Humans are able to continually learn new information and acquire skills that meet the demands of an ever-changing environment. Yet, this new learning does not necessarily occur at the expense of old memories. The specialised biological mechanisms that permit continual learning in humans and other mammals are not fully understood. Here I explore the possibility that neural inhibition plays an important role. I present recent findings from studies in humans that suggest inhibition regulates the stability of neural networks to gate cortical plasticity and memory retrieval. These studies use non-invasive methods to obtain an indirect measure of neural inhibition and corroborate comparable findings in animals. Together these studies reveal a model whereby neural inhibition protects memories from interference to permit continual learning. Neural inhibition may, therefore, play a critical role in the computations that underlie higher-order cognition and adaptive behaviour. Current Opinion in Neurobiology 2021, 67:85–94Neurobiology of learning and plasticityThis review comes from a themed issue on Tara Keck and Sheena A JosselynEdited by Issue and the EditorialFor a complete overview see the Available online 28th October 2020https://doi.org/10.1016/j.conb.2020.09.007http://creativecommons.org/licenses/by/4.0/).0959-4388/© 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license , the principle inhibitory neurotransmitter.in vivo are restricted to invasive recordings which, except in rare circumstances, can only be performed in animal models. By contrast, most non-invasive tools for measuring human brain activity, including functional Magnetic Resonance Imaging (fMRI), aggregate the response from excitatory and inhibitory signals across large neural populations [1H magnetic resonance spectroscopy (1H MRS); (2) brain stimulation; and (3) oscillatory signatures.Box 11H MRS: 1H MRS provides a non-invasive means to detect and quantify chemical compounds within living tissue. In humans, MRS can be used to quantify the concentration of GABA within discrete regions of the brain, together with other neurochemicals such as glutamate  can provide a non-invasive readout for GABAergic inhibition in the human brain. Inhibitory ppTMS involves delivering a conditioning stimulus shortly before a test stimulus, with the two pulses separated by either 2−4 ms  or 100−200 ms . When applied to primary motor cortex (M1), the test stimulus in an inhibitory ppTMS protocol elicits a suppressed motor evoked potential (MEP) in the target muscle, relative to an unconditioned test stimulus  ,5. NeuraIn the human brain, a non-invasive signature of this process can be acquired using MRS a, Box 1.Box 2Brain stimulation: Trans-cranial direct current stimulation (tDCS) is a non-invasive method used to modulate the concentration of cortical GABA [cal GABA . During cal GABA  or usingcal GABA  , learning dependent changes in MRS-derived GABA reveal disinhibition during a target-detection task but increased inhibition during feature discrimination . AnotherDespite fluctuations in neural inhibition with new learning, empirical work in animal models ,29 togetTheoretical models suggest various plasticity mechanisms work in concert to provide homeostatic control . BroadlyBy combining non-invasive imaging techniques with brain stimulation and computation modelling, it is now possible to assess the consequences of selective inhibitory synaptic potentiation in the human brain. After participants learn to associate pairs of abstract visual stimuli, the neural expression of these learned associations can be probed using fMRI repetition suppression, a tool used to index neural representations . ImmediaThese results suggest that following learning, inhibitory plasticity restores network stability by forming inhibitory replicas of newly modified excitatory synapses. Memories in cortex may, therefore, be stored in a dormant state, and only expressed when latent inhibitory connections are unmasked. Spiking activity recorded in the songbird corroborates this view: in juvenile birds, the ability to learn a tutor song predicts maturation of synaptic inhibition, while in adult birds premotor neurons remain silent but can be released from inhibitory control via application of the GABA antagonist gabazine . TogetheInhibitory plasticity may, therefore, be considered a key homeostatic mechanism that facilitates stable memory storage. Yet, this model implies that memory retrieval or reinstatement occurs when neural inhibition is reduced, either locally or at a network-wide level. Indeed, theoretical models suggest retrieval may be achieved by perturbing local changes in synaptic input, effectively gating the expression of neural activity via a transient break in EI balance .In humans, it remains difficult to measure dynamic changes in EI balance. However, resting levels of GABA and glutamate may be related to cognitive variables, such as memory retrieval. A particularly effective approach involves using behavioural modelling. For example, during a decision-making task, choices made by each participant can be explained by several parameters in a computational reinforcement learning model. Together with MRS data recorded from the anterior cingulate cortex (ACC) at rest, this reveals a positive relationship between the model parameter that describes ‘use of learned information’ and glutamate in ACC, with an equivalent negative relationships for GABA •• Figurb. This rInvasive methods available in rodents now provide sufficient spatiotemporal resolution to track dynamic changes in inhibition during memory retrieval. Studies employing these methods suggest latent memories are released from inhibitory control during memory retrieval using the same disinhibitory circuit motif that supports learning . TherefoThe functional significance of incorporating selective neural inhibition in cortical memories may be fully realised when considering how humans and other mammals continue to learn new information throughout their lifetime. While experiences often overlap with each other in content or sensory information, we can selectively recall memories for different experiences. This ability to continually learn new information while protecting memories from interference may in part be supported by the inhibitory component of a memory.et al. trained human participants to encode two overlapping but context-dependent memories. Training was performed across two consecutive days [To test this hypothesis in the human brain, Koolschijn ive days ••. On thive days .Figure 4Neural inhibition may therefore play a key role in promoting continual learning by protecting memories from interference. This view is further supported by evidence in humans showing that extensive training, or ‘overlearning’, on a visual task prevents memory interference and shifts the neurochemical profile in visual cortex from glutamate-dominated excitation to GABA-dominated inhibition •• Figurb.While inhibitory rebalancing following learning promotes network stability, the ability to generalise may be predicted by the relative instability of memory. Behavioural studies in humans show that generalisation from a motor to a word list task is prevented if learning on the two tasks is separated by a sufficiently long time interval . IntriguAlthough continual learning can be observed in humans and some animals, this contrasts with artificial neural networks which typically struggle to avoid overfitting and show catastrophic forgetting when trained on multiple tasks. Artificial neural networks may therefore be used as a testing ground to identify those biological constraints necessary for continual learning. This approach reveals the importance of several key biological features, including synaptic complexity , ‘experiCurrent available methods for indexing neural inhibition in the human brain (MRS and TMS) are typically deployed within a single brain region. This makes it difficult to assess how interactions between different brain regions might affect the contribution of neural inhibition to learning and memory. One possibility is that brain regions that reside higher in the cortical hierarchy regulate neural inhibition at lower levels . For exaCognitive tasks that investigate suppression of unwanted thoughts (e.g. ‘think/no think’ paradigms) further support a framework in which the hippocampus together with the prefrontal cortex mediates inhibitory control over memory retrieval. For example, higher resting levels of MRS-derived GABA in the hippocampus predict superior suppression of unwanted thoughts ••. MoreoNon-invasive measures of neural inhibition in the human brain suggest inhibitory interneurons gate learning and memory. During multi-trial learning, measures of cortical inhibition acquired using MRS reveal a rapid, reversible decrease in neocortical GABA, where a larger drop in GABA predicts superior learning. Network stability is then restored via homeostatic mechanisms that likely include inhibitory plasticity. This promotes stimulus selective inhibition which holds memories dormant unless latent inhibitory connections are unmasked. Resting levels of GABA measured using MRS can predict how memories are used to guide decision-making, while a decrease in GABA, induced using anodal tDCS, leads to an increase in memory interference. Together these findings describe a model where transient decreases in neural inhibition gate new learning, memory retrieval and even generalisation, while inhibitory rebalancing mitigates against catastrophic forgetting. Information in neural circuits is, therefore, not simply remembered or forgotten but rather subject to an inhibitory brake that provides the mechanistic flexibility necessary for continual learning. This suggests inhibitory processing plays a key role in the neural computations responsible for adaptive cognition and behaviour.Nothing declared.• of special interest•• of outstanding interestPapers of particular interest, published within the period of review, have been highlighted as


