavonoid concentrations (x axis)  of the tested flavonoids was performed using the nonlinear regression Michaelis–Menten enzyme kinetics and the corresponding Lineweaver–Burk double reciprocal plots for each concentration of the inhibitor and substrate. The Ki values were calculated with GraphPad Software 6.0 by plotting the reciprocal of maximum velocity (1/(x axis) 38–40.The type of inhibition parameters were all calculated with GraphPad Software 6.0. The obtained results represent at least three independent experiments.Saccharomyces cerevisiae α-glucosidase  are unavailable. There are, however, X-ray structures available for the isozyme isomaltase/α-methylglucosidase (EC 3.2.1.10). The isomaltase/α-methylglucosidase with the PDBSaccharomyces cerevisiae α-glucosidase MAL32 (UniProt entry P38158)3D structures of We identified several differences between residues lining the binding pockets of α-glucosidase and isomaltase, such as the pairs Asp307/Glu204, Asp408/Glu411, Phe157/Tyr158, Thr215/Val216, Ala278/Gln279, Val303/Thr306 or Ala178/Cys179, respectively for α-glucosidase and isomaltase. Most of these differences involve very similar residues, but considering the differences in the size of the side chains  and the high confidence entrusted by the extremely favourable sequence identity, we opted to build a high accuracy homology model of α-glucosidase using isomaltase as template.Supplementary Figure S1 provides a summary of the homology modelling parameters and Supplementary Figure S2 the Ramachandran plot analysis of the homology model.Using MAL32 target sequence and the structure with the PDB ID 3AJ7 as template, we built the α-glucosidase homology model in SWISS-MODELKa at pH 7.The protonation states of the enzyme were predicted using the Protoss serverThe substrate α-maltose was modelled inside the binding pocket and subsequently, to remove potential clashes and relax the structure of the model, Molecular Mechanics minimisation followed by classical MD simulations were done. This protocol was performed within the AMBER 12To validate the receptor for the docking protocol we extracted the last step from the MD simulation and re-docked the substrate α-maltose and also the inhibitor acarbose. Since the predicted binding modes, both for α-maltose and acarboseSupplementary Material for details), such as the exhaustiveness of the global search = 8 or the maximum number of output poses = 9. The search space was confined to a box of circa 15 × 20 × 17 centred on the site occupied by the second glucose in maltose. The receptor was treated as a rigid body and prepared using openbabelAutoDock VinaSubsequently, the poses generated by AutoDock Vina were rescored using the scoring function ChemPLP, which is excellent for oligosaccharidesThe inhibitory effect of flavonoids against α-glucosidase activity is shown in A5 was noticeably the most effective, presenting an IC50 of 54 ± 3 µM. For the other tested compounds, it was not possible to achieve the IC50 value up to the concentrations of 100 µM (A6) or 200 µM (A1–A4). These results indicate that the presence of a 3-OH group in the C-ring is relevant for the inhibitory activity of flavonoids. Moreover, when comparing the flavonoid A5 and the flavonoid A6, we may conclude that the replacement of the 3′,4′-(OH)2, in the B-ring, by methoxy (–OMe) groups withdraws the inhibitory activity of compound A5.From all the flavones from A group, flavone B1 was the less active. Comparing the activities of B1 (with a 5-OH in A-ring) and A1 (without substitutions) it is shown that the presence or absence of 5-OH in A-ring, per se, is indifferent for the inhibitory activity. However, the 5-OH substitution in A-ring, together with the 4′-OH (B3) or 3′,4′-(OH)2 (B4) in B-ring, increases the activity, comparing with the related compounds, from A group, which do not have 5-OH substitution.From B group, flavone C7 with an IC50 = 7.6 ± 0.4 µM, which is almost 80 times lower than the IC50 of the positive control, acarbose (IC50 = 607 ± 56 µM). Comparing the flavones C5 and C4, it is clear that the presence of –OH groups in the B-ring is more relevant in the 3′-position  than in the 4’-position . Flavone C1, with just one 7-OH group, did not achieve the IC50 value up to the maximum tested concentration (200 µM). However, adding another 3′-OH at the B-ring (C2) is enough to increase the inhibitory activity of the compound (IC50 = 53 ± 4 µM). In turn, increasing the complexity of the structure by the addition of a –OMe and/or a benzyloxy (–OBn) group (C8–C13) did not favour the activity.C group has the most active flavonoid, D8 (quercetin) with an IC50 = 15 ± 3 µM. The substitution in 3-position of the C-ring seems to be advantageous for the intended activity, since D7 (luteolin), with no substitution in 3-position, had a higher IC50 (46 ± 6 µM). Moreover, the relevance of the existence of a 3-OH substitution in the C-ring was evidenced by the low activity of D18 (rutin), which had a rutinose in this position. In turn, the close IC50 values of D8 (quercetin) and D10 (IC50 = 21 ± 2 μM) indicate that the presence of a 3-OH group or a 3-Cl atom in the C-ring is indifferent for α-glucosidase inhibition. The relevance of the presence of a catechol moiety in the B-ring is shown by the different IC50 values found for D7 (luteolin) (46 ± 6 µM), which have a catechol group in 3′,4′- positions, and the compounds D5 (apigenin) (IC50 = 82 ± 6 µM) and D4 (IC50 = 89 ± 3 µM), which had just one OH in the 4′- or 3′-positions, respectively. Similarly to the obtained results in C group, results of D group indicate that the methylation of the catechol arrangement in the B-ring (D15–D17) did not show any advantage for the studied activity.In what concerns D group, the most effective compound was 50 of flavonoids from E group, E3 (taxifolin) had the best inhibitory activity. Nonetheless, its IC50 was approximately 200 μM, the maximum tested concentration.Analysing the ICA5, B3, C7, D8 (quercetin), D10, E3 (taxifolin) and acarbose was deducted from the calculation of Km and maximum enzyme velocity (Vmax). These parameters were obtained by the nonlinear regression Michaelis–Menten enzyme kinetics and complemented with Lineweaver–Burk double reciprocal plots. As observed in A5  (acarbose (D10 (E3 (taxifolin) , ed in A5  and B3 (n A5 (B3  showed aercetin)  and the acarbose  have shoose  (y axis) against the flavonoid concentrations (x axis) (The (x axis) .acarbose, A5, B3, C7, D8 (quercetin), D10 and E3 (taxifolin)]. It is important to realise that a few of them may bind in more than one sugar pocket, even predominantly in other sugar pockets that line the active site cavity , and in a lesser extent at the deeper, reactive pocket of the active site. However, for the purpose of drug discovery, the deep reactive pocket is by far the most relevant, because it is the one for which the drugs are always optimised to bind . Therefore, it was the binding to this deep pocket that we explore in this section.We have investigated the binding poses of a selection of inhibitors within the reactive pocket of the active site of α-glucosidase , which presents an IC50 of 44 ± 3 μM. Nevertheless, and corroborating our results, the activity increased substantially with the hydroxylation at the B-ring. In addition, comparing the results obtained for C4 and C5 it is possible to infer that the presence of the –OH group at the 3′-position (C4) is more relevant than at the 4′-position (C5) of the B-ring, as C4 is more active than C5. In turn, with the basic structure studied by Gao et al.C8–C13).We followed our rationale of increasing the complexity of the structure, by the addition of a catechol group in the A-ring. The most effective compound was D1) structure, in the D group. Once again, our results are in agreement with Gao et al.D1), giving apigenin (D5), increased the activity reaching an IC50 = 82 ± 6 µM. With this basic structure, the presence of an –OH group at 3′- (D4) or 4′- (D5) positions of the B-ring seems to be indifferent for the inhibitory activity.We also tested another group of flavonoids, based on chrysin  and D8 (quercetin) or D5 (apigenin) and D7 (luteolin), it is possible to conclude that the presence of the catechol group in the B-ring increased considerably the activity of the flavonoids. Additionally, our results indicate that D8 (quercetin) is a stronger inhibitor when compared with D9 (morin). These findings clearly demonstrate that the ortho –OH groups might be responsible for the higher inhibitory effect, which seems to promote the interaction between α-glucosidase and flavonoids, while the meta-position of –OH groups decreases the electron cloud density of band I resulting in a lower inhibitory activity,D8 (quercetin) (IC50 = 15 ± 3 μM) and D7 (luteolin) (IC50 = 46 ± 6 μM), we can conclude that the presence of a 3-OH group in the C-ring increases the ability to inhibit α-glucosidase. This result is corroborated by Nicolle et al.It has been postulated that –OH groups in the B-ring are favourable for the inhibitory activity of the compounds,D3 , which has –OH groups at 5-, 6- and 7-positions of A-ring, is more effective than D2  and D1 (chrysin), with only –OH groups at 5- and 7-positions. These results are in agreement with Gao et al.50 of D6 (kaempferol) is almost three times lower than the IC50 of C5. Wang et al.It is currently accepted that modification of any of the –OH groups in the A-ring diminishes the α-glucosidase inhibitory effects of flavonoids50 of D5 (apigenin) and D8 (quercetin) when compared with D6 (kaempferol) and D7 (luteolin), respectively. Accordingly, Silva et al.2 groups of the B-ring together with a 3-OH group in the C-ring are crucial for the inhibitory effect of flavonoids.Our results illustrate that the presence of a 3-OH group in the C-ring is also relevant, evidenced by the lowest ICD10–D14) for the first time. It has been postulated that under diabetic conditions, the inflammatory process is exacerbated and consequently there is an excessive production of reactive species, namely, HOClD10 was one of the most active compounds tested in this work, presenting an IC50 similar to the one obtained for D8 (quercetin). Moreover, comparing compounds D10 and D8 (quercetin) it is possible to conclude that the substitution of a 3-OH group or a 3-Cl atom at the C-ring is almost indifferent for the inhibitory effect. In contrast, the results obtained with D18 (rutin), reveal that the addition of a larger group, in this case rutinose, at the 3-position of the C-ring, weakens the activity of the flavonoidIn the panel of flavonoids in this work, we tested a series of chlorinated flavonoids  or 8 (D12) position of the A-ring, the inhibition of the enzyme decreased considerably.Following the order of potency of the chlorinated flavonoids, it is possible to see that the second most effective compound was flavonoid D15–D17), and, corroborating the results obtained in A and C groups, the inhibitory effect on α-glucosidase was reduced by the introduction of an –OMe group, independently of the ring. According to the literature, the contribution of –OMe groups to the inhibitory effect of flavonoids depends on the basic structure of the flavonoid and/or with the type of substrate usedWe also tested a group of methoxylated compounds  with E1 (naringenin), D7 (luteolin) with E2 (eriodictyol), D8 (quercetin) with E3 (taxifolin), it is clear that the lack of C2 = C3 double bond weakens the flavonoids activity. This finding is in agreement with other reports about this issue,It is known that the C2 = C3 double bond of flavonoids is crucial for their anti-inflammatory activityA5, B3, C7, D8 (quercetin) and E3 (taxifolin). We also analysed the chlorinated flavonoid D10, since it was one of the most effective compounds, presenting an IC50 value similar to the one found for D8 (quercetin). Lineweaver–Burk double reciprocal plots were employed to investigate the inhibition kinetics of the tested compounds against α-glucosidase activity. As observed in A5 , which means that binding of the substrate or the inhibitor affects their enzyme binding affinity. In the mixed type inhibition, the inhibitor did not bind to the enzyme in the active site and both inhibitor and substrate can simultaneously be attached to the enzyme. However, the binding affinity for the substrate is decreased when the inhibitor is present. Concerning flavonoid C7, y axis. Moreover, the result obtained by docking calculations for flavone C7 suggested that the binding pose prediction was directed to the active site. Considering the in vitro and docking studies, the results indicate that C7 presents a competitive inhibition type. Flavonoid D8 (quercetin)  , which means that they can bind with equal affinity to both the free enzyme and the enzyme–substrate complex. In this inhibitory mechanism, the inhibitor binds to the enzyme at a site that is distinct from the substrate binding site (active site of the enzyme). A non-competitive inhibitor binds to an enzyme whether the substrate is at low or high concentrationsD8 (quercetin), which was already reported as a mixed type inhibitor,,Once defined the effectiveness of the tested flavonoids, it was our aim to evaluate the type of α-glucosidase inhibition of the most active flavonoids from each