Studies on multisite phosphorylation networks of cyclin-dependent kinase (CDK) targets have opened a new level of signaling complexity by revealing signal processing routes encoded into disordered proteins. A model target, the CDK inhibitor Sic1, contains linear phosphorylation motifs, docking sites, and phosphodegrons to empower an N-to-C terminally directed phosphorylation process. Here, we uncover a signal processing mechanism involving multi-step competition between mutually diversional phosphorylation routes within the S-CDK-Sic1 inhibitory complex. Intracomplex phosphorylation plays a direct role in controlling Sic1 degradation, and provides a mechanism to sequentially integrate both the G1- and S-CDK activities while keeping S-CDK inhibited towards other targets. The competing phosphorylation routes prevent premature Sic1 degradation and demonstrate how integration of MAPK from the pheromone pathway allows one to tune the competition of alternative phosphorylation paths. The mutually diversional phosphorylation circuits may be a general way for processing multiple kinase signals to coordinate cellular decisions in eukaryotes. The decision of whether and when a cell divides is tightly controlled. Here, the authors show in yeast that there is a multi-step competition between different phosphorylation states and sites in the S phase CDK-Sic1 complex, which controls Sic1 degradation and coordinates the precise timing of the G1/S transition. As the master regulators of the cell division cycle, CDKs use a combination of different docking interactions and activity thresholds to temporally order the phosphorylation of hundreds of their targets in the cell cycle13. Most of the CDK targets are phosphorylated at multiple sites distributed along disordered regions within these proteins14. In G1 phase, Cyclin-dependent Kinase Inhibitors (CKIs), like disordered protein Sic1 (Substrate Inhibitor of Cdk)16 in yeast, or p27 in humans17, ensure that S- and M-phase promoting CDK activity is suppressed to allow origin licensing and other G1 processes that require low CDK activity18. Interestingly, both Sic1 and p27 have the seemingly controversial functions of acting simultaneously as an inhibitor of and a substrate for CDK. Although its inhibitory function keeps CDK activity low via tight nanomolar affinity, the substrate function involves phosphorylation-induced degradation of CKI via the ubiquitin–proteasome pathway at G1/S, to unleash free S-phase CDK activity20.Recent works on multisite phosphorylation networks of cyclin-dependent kinase (CDK) have revealed a new level of signal processing complexity that is based on signaling routes encoded into disordered proteins or disordered regions of proteins21 causes a release of free S-CDK, which, in turn, further accelerates the phosphorylation and degradation of Sic1 by positive feedback25. The resulting burst of free S-CDK triggers the S phase. The first steps of the Sic1 multisite phosphorylation process are primed by G1-CDK, a complex that is not inhibited by Sic127. The phosphates are added stepwise via docking by phospho-adaptor Cks1 in the N-to-C-terminal direction with the help of a complex network of interactions  still efficiently phosphorylates Sic1. We found that this intracomplex multisite phosphorylation network contains two competing pathways, one leading to degron phosphorylation, and another that functions as a diversionary path that terminates the degron route. Using fluorescent live-cell microscopy, we demonstrate how diversionary phosphorylation by S-CDK within the inhibitory complex prevents the kinase signal leakage via the phospho-degron route. We also show that the diversionary route can be primed by MAPK from the pheromone pathway to ensure G1 arrest, or alternatively, the diversion can be outcompeted by priming phosphorylations within the phospho-degron route by the G1-CDK complex, which accumulates after the Start decision. This paper will lay out a mechanism that enables the disordered Sic1 molecule, with its linearily encoded phosphorylation pattern, to act as a processor that integrates multiple kinase inputs.32P autoradiography. We observed that within a reconstituted complex, while being inhibited toward a control target histone H1, the S-CDK complex (Clb5–Cdk1–Cks1) still efficiently phosphorylates Sic1 46 46 Fig. . As pred)46 Fig. , we can )46 Fig. . Convers)46 Fig. , indicat2 to the N terminus . We observed fast post-Start accumulation of Sic1 in case of the VLLPP, T2,5S-VLLPP, and 9SP mutations  priming by G1-CDK, (ii) an intracomplex step driven by inhibited S-CDK, and (iii) the positive feedback-driven extra-complex step driven by free emerging S-CDK.To analyze whether the S-CDK catalyzed intracomplex process has a role in Sic1 degradation also in freely cycling cells, we analyzed the same set of mutants both in case of full-length Sic1 (Sic1) and C terminally truncated non-inhibitory Sic1 (Sic1ΔC) in an unperturbed cell cycle (without α-factor). In the case of full-length Sic1, the T2,5S, VLLPP, T2,5S-VLLPP, and RXL mutations all delayed the degradation timing Fig. . In contIn stress-free conditions, the T173S mutation did not affect Sic1 degradation timing but resulted in consistently lower Sic1 levels in case of full-length Sic1, indicating that the declining M-CDK and accumulating S-CDK signal leak was prevented via intracomplex phosphorylation of T173 by CDK and diversional termination , preventing the leak is not a priority any more, and rapidly accumulating G1-CDK efficiently primes the N-terminal sites in Sic1, and the diversional lock is overrun because the probability of pT173-dependent termination is low due to the fewer number of steps required for S-CDK to reach the degrons Fig. . On the 57.In the present study, we made an attempt to explain a design paradox of the CKI-CDK double-negative feedback mechanism: high phosphorylation specificity of CDK toward CKI may compromise tight control over G1, and cause a phosphorylation-dependent degradation leak of CKI, thereby making it difficult to maintain the G1 state. We observed that high intracomplex phosphorylation activity manifested in processive phosphorylation of Sic1 within the inhibitory complex. As in human CKI p27, intracomplex phosphorylation by CDK mediated degradation of the inhibitor58. On the other hand, after the Start decision, the lock is overrun because the balance between the two routes shifts in favor of the N-terminal degron route due to efficient priming phosphorylation by rapidly accumulating G1-CDK.We describe here mechanism of two competing intracomplex phosphorylation routes within the Sic1–Clb5–Cdk1–Cks1 inhibitory complex: the diversional site pT173 is able to prevent the leak via the alternative degron phosphorylation route. We also demonstrate that this diversional lock can be strengthened by extra input from MAPK Fus3 in case of pheromone pathway activity, a functionally overlapping role of Sic1 and Far1 to allow robust G1 arrestThis study reveals yet another complexity level of multisite phosphorylation mechanisms of CDK targets. Intricate network of phosphorylation sites and linear docking motifs along the disordered sections of Sic1 enable differential processing of at least four kinases with alternative input sites: G1-CDK as a primer for the N-terminal sites at G1/S transition, S-CDK as driver of priming-dependent phosphorylation of the degron route, and also as a diversionary input at T173, MAPKs as external inputs for the diversionary site T173, and a bulk of proline-directed kinases as constantly acting primers of site T5 Fig. .63. In this case, however, the problem of leakiness is solved by orthogonality of tyrosine and serine–threonine kinase (CDK) prevents degron phosphorylation by the tyrosine kinase and enables a conditional AND gate-like trigger of G1/S only when both kinase signals are individually present above critical thresholds. Tyrosine kinase signaling is not as highly evolved in yeast as in higher eukaryotes, and the role of proliferation signals in yeast is fulfilled by the G1-CDK complexes, which link the cell cycle Start to the cell size control system, or alternatively, by the decline of MAPK Fus3 activity upon release from mating arrest  phosphorylationest Fig. . The ind65. For quantification of endogenous Whi5, Sic1, and Clb5 proteins the corresponding loci were recombined at 3′ end by PCR-derived mCherry, EGFP or Citrine tag ORF and the selection marker cassette65. Similar approach was used to tag Sic1 with C-terminal 6xHA for western blotting analysis in Supplementary Fig. 2a65. To introduce genomic replacements of SIC1 gene with mutated versions, PCR-derived DNA fragments were used to generate exact genomic replacements over sic1::URA3 using 5FOA selection. To generate strains for Sic1ΔC time-lapse microscopy, pRS306 vector with PSIC1-Sic1ΔC-EGFP cassette was integrated into URA3 locus of the strain with WHI5-mCherry:SpHis5 background. To generate strains for Sic1ΔC western blotting, the pRS316 vector with PADH1-Sic1ΔC-3xHA cassette was used. For Supplementary Fig. 66. For Supplementary Fig. PSIC1-Sic1ΔC-6xHA was used for integration into URA3 locus. Copy number of integrated pRS306 constructs carrying Sic1ΔC-EGFP was assessed by measuring quantitative levels of EGFP expression and qPCR analysis of genomic DNA. Please see Supplementary Table All used yeast strains are described in Supplementary Table E. coli BL21RP and purified using cobalt2+ affinity chromatography. Cyclins were copurified together as stoichiometric complexes with active Cdk1 from Δsic1 yeast strain using tandem affinity purification (TAP)69. Cks1 was expressed and purified from E. coli BL21RP as described previously70. Swe1 was expressed and purified from yeast cells using TAP-tag affinity purification67.Recombinant N-terminally 6xHis-tagged Sic1 proteins were expressed from pET28a(+) plasmid in 2, 150 mM NaCl, 0.1% NP-40, 20 mM imidazole, 2% glycerol, 2 mM EGTA, 0.2 mg ml−1 BSA, 250 μM ATP, and 500 nM Cks1. Wild-type or phosphate-binding pocket mutant Cks171 was added to the reactions containing Cks1, and mock buffer was used for reactions without Cks1. For quantitative intracomplex phosphorylation assays, 0.02 μM Clb5–Cdk1 complex and 1 μM recombinant Sic1 was used. The reactions containing a reference substrate were supplemented with 1 μM bovine histone H1 (Sigma-Aldrich). For phosphate labeling, 0.5 μCi μl−1 radiometric label [γ-32P]-ATP  was added 5 min after the enzyme was added to the reaction buffer containing unlabeled ATP. Finally, the reaction was initiated by adding Sic1 or Sic1 and histone H1 mixture.The general composition of phosphorylation reaction mixture was as follows: 50 mM HEPES-KOH pH 7.4, 5 mM MgCl32P]-ATP label was performed as described above. After 6 min of phosphorylation reaction, 5 mM DDT and 6 mM MnCl2 was supplemented followed by addition of recombinant lambda phosphatase (Santa Cruz Biotechnology) in 50 U µl−1 final concentration. All reactions were carried out at room temperature and aliquots were removed into SDS–PAGE sample buffer.For quantitative phosphorylation assays presented in Figs. 2 was used according to the instructions from the manufacturer28. Unless noted otherwise, 10% SDS–PAGE was used. The identification of different Sic1 phospho-isoforms using Phos-tag SDS–PAGE is described in ref. 1For separating the phospho-isoforms of Sic1, 10% SDS–PAGE supplemented with 100 μM Phos-tag and MnClAfter SDS–PAGE, gels were subjected to vacuum drying and subsequent autoradiography using Amersham BAS-IP MS 2040 E screen, Biomolecular Imager Typhoon 5 (Amersham), and ImageQuant TL software .32P signal in the reference mixture corresponds to one phosphate added to Sic1 per inhibited complex.To estimate Clb5–Cdk1–Cks1 intracomplex activity in units of phosphate numbers per Sic1 molecule bound to the complex, the same concentration of CDK complex was phosphorylated by Swe1 in a parallel reaction for reference. As Cdk1 is monophosphorylated by Swe1, the Cdk1 2.To determine the phosphorylation sites of Sic1 intracomplex, 10 µg of purified 6xHis-Sic1 recombinant protein was phosphorylated using 20 nM Clb5–Cdk1–Cks1. After incubating the kinase reaction for 6 min at room temperature the reaction was stopped in SDS–PAGE sample buffer. The proteins were separated using 10% SDS–PAGE, the gels were stained with Coomassie brilliant blue G-250 (Sigma), and Sic1 protein band was excised from the gels. 1 µg of Sic1 was used for the analysis. Subsequent in-gel digestion, peptide separation, MS analysis and Mascot search was performed as described previouslyFor western blotting, 10–30 µg of pelleted cells were snap frozen in liquid nitrogen and subsequently lysed by silica bead grinding in lysis buffer containing urea. The lysate was cleared by centrifugation, the supernatant was normalized over total protein content determined by Bradford assay (BioRad), subjected for gel electrophoresis. For detecting HA epitope the blotting was carried out by using anti-HA  and anti-mouse-IgG HRP conjugated antibody . For detecting Cdk1 anti-Cdk1 antibody  and anti-goat-IgG HRP conjugated antibody  were used. For electroblotting of Phos-tag SDS–PAGE gels, a dry system iBlot (Invitrogen) was used. Conjugated HRP signal was detected using Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific).CLB5–TAP in 2-μm plasmid was overexpressed using GAL1 promoter. The expression was induced in strains containing endogenously expressed Sic1–6HA or T173A–Sic1–6HA. For co-immunoprecipitation, Clb5–TAP purification using 5–7 g of cell pellet was performed as described above. The presence of Sic1–6HA and Cks1 in final eluate was assessed by western blotting using anti-HA  or anti-Cks1 , and anti-mouse-IgG or anti-rabbit-IgG HRP conjugated antibodies .To carry out the co-immunoprecipitation of Sic1–Clb5–TAP–Cdk1–Cks1 complex, recombinant For quantitative western blotting, the enhanced chemiluminescence (ECL) signal was detected by ImageQuant RT ECL Imager 3088E camera (Amersham) with highest resolution and in variable shutter speed to secure all pixels were below signal saturation threshold. The uncropped images of Typhoon scans, ImageQuant RT ECL images, and all quantitated areas on these images are presented in Source Data files together with the signal values obtained for each area.−1 (~5.9 mM) stock solution in DMSO was used. The final α-factor concentration was 0.59 µM for bar1− strains and 5.9 µM for BAR1+ strains. For arresting cells with α-factor, the appropriate peptide concentration was added at OD 0.3 for 3 h. G1 arrest was confirmed by visually observing shmoo morphology. To induce G1 arrest in Δcln1,2 cln3::PGAL1,10-CLN3 background strains via repression of CLN3 mRNA synthesis, 2% glucose was added for 3.5 h.For western blotting, log-phase cultures grown in selective synthetic complete (SSC) or synthetic complete (SC) media were used. For α-factor synchronization, >95% pure synthetic α-factor peptide  as a 10 mg ml44. Before the switch to α-factor or glycerol–ethanol media, cells were allowed to grow unperturbed under the flow of SC 2% glucose media for at least 60 min. Constant media flow at pressure 5 psi was used through the experiment.For time-lapse microscopy, log-phase yeast culture grown in SC media was used. Before subjecting cells to Y04C type CellASIC ONIX microfluidic plates (EMD Millipore) the culture was sonicated until cell aggregates were separated into single cells. Cells were loaded into the growth chip as described in CellASIC online protocol using SC media supplemented with 2% glucose. The media containing 240 or 2 nM α-factor used in the pulse experiments, or media containing 2% glycerol and 1% ethanol instead of glucose, for long G1 experiments, was supplied from separate wells. For the α-factor pulse and glycerol–ethanol experiments, a previously described protocol was used6, 5 × 105, 105, 104, 5 × 103 cells ml−1. Subsequently, 5 µl of culture was used for spotting.In the yeast viability assays in Fig. 43. The images were collected and exported in raster image formats (.JPG) by using ZEN software  and were subjected to automated cell tracking and fluorescence signal quantitation process using custom MATLAB (compatible with version 2016a) scripts as described previously72. For Sic1-EGFP signal quantification, minor custom changes were made.Time-lapse imaging of cells growing in the microfluidic chips was carried out with Zeiss Axio Observer Z1 inverted microscope using motorized stage and phase contrast imaging combined with Colibri LED module to excitate mCherry, EGFP or Citrine fluorescence. For excitation of EGFP, 470 nm LED for 15 ms, for Citrine 505 nm LED for 700 ms and for mCherry 555 nm LED for 750 ms were used at 25% power. For experiments containing Sic1-EGFP, a maximum of 10 fields of view were collected. For imaging, Hamamatsu Orca ER CCD Monochrome Microscope Camera was used. The microscopy setup was similar to Dončić et al.45. For quantification, the maximal fluorescence plateau level was measured using the aid of a digital ruler. Next, the half-maximal value of the signal and corresponding point in the time axis was determined. Finally, the program delivers the corresponding time value automatically into an output table. All the software used for microscopy image quantification and analysis are available upon request.The fluorescence time-lapse profiles were subjected to manual assessment for cell tracking and imaging quality and further timing measurementAt least two independent experiments were conducted for all kinase assays and western blots. The number of quantitative replicates used, as well as the raw data for quantification of all conducted experiments are available in Source Data files. For all kinase assays and quantitative western blots, the mean values were derived from at least two replica experiments.In live-cell microscopy, data of each strain were collected from at least two independent cell colonies. On the Sic1 degradation timing distribution plots, the median values as well as 95% confidence interval values are shown. Also, the 95% confidence intervals of the medians are summarily presented in Supplementary Table Further information on research design is available in the Supplementary InformationReporting Summary