group (A–E): ed in A5  and B3 (n A5 (B3  presenteercetin)  and acaracarbose  are alson in D10  and E3 (xifolin)  are non-acarbose, we verified a competitive inhibition type, as observed by the studies of Kim et al.In what concerns Ki values can also be a useful tool to compare the activity of the compounds, being reflective of the binding affinity of the inhibitor for the enzyme. The tested flavonoids presented the following order of Ki values: C7 < D8 (quercetin) < A5 < D10  < B3 < E3 (taxifolin). These results show that flavonoid C7, followed by D8 (quercetin), exhibit the strongest affinity for the enzyme.The A5, D8 (quercetin) and C7 share very similar interactions with the active site and mimic interactions found between the first glucose of acarbose and the active site pocket. Moreover, the predicted mode of binding can rationalise the trends found across the series of studied flavonoids, namely the importance of 3-hydroxylation in the C-ring and the catechol group in the B-ring. The 3-OH substitution provides a hydrogen bond to Asp349, while the catechol in the B-ring hydrogen bonds Asp68, Asp214 and Glu276. Similarly, the inhibitor acarbose presents hydrogen bond interactions with the same active site amino acids.Flavones C7 and D8 (quercetin), which indicates that hydoxylations at 5- and 7- or 8-positions of the A-ring, at 3′ and 4′-positions of the B-ring and at 3-position of the C-ring, as well as the C2 = C3 double bond in the C-ring, are critical for the inhibitory activity of flavonoids (Our results have shown that adequately substituted flavonoids are effective α-glucosidase inhibitors. The flavonoid structure, the position and number of OH groups are determinant factors for the intended effect. The most active flavonoids were avonoids . The res


A review paper is presented on modeling for polymer extrusion for both single screw and twin-screw extrusion. An issue of global modeling is discussed, which includes modeling for solid conveying, melting, melt flow, and co-operation of the screw/die system. The classical approach to global modeling of the extrusion process, which is based on separate models for each section of the screw, i.e., solid transport section, melting and pre-melting sections, and the melt flow section is presented. In this case, the global model consists of the elementary models. A novel continuous concept of global modeling based on CFD  computations is also presented, and a concept of using the DEM (Discrete Element Method) computation coupled with CFD computations is discussed. Extrusion  is the mThere are several important functions of extruding machines, polymer transport (from a hopper to a die), polymer melting, pressure generation, material mixing for thermomechanical and chemical homogenization, and, lastly, product forming. Melting should be quick to provide enough room for good material mixing. Melting and mixing are fundamental in polymer processing and crucial for the development of novel, advanced materials, polymer composites, or polymer blends, as well as for material recycling of plastics.Single screw extruders are generally poor mixers and melting progresses slowly. In twin-screw extrusion, melting progresses faster and mixing action is considerably improved. In order to improve melting and mixing actions, various screw configurations are applied, using elements that intensify these actions, e.g., Maillefer, Barr, Maddock, and many others.The design of polymer processing is currently supported by computer simulations based on the mathematical models of manufacturing processes. Modeling makes it possible to predict the course of these processes on the basis of process data .Several fundamental books or book chapters have been devoted to an extrusion process, e.g., by McKelvey , Tadmor It is a particularly important challenge where the modeling of the two most important technologies are extrusion and injection molding. In these, there is a need for global modeling, i.e., description of the solid transport, polymer melting, and the melt flow. Polymer melt flows are relatively well understood. However, the transport of solid material as well as melting of the polymer are still poorly understood. The right polymer melting model is the basis for developing a comprehensive  model of the process.Solid conveying in the single screw extruder was described first by Darnell and Mol  who elabThis fundamental approach was later extended by other researchers. Schneider  introducAlthough numerous researchers extended the work of Darnell and Mol, the basic analysis remained relatively unchanged and was the basis for modeling the extrusion process.All the models of solid conveying presented so far were based on the assumption that the polymer granules are moving as a solid bed without relative movement of polymer granules. However, some investigations, e.g., performed by Fang et al.  showed tTo solve the problem of modeling the solids’ transport within a single screw extruder, the discrete element method (DEM) was proposed, which is well known within the field of granular mechanics. The first research using this method in the field of solid conveying the extrusion process was performed by Potente and Pohl  who studThe fundamental studies on modeling the solid conveying in the single screw extruder with the use of the discrete element method were performed by Moysey and Thompson ,48 who dA further description of the solid conveying section based on the discrete element method (DEM) DEM was performed by Schöppner et al. ,44 who dThe discrete element method (DEM) is a very useful and powerful tool for modeling the solid conveying section of single screw extruders. However, it has two substantial drawbacks. It is time consuming and the material parameters, i.e., the coefficient of friction (CoF) and coefficient of restitution (CoR), are difficult to determine. Since it is time consuming, this method cannot be used for global modeling of the extrusion process, which requires hundreds of computing iterations.Solid conveying in twin-screw extruders was studied, mainly for co-rotating extrusion, e.g., by Carrot et al. , BawiskaThe first melting tests in the single screw extruder were performed by Maddock and Street ,58 who uThe experiments showed that two steps of melting can be distinguished, including the delay section (or pre-melting), which corresponds to forming and growing the polymer melt layer, and the melting section (or plasticating), which corresponds to the accumulation of the molten polymer at the active flight of the screw.Several years later, Tadmor et al. ,61,62 coMany researchers repeated the “screw