Despite the continuous efforts in the development of BL inhibitors (BLIs), new BLs able to hydrolyze the last developed antibiotics rapidly emerge. Moreover, the insurgence rate of effective mutations is far higher than the release of BLIs able to counteract them. This results in a shortage of antibiotics that is menacing the effective treating of infectious diseases. The situation is made even worse by the co-expression in bacteria of BLs with different mechanisms and hydrolysis spectra, and by the lack of inhibitors able to hit them all. Differently from other targets, BL flexibility has not been deeply exploited for drug design, possibly because of the small protein size, for their apparent rigidity and their high fold conservation. In this mini-review, we discuss the evidence for BL binding site dynamics being crucial for catalytic efficiency, mutation effect, and for the design of new inhibitors. Then, we report on identified allosteric sites in BLs and on possible allosteric inhibitors, as a strategy to overcome the frequent occurrence of mutations in BLs and the difficulty of competing efficaciously with substrates. Nevertheless, allosteric inhibitors could work synergistically with traditional inhibitors, increasing the chances of restoring bacterial susceptibility towards available antibiotics. Antimicrobial resistance (AMR) is critically threatening the treatment of a large range of infections caused by bacteria able to inactivate even last resort antibiotics . In the Among bacteria, resistance is often caused by the overexpression of BLs (β-lactamases) able to hydrolyze and inactivate a vast number of β-lactam antibiotics ,8. BLs aSerratia fonticola resistant to carbapenem BL (SFC-1) and Serratia marcescens enzyme (SME), the plasmid-encoded Klebsiella pneumoniae carbapenemase (KPC) and Guiana Extended Spectrum (GES)-type enzymes; for class C, only the plasmid-mediated AmpC β-lactamase active on cephamycins (CMY-10), for class D, the acquired OXAcillinase carbapenemase (OXA)-type enzymes. For class B, that includes the most clinically-significant carbapenemases, the New Delhi Metallo-beta-lactamase 1 (NDM-1), the IMiPenemase (IMP) and Verona Integron-encoded Metallo-beta-lactamase (VIM) series have been reported worldwide. Class B enzymes are both plasmid- and integron-located, and are able to inactivate all available β-lactams, with the only exception of aztreonam . As. As59]. In stark contrast, NDM-1 enzyme presents an Ala68 residue, not following the proline conservation at position 68 . To finaAllosteric binding events occur when a protein changes its functioning upon ligand binding to a secondary site, distant from the active one. Interestingly, some allosteric sites may be cryptic, as they remain hidden in X-ray apo structures and may open only upon ligand binding. While the clinical benefit of allosteric activators in BLs may not be of relevance, allosteric inhibitors may offer several advantages, the most relevant one being eluding the competition with the substrate. From a drug design point of view, in SBLs, competitive inhibitors are generally conceived for mimicking the tetrahedral transition state, whose charge can be challenging for further ADME optimization. In MBLs, active site inhibitors can act, disrupting the active nucleophile in the active site or, more often, contain zinc coordinating moieties that may exert off-target toxicity in host zinc enzymes and can be limitedly modulated for adjusting the ADME profile . Recent Class A allosteric effectors have been investigated since the discovery of a cryptic site in SHV-1 . In hisi in the high micromolar range . This slight inhibitory activity has been linked to the unzipping of H11 (residues 219–226) and H12 (residues 271–289), drifting away Leu220 and Asn276, and consequently loosening Arg244 from the overall structure packing. Arg244 has a crucial role in class A BL catalysis, and its displacement may moderately affect enzymatic activity phenyl)amino)butanoic acid) has been reported by Grigorenko et al. to inhibit TEM-171 with a K = 88 µM . NotwithLately, Hart et al. performed a structure-based drug design campaign for identifying TEM-1 allosteric effectors . StartinA comparison of class A BLs co-crystallized with allosteric effectors is reported in Allosteric regulation in class A BLs has been further explored by the engineering of TEM-1 BL . In theiin silico simulations have a key role in studying their architecture and behavior in presence or absence of their allosteric effectors. Indeed, many algorithms and approaches have been proposed to identify pockets that, otherwise, might not be visible [As cryptic sites are, per definition, not detectable in apo crystal structures,  visible ,72,73.Specifically, Bowman and Geissler used TEM-1 BL as a proof of concept for demonstrating that allosteric sites may be predicted by equilibrium fluctuations of the apo protein . In factAllosteric regulation of class A BLs has been thoroughly explored in the last decade for helping the design of more potent allosteric effectors, and for a better understanding of how mutations can affect enzyme activity. Meneksedag et al. started by observing that the interaction with the BL inhibitor protein (BLIP) causes flexibility loss in H10, which is part of site A1 . In partLately, Huang et al. published a work demonstrating how increased flexibility of H10 favors the binding of BLIP, and the consequent BL inactivation . This reE. coli, and their increased resistance to meropenem and cefotaxime was tested. The three mutants showing highest resistance were purified and characterized for understanding the underlying allosteric pathway, as the point mutations were distant to the active site. Of these three mutants, only T165W and L48A were successfully crystallized at high resolution , but did not show any change in structural features with respect to the wild-type CTX-M-9 (RMSD = 0.3 Å). After the measurement of the root mean square fluctuation (RMSF) of heavy atoms along the MD simulations, the authors suggest that T165W and L48A mutations may affect the drug–enzyme interaction by increasing the conformational flexibility in loop 103–105 and residue N170, both critical in ligand binding and hydrolysis. Lastly, a machine-learning method was applied to MD simulation snapshots, for investigating the active site residues more sensitive to S281A, T165W and L48A mutations. The results for the acyl-enzyme highlighted a pivotal role for residues Asn104, Glu166 and Lys234, whose position was heavily affected by mutations.A work by Latallo et al. presented interesting highlights regarding alternative allosteric pathways in CTX-M-9 . By compAllosteric sites found in class A BLs cannot be repurposed in class B BLs, nor in class D BLs, because of their different fold that lacks the H10 helix.Allosteric inhibition is the least exploited mechanism to target MBLs, due to two major pitfalls. No allosteric mechanisms have been reported for this class of BLs, and the sequence variability of possible exosites would make the design of small, broad-spectrum molecules nearly impossible. Three azolylthioacetamide derivatives reported by Xiang et al. were characterized as inhibitors with mixed inhibition mechanism (competitive and uncompetitive), however, their uncompetitive inhibition mechanism and binding site has not been reported . At the Allosteric pathways or effectors in class C and D have not been reported in the literature, however, cooperativity correlation areas  across four AmpC enzymes have been reported by Brown et al. Specifically, the authors noticed the existence in AmpC of three flexible correlated regions, the first two lining the catalytic Ser64, the third one being the Ω-loop .As the necessity of new antibiotics is more and more compelling, new design strategies have to be developed. The capability of carbapenemases to hydrolyze nearly all kinds of antibiotics seems to be strictly related to a binding site highly hydrophobic and flexible. Thus, the knowledge of BLs active site dynamics is fundamental to predict the binding of new ligands/inhibitors and to guide their development and optimization. As well, the identification, and prediction, of cryptic sites may open the way for the development of allosteric inhibitors able to affect antibiotic binding, possibly restoring their activity, and to synergize the action of classical inhibitors.in silico studies successfully managed to unravel allosteric mechanisms and pockets, proving that computational techniques can have not only a hypothesis-generating, but also a predictive role in this field. Among all the possible and transient allosteric pockets reported by the literature, only one cryptic but druggable site (here named site A1) has been widely recognized in the α-helical domain of class A BLs. Small molecules binding to this site slightly reduced or, in some cases, enhanced enzymatic activity and, thus, were identified as allosteric effectors. However, given the variety of scaffolds reported, several questions remain open. There is no consensus about the chemical features a ligand should have for behaving as allosteric effector, and the rationale for obtaining allosteric inhibitors (rather than activators) is still missing and requires further investigation.Several Allosteric inhibitors for class A BLs cannot be repurposed for class B and D members, because of their different fold lacking H10, but there might be a chance for targeting class C BLs, which share some structural and dynamic similarities with class A enzymes. On a separate note, macromolecules such as engineered BLIP, nucleic acids, antibodies or polymers may be an alternative way for studying allosteric inhibition of BLs.