pulling out” experiments, and, in the majority of cases, they confirmed the Tadmor melting model. However, there were some exceptions, especially in the case of melting PVC, which were reported by Menges and Klenk  and MennThe fundamental model of Tadmor was later extended by other researchers. Donovan  relaxed Housz and Meijer ,82 pioneThese studies presumed that the beginning of the barrier section coincides with the onset of melting, and the melting rate follows the rate of change of the cross-channel areas for solids or melt, which is not consistent with experiments. Gaspar-Cunha and Covas ,88 develSingle screw extruders may have barrels with a grooved feed zone and a grooved melting zone. Numerous experiments were performed to study the effect of the grooved barrel on extrusion performance, e.g., by Grünschloß ,90, ChunThe “screw pulling out technique” is a relatively time consuming and expensive method of studying melting in extruders. Therefore, other approaches were applied for observing the melting behavior directly in the extrusion process. For example, Zhu et al. ,96 used Similarly, as in the case of Darnell and Mol’s work for solid conveying, even though numerous researchers extended the work of Tadmor and Klein, the basic analysis remained relatively unchanged and was usually the basis for modeling the extrusion process. However, the models of the type presented so far are based on a prior assumed melting mechanism, which are not universal, and cannot be valid for all polymers, various operating conditions, and various screw configurations. These models can be useful only in qualitatively predicting the trends in melting polymers in single screw extruders.Instead of the melting mechanism previously assumed, melting of a polymer in single screw extruders can be simulated by solving the conservation equations of mass, motion, and energy along with a constitutive equation for the polymer being used.This different approach was proposed first by Viriyayuthakorn and Kassahun  who deveThis novel approach based on solving the conservation equations without an assumed melting mechanism seems to be very promising for the future work on modeling the melting, even though it requires very large computational capabilities. Altinkaynak et al.  performeContrary to melting in single screw extruders, the studies on melting in twin-screw extruders were much more limited. These studies involved mainly modular self-wiping co-rotating twin-screw extruders, both experimentally  ,139,140 The first analysis of flow in a single screw machine (screw pump) was performed by Rowell and Finlayson  for viscThese basic studies analyzed one-dimensional flow through a rectangular channel of infinite width. Later, these models were improved by considering the transverse flow caused by the screw flights  or unsteady (cyclic processes), and of distributed parameters or locally lumped parameters. For engineering purposes, the lumped parameter models may be generally sufficient. The main goal of engineering designs is to predict the pressure and mean polymer melt temperature profiles along the machine for a given screw and die geometry as a function of the process operating conditions. In these models, the screw channel is usually divided into short axial segments (increments), where the input temperature and pressure parameters come from the calculation in the previous segment. In addition, the output parameters from the current segment are the input parameters for the next segment. Within each segment, the local parameters are assumed to be constant. The lumped parameter approach becomes particularly useful when dealing with plasticating processes, like extrusion and injection molding, where, in addition to the melt flow, we are faced with the solids’ transport and melting of the material.With immense progress in the computational fluid dynamics, the current trends in modeling polymer processing apply very sophisticated numerical  methods. This includes both two-dimensional and three-dimensional computations of velocity, stress, pressure, and temperature fields with a variety of boundary conditions for shear-thinning and temperature-dependent, sometimes viscoelastic, fluids. In real extruders and injection molding machines, however, there is a lot of other problems that are still unsolved, and, at present, these methods are generally not applied for comprehensive  modeling of these screw processes.The state-of-the-art for composite modeling of screw processes was presented in some fundamental books, e.g., by White and Potente , RauwendTadmor and Klein  developeResearch on the co-rotating twin-screw extrusion was initiated by White and his co-workers. On the basis of the melt flow studies ,174 and Research on the counter-rotating twin-screw extrusion was also initiated by White and his co-workers. Based on the melt flow studies ,201 and Research on the starve fed single screw extrusion was much more limited. Wilczyński et al. based on the polymer melting studies ,127 deveWhen modeling polymer extrusion, it is generally assumed that there is no slippage at the fluid/solid interface, and flowing materials in the screw extruders and dies adhere to the wall. However, there are several materials like filled polymers , elastomers, polymers like poly(vinyl chloride) and high-density polyethylene, and polymer suspensions, which exhibit wall slippage under certain conditions.The phenomenon of wall slippage was studied first by Mooney . AfterwaRecently, Lewandowski and Wilczyński ,251 perfCurrently, it is established that extrusion of wall-slipping polymers results in the reduction of the die pressure, and the screw characteristics changing, which affects the operating point of the extruder, and results in the global modeling of extrusion of wall-slipping polymers. This requires developing models both for the screw (plasticating unit) and for the extrusion die.When modeling polymer extrusion, it is also generally assumed that flowing materials in the screw extruders and dies have no yield stress. However, it is well known that many materials have a yield stress, e.g., filled polymers, composites, and blood, paints, cosmetics, and foodstuffs such as margarine, mayonnaise, butter, and ketchup. These materials were first described by Bingham , and, laCompared to fundamental studies on the viscoplastic flows, much less research was devoted to the viscoplastic flows in the extrusion process. Laval and Kalyon ,240 firsRecently, Lewandowski and Wilczyński ,255 perfExtrusion is a continuous process of co-operation of the extruder (screw) and the die. Physical phenomena occurring in the extruder