Adaptive of trees and its correlation with the climatic are causing changes in tree species performance and distribution, which will change breeding programs and influence forest productivity. To further evaluate the joint influence of climatic factors and provenance on the ring width (RW) and ring density (RD) of Masson pine. We selected 18 provenances at Chun’an (CA) and Taizi Mountain (TZS) test site, which representing four different breeding regions, including the south, west, north and east-central regions. The results showed that the provenance effects were significantly for the RW and RD. The provenances from high temperature and low latitude regions had greater mean RW compared to species from local and cold sources. The geographical genetic variation in wood traits is generally weak. The correlation between RW of Masson pine and precipitation was stronger in the relatively arid TZS site compared with that in relatively wet CA site, as well as the effect of temperature and precipitation on RD was earlier than that in CA test site. The response relationship between establishing the width of tree rings and the environmental variables of provenance indicated that during the transition from the northern and western breeding regions to the eastern and southern breeding regions, the response of RW to climate factors changed from being temperature-based to being precipitation-based. In addition, the response of provenance to the climate of seed sources origin showed their own variation characteristics in each breeding area. Therefore, genetic improvement of big diameter wood and wood density can be gain through selection of provenance and analysis of adaptability. The research showed that trees could adapt to the rapid environmental changes through genetic selection and showed different responses to environmental changes in radial growth and wood density5. In addition, there is strong growth correlation of trees with climate, particularly with temperature and precipitation7. The different tissues cells of wood change at different growing season, affect the ring width (RW hereafter) and ring density (RD hereafter) parameters and involve fast and slow of growth processes8. These xylogenesis phases  translate into varied responses of radial growth and wood property to climate10. The coupling relationship will be found among density parameters or between RW and climate, although such changes often depend on how the RW and wood density change over time as a function of genetics, site condition and climate factors13.Diameter growth and wood density are important selection traits for sawn timber and pulpwood, respectively, which have been part of the long-term breeding program of conifer speciesPinus massoniana Lamb.) in China is located at latitude 21 °41′∼33 °56′N and longitude 102 °10′∼123 °14′E. The distance between the north and south provenances is approximately 1360 km, and the distance between the east and west provenances is approximately 1900 km14. The genetic improvement work of Masson pine have been implemented since the 1970s in China, which is focus on survival, rapid growth, resin production, stem form and wood quality. Among the vast natural distribution regions of Masson pine, the southern provenances of Masson pine grows fast and the provenances in Guangdong and Guangxi have large radial growth and “polycyclic” annual shoots15. Compared with the southern provenances, the northern provenances, which have a relatively short growing season and fewer precipitation, temperature and light resources, especially in regions where soil moisture is relatively low, limits the radial growth and the formation of wood characteristics in Masson pine.The distribution area of Masson pine  analyze the RW and RD among provenances at different testing sites; (2) test the effects of provenances and climate on RW and RD; and (3) assess the influence of climate change on the adaptive of Masson pine in China.Provenance tests and provenance selection are the most basic tools for the genetic improvement of forest trees. Adaptive genetic variations of provenance and their correlations with climatic factors have been investigated in experiments, and reports have suggested that genetic variations should be considered for the provenance selection as an adaptation to climate changeth and 15th year after afforestation. The other trial was in a forest at Taizi Mountain , Hubei Province  of Zhejiang Province, and a total of 49 provenances from 14 provinces were tested in this forest. The experiment used completely randomized block design (8 blocks and 8-tree plots planted with a 2-row layout and 4 trees per row in each blocks). The row spacing of the initial planting was 2 m × 2 m, and thinning was done in the 10The condition of CA and TZS site is different. TZS site is in a relatively arid area with annual precipitation of 1090 mm and an mean annual temperature of 16.4 °C, whereas CA presents annual precipitation of 1530 mm and an mean annual temperature of 17.2 °C. The warmest month in the two study regions is July, and the coldest month is January. The total precipitation in CA occurs mainly from February to August, which accounts for 71% of the annual precipitation. The total precipitation in TZS occurs mainly from April to August, which accounts for 65% of the annual precipitation Fig. . The sam3. Resin composition in the core samples was extracted by Soxhlet extractor37. The cores were removed, dried, and polished step-by-step with different grades of sandpaper . The cell lumina and cell wall of the wood were clearly showed under 20x microscope, and the RW was measured using a LINTABTm6.0 RW measuring instrument (accuracy 0.01 mm) . The X-ray radiographic images were analyzed with density software to determine RD (g/cm3) profiles of each specimen38. The diameter cumulative growth (Dt1.3) was calculated according to the RW3.In November 2016, sampling of the experimental forests was carried out in three blocks at two test sites, respectively. From each provenance, a total of 6 trees in three blocks were selected in each site. 216 trees were selected from the two sites, and 5.15-mm cores were extracted at 1.3 m using a increment borer39, and used the following mixed linear model for variance analysis38:yijklm represented the value of the mth year the lth tree of the kth provenance growing at the jth block within the ith site; and Am, Si, B(S)j(i), P(A)k(m), S*P(A) ik(m), S*Aim and εijklm represented the effects of mth tree age, the ith site, jth block within ith site, mth year within kth provenance, interaction between the ith site and mth year within kth provenance, interaction between the mth tree age and ith site and the error, respectively. B(S)j(i) and εijklm were considered as random factor and the rest was considered as fixed factor. In addition, the affect of provenance-by-block interactions and tree age-by-block interactions was little, which could be ignored in the model40. The least square method was used to estimate the first order auto-correlation of model residuals40.We tested the homogeneity of variance of RW, RD and cumulative stem growth41. The results showed that the main data series covered 30 years from 1987 to 2016 (corresponding to rings 4 to 33). RW and RD were not only affected by environmental and ecological factors but also controlled by their own physiological factors; therefore, the growth trends of trees would be removed before application. The RW and RD series were standardized using ARSTAN software with the negative exponential declination method. This method eliminated the influence of low-frequency variations of the RW and RD42. The RW and RD sequences that removes the growth trend will be used to create the residual chronologies43 (RES). The basic statistics of the remaining chronology of the rings are listed . Jingshan weather station  70 km away from TZS test point and Chun ‘an weather station  10 km away from CA test site were selected, respectively. For the climate stations of provenance, the same climate stations were chosen to provide seed sources in the report.The meteorological data period was 1986∼2016, and was downloaded from the China Meteorological Science data sharing service network  of 0.05.The multivariate regression analysis was done to test the influence of climate on growth of Masson pine. Therefore, to obtain the optimal equation, the stepwise regression method was used to screen the independent variables included in the equation, and adjusted 29.Five temperature, three precipitation and geographic coordinates variables were chosen. They included the following: mean temperatures , mean minimum and maximum daily temperature, annual total precipitation, precipitation in January, mean precipitation from May to August, and geographic coordinates (latitude and longitude). The differences between the above mentioned variables of origin and planting place of each provenance were used to analyze the trend of climate factors after each provenance introduced to the planting place. We calculated Principal Component Analysis (PCA) of width and density characteristics, respectively. The PCA axes which accounted for at least 50% of the total variance were kept in the PCAs1.3 differences between the provenances were statistically significant. The differences in Dt1.3 between the breeding regions changed with the age of the trees , respectively Table . AlthougAt CA test site, the RW of Masson pine showed significant negatively correlations to the average temperature in August. At TZS test site, the correlation between RW and mean temperature in June, was also negative, and showed significant positively correlations to precipitation in May Fig. .Figure 3At CA site, RD showed negatively correlations to precipitation in May and positively correlations to the average temperature in August. At TZS test site, RD was also significant negatively correlated with the precipitation in April and positively correlated with the mean temperature from April to May. At TZS test site, the time period in RD was affected by the temperature, which was earlier than that at the CA test site Fig. .The analysis of the linear relationship between climate variables and RW and RD showed that the slope of the linear relationship between various sources was significantly different, which reflected the different responses of various sources to future precipitation and temperature changes Table . At CA tAccording to the principal components, different provenances were grouped into regions related to climate factors. A scatterplot of the first two principal components was shown in Fig. The latitude, the mean annual temperature, the mean temperature in January, and the mean annual minimum daily temperature of seed-source origin were significantly related to RW, and they explained between 32%–50% and 31%–36% of the growth variations, respectively. At TZS site, RD showed the significant negatively correlations to average precipitation from May to August. The mean temperature from May to August, the mean annual maximum daily temperature were positively correlated with RD, respectively. At CA test site, the correlation between RD and average temperature from May to August of seed-source origin, was also positive . TZS test site was located in a relatively dry area in the central and western regions, and the significant impact of precipitation in May on RW might be partly explained by the high temperature in May and June, which also affected RW. Early wood had a high transport capability during its formation45. Precipitation from May to August was 550 ml, and relatively dry soil conditions limited the growth of Masson pine at TZS site. In June and July, particularly, which were the most active period of growth for conifers, high summer temperature could greatly reduce soil moisture, thereby affected tree growth. The mean temperature in August was the major climatic factor affected the RW of Masson pine. Precipitation from May to August could reach 810 ml at CA test site; thus, the availability of soil water did not the major factor affecting the growth of RW.The study showed that the climatic factors could explain 18.76% and 26.33% of the total variance of the annual RW, which could explain 50.31% and 65.13% of the total variance of the annual RD, and the annual RD was more affected by the climatic environment than the annual RW48. From April to July, particularly, which were the most active period of growth for conifers, high summer temperature could greatly reduce soil moisture. The needles tended to close stomatal and reduce water loss with transpiration increasing. The affect of precipitation and temperature on radial growth changed gradually with the changing of location and drought degree. For example, at high altitudes, the sensitivity of wood density to temperature would decrease due to climate warming, then turned to the coupling relationship between temperature and precipitation50. It was important to predict the influence of precipitation on wood density. The total precipitation in April (TZS sites) and May (CA sites) was increased in RD (majority of provenances). Increasing rainfall in early summer, wood formation might have been delayed, which affected the proportion of early-late wood within the tree-ring and increased late wood proportion. The wood density of P. massoniana was limited by extreme summer heat in the growing season at CA sites. This was consistent with study findings of significantly positive correlations between wood density and the mean maximum temperature and sunshine hours during late summer and early fall that had been revealed in conifers such as Silver fir 29. The net photosynthesis of tree affected the duration and rate of diameter growth as well the number and size of the cells formed in the growing season50. In the early growing season, the climate is mild, the cambium is active, the radial growth rate is high, and more growth-regulating hormones will be produced in the early growth process. The duration of cell enlargement is relatively long but the duration of cell wall thickening is short, which producing a lot of large, thin-walled cells of earlywood. In the late growing season, given the effective and rapid stomatal control of water loss from transpiration, the formation of photosynthetic products and the storage of carbohydrates are reduced, the speed of cell division falls down, while the speed of enlargement duration of cell increases and thickening duration increases. In the end, some narrow, thick-walled cells of dense latewood are produced50.In this study, the major climatic factors affecting RD of Masson pine were the mean temperature in August and the total precipitation in May at CA test site. The main climatic factors affecting RD were the average temperature from April to May and precipitation in April at TZS site. The characteristics of RD showed strong correlations with temperature53. The trend associated with temperature is opposite, while temperature was considered to be an important factor limiting the northward movement of tree species54. The study showed that there was a complex relationship between the tree-ring parameters and temperatures during the growing season and across zones. For example, the response of the RW parameter to temperature changed from a positive correlation in high latitudes (late summer) to a negative correlation or no correlation in low latitudes (late spring or early summer)50.The results also suggested that species from high temperature and low latitude regions had greater RWs compared to species from local and cold sources. One reason: The southern provenances showed faster radial growth, which might be due to adapting to the warm growing environment of seed-source origin. When introduced to the planting site, they still maintained a longer growing period. Another reason: Growth differences between provenances were generally genetically controlled and had certain laws of geographic variatio. For example, the high latitude provenance grows slowly and has strong resistance to snow pressure, wind damage and freezing damage. When low-latitude provenances are shifted to the north, the properties of these provenances, including fast growth and low resistance to snow pressure, freezing damage, and wind damage, are essentially maintained. The growth of trees in southern provenance requires more precipitation compared with that in the northern areas29. We have the following explanation for these results. When many provenances were planted at CA site, the relatively warm and humid climatic conditions of this site benefited the continued formation of late wood because of abundant precipitation and the relatively high temperature during the late growing season. Therefore, late wood density at this site was relatively high. For the southern provenances, because precipitation was inadequate, and temperature was relatively low during the late growing season, late wood density was relatively low due to suppressed growth. After introduced to TZS planting site, the provenances  showed high dependence on precipitation. The experimental site at TZS had a relatively dry climate, which was similar to what the western and central provenances experienced. Temperature during the late growing season at this site was relatively high, which benefited the continued formation of late wood and resulted in the higher late wood density of provenances in these regions. Because summer precipitation at this site was relatively low, and the temperature during late summer and early fall was also relatively low, the growth of the southern provenances was suppressed, resulting in the lower wood density observed for these provenances.However, the geographical genetic variation in wood traits is generally weak or nonexistent. Principal component analysis showed that after introduced to CA planting site, the provenance in the breeding regions in the west and north of China showed better RD in the regions with abundant precipitation, which was gradually higher than that in the breeding regions in the east and south (shown in Fig. Supplementary Information.


Apis mellifera, Polybia paulista and Solenopsis invicta venoms. We also distinguished, using sera adsorption procedures, the cases of CCD cross-reaction using Brl as a marker and inhibitor of CCD epitopes. The presence of reactivity for bromelain (24–28 kDa) was obtained in 43% of the patients, in which 64% presented reactivity for more than one Hymenoptera venom in radioallergosorbent (RAST) tests, and 90% showed reactivity in immunoblot analysis to the major allergens of Apis mellifera, Polybia paulista and Solenopsis invicta venoms. Sera adsorption procedures with Brl lead to a significant reduction in patients’ sera reactivity to the Hymenoptera allergens. Immunoblotting assay using pre- and post-Brl adsorption sera from wasp-allergic patients blotted with non-glycosylated recombinant antigens  from Polybia paulista wasp venom showed no change in reactivity pattern of sIgE that recognize allergen peptide epitopes. Our results, using Brl as a marker and CCD inhibitor to test sIgE reactivity, suggest that it could complement diagnostic methods and help to differentiate specific reactivity to allergens’ peptide epitopes from cross-reactivity caused by CCD, which is extremely useful in clinical practice.Allergic reactions to Hymenoptera venom, which could lead to systemic and even fatal symptoms, is characterized by hypersensitivity reactions mediated by specific IgE (sIgE) driven to venom allergens. Patients multisensitized to sIgE usually recognize more than one allergen in different Hymenoptera species. However, the presence of sIgE directed against Cross-Reactive Carbohydrate Determinant (CCD), which occurs in some allergens from Hymenoptera venom, hampers the identification of the culprit insects. CCD is also present in plants, pollen, fruits, but not in mammals. Bromelain (Brl) extracted from pineapples is a glycoprotein commonly used for reference to sIgE-CCD detection and analysis. In sera of fifty-one Hymenoptera allergic patients with specific IgE ≥ 1.0 KU/L, we assessed by immunoblotting the reactivity of sIgE to the major allergens of  Allergic reaction to insect venom is one of the most common causes of anaphylaxis  and may Allergenic proteins present in Hymenoptera venoms may trigger a type I hypersensitivity reaction, mediated by antigen-specific immunoglobulin E (IgE), which is a risk factor for subsequent systemic reactions (SR) . However3F6), being epitopes highly immunogenic and capable of inducing the production of IgE antibodies, [CCDs are present in glycosylated proteins, and the glycosylation process occurs in the endoplasmic reticulum and in the Golgi complex by the addition of carbohydrates to