determine the flow in the extrusion die and vice versa. The flow in the die determines the phenomena occurring in the extruder. Any change in the processing conditions in the extruder causes a change in the processing conditions in the die and vice versa. Modeling of the extrusion process cannot be limited to the modeling of flow in the extruder and must include the extrusion die.The term global modeling means modeling the interacting phenomena occurring in the extruder and the die, which entails modeling the extruder/die system (or screw/die system).The classical global modeling of the extrusion process is based on the separate models for each section of the screw, i.e., solid transport section, melting and pre-melting sections, and melt flow section, and the global model consists of these elementary models . In thisGlobal modeling requires the use of a computation algorithm appropriate for a given type of extrusion. For classical (flood fed) extrusion, the forward scheme of computation is suitable. In this case, the flow rate of the material is not known, and is the result of the screw/die co-operation and must be determined in multiple iterative computations. For extrusion with starvation, the backward scheme of computation is applied, i.e., the inverse computation algorithm since the pressure profile is not continuous in this scenario, and there is no continuous flow rate/pressure relation. In this case, the flow rate is established and equal to the flow rate of the material metered by the dosing device .Simulation schemes for flood fed single-screw extrusion are relatively well known ,215,216.In the case depicted in Simulation schemes for starve fed single-screw extrusion are much less known ,205,206.In the case depicted in Using this inverse computation approach, the authors developed the composite models for closely intermeshing counter-rotating twin-screw extruders ,231,232.In the extrusion process, both single-screw extrusion and twin-screw extrusion (co-rotating and counter-rotating), flood fed or starve fed, the modeling of polymer melting consists of performing an experiment to get to know the melting mechanism, then proposing the physical model of this mechanism, and, lastly, developing the mathematical model. Thus, these models are not overall models, and are limited to the specific extrusion method.These models are also not general in nature because of the material being processed. For example, melting of polymers that exhibit slippage during the flow does not follow the Tadmor model because the molten material does not accumulate at the active flight of the screw, but flows toward the passive flight. These models are also not general in view of the operating and geometrical parameters of the process. For example, the melting mechanism may vary substantially depending on the screw geometry  as well In conclusion, the currently available extrusion models are not general in nature due to limitations of the extrusion type, specific material, and specific operating and geometrical conditions of the process. Therefore, the question arises whether it is possible to solve the problem of global modeling of the extrusion process without referring to the specific solid/melt flow mechanism, which is determined by the extrusion method, the material type, and the process conditions. So far, there is no global model of the extrusion process, which would not be limited in this range. Therefore, the concept may be proposed to solve this problem on the basis of fluid mechanics using CFD  computation procedures. Instead of the specific solid/melt flow mechanism assumed, the polymer flow in the extrusion process can be simulated by solving the conservation equations of mass, motion, and energy along with a polymer constitutive equation, which was proposed by some researchers for the melting section ,104,105.This novel approach based on solving the conservation equations without an assumed polymer flow mechanism seems to be very promising for the future work on modeling the extrusion process, even though it requires very large computational capabilities. The global modeling of the extrusion process, however, requires iterative computations to define the process operating point, and the question arises regarding how to implement the continuum computations into the global model of the process. This might be solved by developing a novel concept of the total (continuous) screw characteristics, which is schematically depicted in The most promising approach would be the coupled DEM/CFD modeling. Very recently, a coupling between EDEM, a DEM software, and OpenFOAM, which is an open-sourced computational fluid dynamics CFD software, has been developed and is now under testing. The initial version of this coupling allows for momentum transfer between EDEM and OpenFOAM, with a heat transfer capability to be implemented. The coupling overcomes one of the common limitations with DEM/CFD coupled simulations, namely that particles must be smaller in volume than the mesh cells they occupy, which allows for a wide range of application types that were previously not possible.An issue of global modeling of polymer extrusion was reviewed and discussed, which includes modeling for solid conveying, melting, and melt flow, as well as co-operation of the screw/die system.It was observed that the basis of computer extrusion models are still the early models of solid conveying and polymer melting, and no significant progress has been made in recent years. Extrusion modeling using a discrete element method (DEM) for solid conveying, and computational fluid dynamics (CFD) for polymer melting may be promising in this respect.It was also observed that the extrusion models are generally limited to neat polymers. When modeling the polymer extrusion, it is assumed that there is no slippage at the fluid/solid interface, and flowing materials in the screw extruders and dies adhere to the wall. It is also assumed that flowing materials have no yield stress. However, there are many materials like filled polymers  that have a yield stress and exhibit wall slippage under certain conditions.Limitations of the traditional approach to modeling based on an assumed polymer solid/melt flow mechanism were presented, and the novel continuous concept of global modeling based on solving the conservation equations without an assumed polymer flow mechanism was proposed. A concept of the total (continuous) screw characteristics was discussed to solve the problem of implementing the continuum computations into the global model of the process. Promising progress in coupled DEM/CFD modeling was indicated.