specific amino acid residues of proteins . In hymeibodies, ,12,13,14Although CCD is considered an immunogenic molecule, it is clinically irrelevant, because there are no reports of allergic reactions caused as a result of immune response triggered to these epitopes . This laApis mellifera [Solenopsis invicta [Polybia paulista allergenic proteins, phospholipase A1 and antigen 5 are glycosylated proteins, so they do not have CCD epitopes, such as antigen 5 from Solenopsis invicta [Advances in the analysis of hymenopteran venoms proteins showed that some allergens, such as the Hyaluronidase  and phosellifera  and phos invicta , present invicta ,21.Ananas comosus), which has two N-glycans, β1,2-xylose and α1,3- fucose, with a structure called MUXF3. Due to the similarity between this structure and the CCD epitopes present in hymenopteran allergens, Brl can be a useful tool to differentiate patients that present only anti-CCD IgE from those that present specific recognition of protein epitopes [Apis mellifera, Polybia paulista and Solenopsis invicta venom.CCDs are molecular structures also found in bromelain (Brl), an enzyme of approximately 24 kDa obtained from the crude and aqueous extract of the pineapple : 34 kDa and Antigen 5 (Ag 5): 23 kDa from Polybia paulista; and Phospholipase A1 (PLA1B): 35 kDa and Antigen 5 (Ag 5): 24 kDa from Solenopsis invicta. Electrophoresis showed the Brl as a band around 24–28 kDa. Additionally, we performed electrophoresis using the recombinant forms of PLA1 and Ag 5 since they were used in some blotting experiments described in item 2.5 : 45 kDa; Phospholipase A2 (PLA2): 16 kDa and trypsin inhibitor rich in cysteine (Api m6): 8 kDa from item 2.5 .Apis mellifera venom extract. The other fifty showed reactivity to PLA2 protein. Additionally, twelve of them also reacted to Hyal., and thirty-eight reacted to the Api m6 protein. Only ten patients  presented reactivity to all three allergens  did not show serum reactivity to allergens presented in the llergens .Polybia paulista venom extract, we observed that just three patients  did not have serum reactivity to either of the two studied allergens (PLA1 and Ag 5). Furthermore, thirty-four patients showed serum reactivity to both allergens, ten patients presented serum reactivity to PLA1 , and four patients had serum reactivity only for Ag5   .Solenopsis invicta venom extract, we observed that sera from only three patients  did not react to any of the studied proteins (PLA1 and Ag5), forty-one patients had serum reactivity to both allergens, six patients had serum reactivity to PLA1B only , and one patient had serum reactivity only to Ag5 (pt 9)  .Twenty-two sera from the studied population recognized Brl in the immunoblot assays in opposite to twenty-nine sera that did not react. Among the patients’ serum that recognize Brl, 64% (14/22) showed reactivity for more than one Hymenoptera venom, and 36% (8/22) presented reactivity for just one, as demonstrated by the presence of sIgE in the RAST tests. On the other hand, in patients’ sera without recognition of Brl, 48% (14/29) showed reactivity for more than one Hymenoptera venom, and 51.7% (15/29) presented reactivity for just one . The resHowever, the analysis of immunoblot assays showed that 90% (20/22) of patients with anti-CCD antibodies in the sera showed reactivity for all Hymenoptera venoms tested , whereasApis mellifera, Polybia paulista and Solenopsis invicta venom extracts. The intensities of the pre- and post-adsorption bands are presented in To eliminate non-specific sIgE reactivity, due to the presence of anti-CCD antibodies presented in patient’s serum, we used an adsorption protocol incubating sera with Brl. Immunoblotting procedures were then conducted with serum previously adsorbed and blotted with Apis mellifera venom, post-adsorption sera from pt 1, pt 4, pt 5, pt 9, pt 15, pt 16, pt 17 and pt 19 did not react to any of the allergens studied and pt 22 remains negative. The other patients maintain reactivity to the allergens, despite the change in the intensity of the bands. Similarly, in immunoblot assays with Polybia paulista and Solenopsis invicta venom, there is also a reduction in the intensity of the bands in the post-adsorption sera and the pt 1, pt 4, pt 5, pt 6, pt 8, pt 11, pt 12, pt 13, pt 14, pt 16 and pt 19 sera stopped reacting to the venom allergens from Polybia paulista and the sera from pt 4, pt 5, pt 6, pt 8, pt 17, pt 20 and pt 21 no longer react to the allergens of Solenopsis invicta venom. Interestingly, the sera from pt 4 and pt 5 stopped reacting to all venoms studied after adsorption with bromelain, although both patients have sIgE for the three venoms, as shown in RAST results  and rPoly p 5 (Ag 5). As shown in To verify an eventually allosteric binding effect in sIgE that recognize peptides epitopes, during the adsorption of patient’s sera with Brl, sera of the six patients with IgE reactive to Apis mellifera, Polybia paulista and Solenopsis invicta venoms pre- and post-adsorption with Brl, a plant glycoprotein known as a source of N-glycans that mimics the CCDs presented in some allergens’ composition of the Hymenoptera venoms.The presence of cross-reactive carbohydrates (CCD) in the allergens of Hymenoptera venoms is considered essential to serum antibody cross-reactivity that could be misinterpreted as multiple sensitizations hampering the correct diagnosis of the culprit insect in allergic patients ,10,11. TApis mellifera venom extract, PLA1 (34 kDa) and Ag 5 (23 kDa) in Polybia paulista venom, and PLA1B (35 kDa) and Ag 5 (24 kDa) in Solenopsis invicta venom  have sIgE able to recognize allergens of more than one species. In contrast, RAST data showed that only twenty-eight patients (55%) showed this same reactivity pattern. The difference in the sIgE reactivity found when we are comparing both sIgE detection methods could be explained by the sensitivity of each method. Consistently with this, Mosbech et al.  demonstr3 (glycoprotein structure extracted from Brl), being present in 43% of cases of double-sensitization to bee and wasp venom. Jappe et al. [Bromelain has been used in several studies to elucidate the presence of CCD in Hymenoptera allergens and to identify anti-CCD antibodies in sera from allergic patients ,32,33,34Anisakis spp. allergens and those from wasp venom. Using bromelain to inhibit anti-CCD IgE, through the previous incubation of the sera with this source of CCDs, it was demonstrated in the immunoblot assay the loss of the patient’s serum reactivity in cases where CCD was previously detected in the chemical structure of the allergens. Hemmer et al. [3) to investigate the cases of double-sensitization, showed in immunoblot assays a significant change in sera reactivity after inhibition procedures, demonstrating that previous serum adsorption procedures with CCD inhibitors could be a promising strategy for the tracing of CCD cross-reactivity. Consistently with this, in our study, we observed a significant reduction in the sera reactivity to all allergens studied, after the sera adsorption with Brl.Using a similar strategy, Rodríguez-Pérez et al.  analyzedr et al. , using CPolybia paulista or to Ag5 from Solenopsis invicta after Brl adsorption procedures. Perez-Riverol et al. [Polybia paulista are glycosylated proteins and therefore do not have CCD, as in the Ag 5 from Solenopsis invicta [The change in sera reactivity pattern after Brl adsorption procedures, observed in our study, demonstrated that there was a partial or total reduction not only in the 22 patients reactive to Brl but also in the group of patients who did not react previously to Brl. Interestingly, some serum stopped reacting to PLA1 and Ag 5 from l et al.  in recen invicta . Thus, tApis mellifera and pt 33, pt 44 and pt 48 with PLA1 from Polybia paulista), after the adsorption procedure with Brl.We observed that the loss of sera reactivity, after adsorption with Brl, occurs in those that previously had low reactivity (+) to the allergen, so after the adsorption procedure, this reactivity tends to disappear, probably due to an allosteric effect, as mentioned above. This effect can also explain the fact that in some patients’ sera  there was an increase in sera reactivity, or they even started to react to allergens that had not been previously detected in the immunoblot assay  to the allergens, the signal decreases after adsorption with Brl, which should probably be associated with the inhibition of anti-CCD antibodies, as described by Hemmer et al.  and in oAdditionally, we observed no change in the serum reactivity pattern to the recombinant allergens when comparing pre- and post-adsorption sera. Since the recombinant allergens are non-glycosylated proteins, we demonstrated that the adsorption procedures did not interfere with the sIgE antibodies that recognize peptides epitopes. Finally, despite the reduction in sIgE reactivity intensity, several patients still maintain reactivity to the allergens from the three Hymenoptera venoms, even after adsorption procedures, indicating that these reactivities may be caused by another reason, such as similarity of peptides epitopes or cases of multiple independent sensitivities, and not exclusively by CCD recognition.The anti-CCD IgE presence, despite being clinically irrelevant, can interfere with the proper diagnosis, considering the reactivity to the venom, and consequently with the prescription of adequate immunotherapy. The diagnosis of Hymenoptera venom allergy is dependent on several factors, such as a clinical classification of anaphylaxis, skin tests and specific serum IgE quantification. Our results, using bromelain as a marker and CCD inhibitor, alter the IgE reactivity. Thus, it supplements diagnostic methods and helps to differentiate clinical cases with cross-reactivity caused by CCD, being useful and promoting improvement in clinical practice.