Based on the ODC inhibition and AZ-ODC binding studies, we demonstrated that amino acid residues reside within the α1 helix, β5 and β6 strands, and connecting loop between β6 and α2 (residues 142–178), which is the posterior part of AZ95–228, play crucial roles in ODC binding and inhibition. We also identified the essential elements determining the ODC-degradative activity of AZ; amino acid residues within the anterior part of AZ95–228 (residues 120–145) play crucial roles in AZ-mediated ODC degradation. Finally, we identified the crucial factors that govern the differential binding and inhibition of AZ isoforms toward ODC. Mutagenesis studies of AZ1 and AZ3 and their binding and inhibition revealed that the divergence of amino acid residues 124, 150, 166, 171, and 179 results in the differential abilities of AZ1 and AZ3 in the binding and inhibition of ODC.Antizyme (AZ) is a protein that negatively regulates ornithine decarboxylase (ODC). AZ achieves this inhibition by binding to ODC to produce AZ-ODC heterodimers, abolishing enzyme activity and targeting ODC for degradation by the 26S proteasome. In this study, we focused on the biomolecular interactions between the C-terminal domain of AZ (AZ Antizyme (AZ) is a central player in the regulatory circuit that controls cellular levels of polyamines by regulating ornithine decarboxylase (ODC) degradation and polyamine uptake activity ,2,3,4. AAZ was the first protein shown to utilize translational frame shifting to regulate mammalian mRNA ,33. Poly95–228) was sufficient for binding and inhibiting ODC [95–228-ODC complex  and ODC to identify the functional elements of AZ that are essential for binding, inhibiting and degrading ODC. Furthermore, we identified the crucial factors that govern the differential binding and inhibition of AZ1 and AZ3 toward ODC.Our previous studies have shown that the N-terminus of AZ is not essential for the binding and inhibition of ODC, and the C-terminal AZ peptide from residues 95 to 228  to express the target proteins. ODC or AZ proteins were overexpressed with 1 mM isopropyl-1-thio-β-D-galactoside (IPTG) induction in JM109 cells for 20 h at 25 °C. After harvesting the cells, the total cell extract was applied to a His-Select™ nickel affinity column  for further purification. The protocol for the protein purification of ODC or AZ was followed as described in Hsieh et al. [Human ODC, AZ, and a series of AZ mutant proteins were subcloned into an N-terminal His6-tagged pQE30 vector . The expression vector with ODC or AZ gene was introduced to JM109 h et al. . First, E. coli strain. Finally, the DNA sequence with the desired mutation was confirmed by auto sequencing. Site-directed mutagenesis on AZ1 or AZ3 was carried out with a QuikChange™ kit to generate the AZ1 and AZ3 mutants . The mutagenic primers with the desired mutations were approximately 35–45 bases. The mutations in this study are shown in The continuous ODC enzyme activity was measured by the reactions that were coupled with the phosphoenolpyruvate carboxylase and malate dehydrogenase, and the ODC enzyme (0.38 μM) was inhibited with various quantities of AZ proteins . The ass2 was accompanied by the oxidation of 1 mmol of NADH analog in this coupled reaction. For the NADH analog, an extinction coefficient of 2410 cm−1 mM−1 was used in the calculations. The IC50 value of each inhibition plot was calculated with the following equation:50 value denotes the AZ concentration that is required for inhibiting 50% of the ODC enzyme activity. All calculations were carried out with the SigmaPlot 10.0 software program .The reaction was traced at the absorbance decrease at 405 nm using a Perkin–Elmer Lamba-25 spectrophotometer, and the production of 1 mmol of COp = 0.95 and a resolution N of 200.Sedimentation velocity experiments were carried out at 20 °C with a Beckman Optima XL-A analytical ultracentrifuge. In a buffer of 30 mM Tris-HCl (pH 7.4) and 25 mM NaCl, the concentration of ODC was fixed at 0.3 mg/mL with AZ concentrations AZ ranging from 0.02 to 0.19 mg/mL (the molar ratio of AZ/ODC ranged from 0.25 to 3). In the centerpiece, the reference and sample sectors were filled with buffer (400 μL) and sample protein (380 μL), respectively, and the whole module was then set up in an An-50 Ti rotor. Sedimentation velocity experiments were implemented at 20 °C with a rotor speed of 42,000 rpm using the absorbance at 280 nm to measure every 420 s with a step size of 0.002 cm. At different time points, a total of 20–30 scans were collected and fitted to a continuous size distribution model with the program SEDFIT ,50. All dK) of the AZ-ODC complexes, sedimentation velocity experiments were carried out with different concentrations of AZ proteins at a constant concentration of ODC protein. The dK values of the AZ-ODC complexes were calculated by totally fitting all data sets with the AB hetero-association model in the SEDPHAT program [To determine the dissociation constant , 1.5 mM ATP, 10 mM creatine phosphate , and 1.6 mg/mL creatine phosphokinase  for 2 h at 37 °C. The degradation reaction was stopped after adding the 2× protein sample dye. After the proteins were separated by 13.5% SDS-PAGE, they were transferred to a polyvinylidene difluoride (PVDF) membrane for immunoblotting using anti-ODC , anti-AZ , and anti-GAPDH  antibodies as probes, and the protein bands of ODC, AZ, and GAPDH  were monitored by ImageQuant™ LAS 4000 . The degradation ratio of ODC at 2 h was quantitated by the program ImageJ when the amount of ODC at 0 h was defined as 1 [AZ-ODC proteins at a fixed molar ratio (1:1) were incubated with a rabbit reticulocyte lysate  mixture including 40 mM Tris-HCl (pH 7.4), 5 mM MgClned as 1 .95–228 heterodimer, we selected 18 positively or negatively charged amino acid residues in the C-terminal domain of AZ1 (AZ95–228) to bind and inhibit ODC  values of these mutants were approximately 1~1.3-fold. In contrast, the IC50 values of AZ95–228_E142A, AZ95–228_K153A, AZ95–228_D154A, AZ95–228_E161A, AZ95–228_E164A, AZ95–228_D165A, AZ95–228_H171A, and AZ95–228_K178A were higher than those of AZ95–228_WT  were approximately 1.6~2.5-fold.In the ODC inhibition studies, the AZn of ODC . The inhAZ95–228 . The IC5 0.16 μM ; the IC55–228_WT , and the , was created to examine its ability to inhibit ODC , was generated, and this AZ95–228-5Mα1 mutant completely lost its inhibition of ODC enzyme activity , β6 (His171), and connecting loop between β6 and α2 (Lys178) also play a role in ODC inhibition . Adding ely lost . 95–228-5Mα1 and AZ95–228_E142A/H171A/K178, respectively, and the hybrid octuple mutant AZ95–228_E142A/K153A/D154A/E161A/E164A/E165A/H171A/K178A (AZ95–228-8M) had no inhibitory effects on ODC  exceeded 1.5  between AZ95–228 and ODC  was 12.44 μM, higher than that of all of the mutants of AZ95–228-ODC  were shown to play crucial roles in ODC binding and inhibition.The ODC binding affinities of the following quadruple mutants, AZ3Mα1-ODC . FurtherAZ95–228 . The Kd,–228-ODC , which i95–228, the determinants for ODC degradation induced by AZ may reside within the anterior part of AZ95–228.Since we found that the critical ODC-binding domain of AZ resides within the posterior part of AZ34–228 and AZ95–228 mutants were performed under a reticulocyte lysate-based system. Basically, AZ34–228 and AZ95–228 peptides had similar degradative activity toward ODC  play crucial roles in AZ-mediated ODC degradation.An earlier study showed that the region containing residues 130–145 of AZ might be necessary for ODC degradation . We idenward ODC . Mutatioward ODC . Neverth peptide B–D. The  peptide D. Overal peptide B, may goWe have identified the amino acid residues in AZ, such as Glu142, Lys153, Asp154, Glu161, Glu164 and Glu165, His171 and Lys178, that function in the binding and inhibition of ODC . Among t50 values than AZ3-WT  and AZ3_S124D/Q150E/K166Q/S171H/D179N (AZ3_5M) were 0.25 μM and 0.29 μM, respectively  were reduced to levels similar to those of AZ1_WT , indicating that AZ3_5M is similar to AZ1_WT in ODC binding and inhibition  and AZ1_D124S/E150Q/Q166K/N179D/H171S (AZ1_5M) were 0.55 μM and 0.49 μM, respectively  were elevated to levels similar to those of AZ3_WT , indicating that AZ1_5M is similar to AZ3_WT in ODC binding and inhibition  determine the isoform-specific characteristics . Lys178 95–228 (We have also identified the essential elements that determine the ODC-degradative activity of AZ. We identified at least four amino acid residues in AZ, including Ser120, Asn129, Gly137, and Gly145, which are essential for AZ-mediated ODC degradation . These r95–228 . In the 95–228 . Therefo


Ionizing radiation (IR) and epidermal growth factor (EGF) stimulate Y-box binding protein-1 (YB-1) phosphorylation at Ser-102 in KRAS wild-type (KRASwt) cells, whereas in KRAS mutated (KRASmut) cells, YB-1 is constitutively phosphorylated, independent of IR or EGF. YB-1 activity stimulates the repair of IR-induced DNA double-strand breaks (DSBs) in the nucleus. Thus far, the YB-1 nuclear translocation pattern after cell exposure to various cellular stressors is not clear. In the present study, we investigated the pattern of YB-1 phosphorylation and its possible translocation to the nucleus in KRASwt cells after exposure to IR, EGF treatment, and conditional expression of mutated KRAS(G12V). IR, EGF, and conditional KRAS(G12V) expression induced YB-1 phosphorylation in both the cytoplasmic and nuclear fractions of KRASwt cells. None of the stimuli induced YB-1 nuclear translocation, while p90 ribosomal s6 kinase (RSK) translocation was enhanced in KRASwt cells after any of the stimuli. EGF-induced RSK translocation to the nucleus and nuclear YB-1 phosphorylation were completely blocked by the EGF receptor kinase inhibitor erlotinib. Likewise, RSK inhibition blocked RSK nuclear translocation and nuclear YB-1 phosphorylation after irradiation and KRAS(G12V) overexpression. In summary, acute stimulation of YB-1 phosphorylation does not lead to YB-1 translocation from the cytoplasm to the nucleus. Rather, irradiation, EGF treatment, or KRAS(G12V) overexpression induces RSK activation, leading to its translocation to the nucleus, where it activates already-existing nuclear YB-1. Our novel finding illuminates the signaling pathways involved in nuclear YB-1 phosphorylation and provides a rationale for designing appropriate targeting strategies to block YB-1 in oncology as well as in radiation oncology. YBX1 gene. The N-terminal domain, the hydrophilic C-terminal domain, and the CSD are the main YB-1 domains [Y-box binding protein-1 (YB-1) is a member of the cold-shock domain (CSD) protein superfamily encoded by the  domains , and det domains ,4. YB-1, domains , is consYB-1 can be phosphorylated at several serine residues. To exert the described functions, YB-1 must be phosphorylated at serine 102 (Ser-102) within the CSD. The expression statuses of YB-1 and Ser-102-phosphorylated YB-1 have been described to be reversely correlated with clinical outcomes, such as in diffuse large B-cell lymphoma patients . BesidesYB-1 is generally expressed in the cytoplasm, but it is also transported to the nucleus at the end of G1-phase before S-phase entry  followinIonizing radiation (IR) induces a variety of DNA damage, including single-strand breaks and double-strand breaks (DSBs). DSBs are the most lethal lesions and the main cause of radiotherapy-induced cell death. Radiation-induced DSBs are repaired by either homologous recombination or nonhomologous end-joining ,17. YB-1Previously, we demonstrated that exposure to clinically relevant doses of ionizing radiation induces phosphorylation of YB-1 in KRASwt but not in KRASmut breast cancer cells . To dateEpidermal growth factor (EGF) induces YB-1 phosphorylation in KRASwt cells based on analysis of total cell lysates . The datBased on these data, we proposed that EGFR kinase activity stimulates accumulation of activated RSK in the nucleus, whereas RSK stimulates phosphorylation of nuclear YB-1. We investigated this hypothesis in cells pretreated with the EGFR tyrosine kinase inhibitor erlotinib (10 µM for 2 h) and those stimulated with EGF . The data presented in Growth-factor-induced YB-1 phosphorylation in KRASwt cells  is mainlExposure to ionizing radiation concomitantly induces YB-1 phosphorylation in both cytoplasmic and nuclear fractions of KRASwt cells. Thus far, the data presented demonstrate that phosphorylation of YB-1 in the nuclear fraction of KRASwt cells is not due to acute translocation of phospho-YB-1 or YB-1 to the nucleus. KRAS mutation leads to constitutive phosphorylation of YB-1 . Here, wThe RSK family, important downstream effectors of the RAS/MAPK pathway, consists of four isoforms (RSK1–4) that regulate key cellular processes. We investigated whether RSK inhibition affects KRAS(G12V)-induced YB-1 phosphorylation in the nuclear fraction. The data shown in The activity and subcellular distribution of YB-1 are two important parameters for the function of YB-1. Currently, it is not known whether acute phosphorylation of YB-1 leads to its translocation to the nucleus, where it performs the majority of its functions. The importance of YB-1 localization to the nucleus is supported by the fact that the level of nuclear YB-1 is correlated with poor prognosis in various human cancers ,13,14,15YB-1 through its cold-shock domain interacts with double-stranded DNA . PreviouRSK is the major kinase that phosphorylates YB-1 ,22 by inThe signaling pathway described in RAS GTPases, after mutation or external stimuli, localize to cell membranes for activation . In the In conclusion, as depicted in ® HTB-26™), KRAS(G12V)-mutated non-small cell lung cancer cell line A549 , KRAS (Q61H)-mutated non-small cell lung cancer cell line H460 , and KRASwt breast cancer cell lines MCF7 (ATCC® HTB-22™), SKBR3 (ATCC® HTB-30™), and HBL-100 , penicillin (100 U/mL), streptomycin (100 μg/mL), puromycin (5 μg/mL), and blasticidin (5 μg/mL). All cells were incubated in a humidified atmosphere with 93% air and 7% CO2 at 37 °C.The KRAS (G13D)-mutated breast cancer cell line MDA-MB-231 (ATCCman milk ) were usAntibodies against phospho-YB-1 (Ser-102) (cat. # 2900), YB-1 (cat. # 4202), phospho-p90RSK (cat. # 9344), RSK1/2/3 (cat. # 9355), RSK1 (cat. # 9333), RSK3 (cat. # 9343), P-Akt (Ser-473) (cat. # 4060), P-ATM (Ser-1981) (cat. # 4526), and ATM (cat. # 5883) were purchased from Cell Signaling . RSK2 was purchased from ThermFisher Scientific (cat. # PA5-29280) . Antibodies against K-RAS (cat. # ab157255) and Lamin A/C (cat. # ab40567) were purchased from Abcam . The anti α-tubulin antibody (cat. # CP06) was purchased from Calbiochem . Epidermal growth factor (EGF) was purchased from Sigma-Aldrich . The EGFR inhibitor erlotinib was provided by Hoffmann-La Roche . LJI308 (cat. # S7871) was purchased from Selleckchem .Irradiation was performed at 37 °C using a Gulmay RS225 X-ray machine  at a dose rate of 1 Gy/min with the following exposure parameters: 200 kVp, 15 mA, and 0.5 mm copper additional filtering. At the indicated time points after EGF treatment (100 ng/mL) or irradiation, YB-1 phosphorylation was analyzed according to the design of each experiment. The inhibitors were dissolved in DMSO to create 10 mM stock solutions and stored at −20 °C. The inhibitor solutions were diluted in cell media to appropriate working concentrations just prior to use. Control cells received media that contained appropriate concentrations of the solvent DMSO.Isolation of nuclear and cytoplasmic fractions was performed as previously described ,39. Totat-test was performed to compare data between groups. A p-value less than 0.05 was considered to indicate a significant difference. Densitometric quantification analyses of immunoblots was performed using Image Studio Light Ver 5.2 from LI-COR Biosciences system .Student’s