°C until use.Sera from 51 Hymenoptera venom-sensitized patients were selected considering specific IgE ≥ 1.0 kU/L , regardless of sex or age. They were obtained from the Ambulatório de Anafilaxia—Hospital de Clínicas, Faculdade de Ciências Médicas, Universidade Estadual de Campinas—UNICAMP. The FCM-UNICAMP Ethics Committee approved the study of under n. 80822817.3.0000.5404, Date: 2 January 2018. Informed consent was obtained in written form from all participants of the study, and participation was voluntary. The samples were stored at −30 Polybia paulista and Solenopsis invicta were authorized by SISBIO under number 60025-2 and duly registered in SISGEN under number AEE0F92. Venom glands were extracted from the Polybia paulista specimens and pulverized in liquid nitrogen, and then diluted in saline (NaCl)/ bicarbonate (NaHCO3) solution (85 mM/33 mM). Solenopsis invicta venom was obtained according to the protocol previously described by Fox et al. (2013) [Apis mellifera (apitoxin) venom was obtained commercially from the National Cooperative of Beekeepers of Minas Gerais  and 10 g of the extract were diluted in 40 mL of saline (NaCl)/bicarbonate (NaHCO3) solution (85 mM/33 mM). The lyophilized bromelain was obtained commercially , and extracts were prepared by diluting 10 mg in 1 mL of deionized water. Protein quantifications were performed using the colorimetric methods of Hartree [Apis mellifera and Polybia paulista) and Bradford [Solenopsis invicta).The collection of specimens . (2013) . Powdere Hartree  (Apis meE. coli BL21 (DE3) cells using the previously described protocol [P. pastoris X-33 cells, using the previously described protocol [+2 Sepharose™ High Performance; GE Healthcare, Danderyd, Sweden), according to the manufacturer’s instructions, followed by SDS-PAGE (15%) analysis for monitoring the efficiency of the purification process.The recombinant allergens rPoly p 1 and rPoly p 5 were obtained as described by Perez-Riverol et al. (2016)  and Bazoprotocol . The rPoprotocol . For theThe sera adsorption was carried out in 96-well ELISA plates , previously coated (0.1 µg/well) with bromelain. The plates were blocked with 300 µL/well of the blocking solution  and incubated for 1 h at 37 °C in a humid chamber. After this period, diluted sera (1:50 or 1:100) were incubated for 18 h (100 µL/well) at a temperature of 4 °C in a humid chamber, and subsequently that sera were collected for immunoblotting assays.® Tetra Cell System , followed by staining with either Coomassie Brilliant Blue R-250 (Sigma-Aldrich) or silver stain. Briefly, 100 μg of Apis mellifera or Polybia paulista venom extracts, and 50 μg of Solenopsis invicta and Bromelain, were submitted to SDS-PAGE and electrotransferred to a 0.45 µm nitrocellulose membrane (Bio-rad) using a semi-dry system . After blocking with 20 mM Tris-HCl, 150 mM NaCl, pH 7.4, with 0.5% Tween-20  (TBS-T wash solution) and 3% non-fat dried milk (block solution) for 2 h at room temperature under slow agitation on a Rocker II™ Platform mixer , the membranes were washed (3 times with TBS-T wash solution). The membranes were cut into strips of approximately 5 mm and incubated for 18 h with the patient’s sera diluted at 1:50 or 1:100 in TBS-T. Immunodetection was performed using anti-human IgE (ε-chain specific) peroxidase conjugate antibody (Sigma-Aldrich) (1:3000). The bands were visualized in Image Quant 400  using the chemiluminescent substrate Luminata™ Forte Western HRP substrate . The results of the immunoblots were analyzed qualitatively by determining the intensity of the venom and bromelain protein (CCD) bands, established from a pre-established model and represented by +; ++, and +++, as shown in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) 10–20% was performed according to Laemmli, 1970 , using a


The use of slip-resistant winter footwear is crucial for the prevention of slips and falls on ice and snow. The main objective of this paper is to evaluate a mechanical testing method to determine footwear slip resistance on wet and dry ice surfaces and to compare it with the human-centred test method introduced by researchers at KITE -Toronto Rehabilitation Institute-University Health Network. Phase 1 of this study assessed the repeatability and reproducibility of the mechanical method by evaluating ten different occupational winter boots using two SATRA Slip resistance testers . One tester is located in Toronto and one in Montreal. These boots were chosen based on the needs of the IRSST , who were primarily interested in providing safe winter footwear for police, firefighters and municipal workers. In Phase 2, the results of the human-centred test approach were compared with the mechanical results. In Phase 3, two of these boots with conflicting results from the previous phases were tested using a second human-centred method. In Phase 1, the mechanical testing results obtained in the two labs showed a high linear correlation (>0.94) and good agreement on both ice surfaces; however, they revealed a bias (~0.06) between the two labs on the dry ice condition. The mechanical and human-centred tests (phase 2) were found to be better correlated in the wet ice condition (R = 0.95) compared to the dry ice condition (R = 0.34). Finally, the rating of the footwear slip resistance based on the number of slips counted in phase 3 was consistent with the rating by the human-centred test method (phase 2), but not the mechanical method (phase 1). The findings of this study provide a better understanding of the limitations of the SATRA ice tray for measuring footwear slip resistance and demonstrate that the mechanical method must be further refined to make it more comparable to the human-centred methods to achieve better agreement with real-world performance. Slips and falls on ice and snow are widespread in many winter-experiencing countries ,2,3,4,5.The challenge during gait is that the body’s centre of mass (COM) is continually thrown ahead of its base of support and safe placement of the swinging foot is critical for preventing a loss of balance at each step . FurtherHowever, there are very few available methods for assessing the slip resistance of footwear on icy surfaces. ,18. A huA shoe’s slip resistance can also be assessed mechanically by measuring the COF between the shoe outsole and a test surface ,29. TherSome studies have evaluated the ability of the standard mechanical test method to provide a footwear slipperiness measurement representative of the actual footwear performance experienced by human participants walking on contaminated indoor surfaces ,36,37. HThe goals of this study were to explore and compare existing evaluation methods for slip resistance on ice and to consider new measurement and analysis methods that can help to demonstrate which methods can best reflect real-world performance. The study was conducted in three phases with the following objectives:(A)Develop an alternative ice surface preparation protocol for the mechanical method based on the existing SATRA TM144 protocol. In order to compare the mechanical method with the MAA, the ice surface conditions in these two methods had to be comparable.(B)Evaluate the repeatability and reproducibility of the mechanical method for measuring footwear slip resistance on the ice surfaces at two different laboratories: KITE and Institut de Recherche Robert-Sauvé en Santé et en Sécurité du Travail in Montréal (IRSST).Phase 1:Phase 2:Compare the results from the mechanical method with the MAA human-centred method for the evaluation of footwear slip-resistance performance on ice surfaces, using two methods for calculating the MAA score to determine which method is most appropriate for consumers.Phase 3:In the case of inconsistency between the results of mechanical and human-centred methods, investigate which method is more reliable for ranking footwear by using another human-centred test that is based on walking on a level ice surface to more closely reflect real-world conditions with a subset of two types of footwear.One SATRA STM 603 Slip resistance tester and one STM 603ICE were used in both labs (KITE and IRSST) to generate mechanical test results, as shown in The inter-laboratory reproducibility was evaluated by comparing results from the two labs using the same protocol. The mechanical test parameters specified by the standard test method ASTM F2913-19  were useTen types of occupational footwear (6 men’s and 4 women’s styles) shown in There was one operator at KITE and two operators at IRSST. Each operator ran the test three times for all footwear on dry and wet ice conditions. The goal was to measure repeatability, inter-lab reproducibility and inter-operator reproducibility. For repeatability analysis, two parameters were used: the pooled standard deviation (_pooled)  and the _pooled) .(1)COF¯mean).The inter-operator reproducibility and the inter-laboratory reproducibility were assessed by different analyses of variance (ANOVA). The intent was to identify what the significant effects were, such as operators, and the effect sizes of different independent variables, especially the laboratory effect. Post hoc Tukey multiple comparison tests were also run to determine the significant differences between boot COFs. The ANOVA and Tukey statistical analyses were performed using SPSS statistical software  . The anaThe MAA test protocol was conducted in WinterLab at KITE. This self-contained lab can be tilted up to 15° as participants walk up and down slopes to test footwear. Eight healthy young adults (range from 20 to 65 years of age) were recruited for testing. Only participants who self-reported that they normally walked outdoors in winter conditions and were not taking any medications or drugs that might affect their ability to balance or walk were recruited. As participants may have adverse medical effects , due to the cold exposure if they had predisposing conditions , participants were asked to decline participation if they had been diagnosed with any of these conditions, or if they had any concerns related to their health as a result of cold exposure. Four female participants were recruited to walk with women’s footwear , and four male participants were recruited to test men’s footwear  in random order. They ranged in age from 20 to 65 years and had no known musculoskeletal dysfunctions or mobility limitations. The demographic information of each participant is shown in The MAA test protocol was approved by the University Health Network Research Ethics Board. Participants provided consent before participating in the study. For safety, participants wore a fall arrest harness and were instructed to use a rope handrail only when they felt unsafe.Two ice conditions were created in WinterLab: dry cold ice and wet melting ice. For the dry cold ice, the entire WinterLab floor surface was flooded with water and cooled to a temperature of −5.5 ± 1.0 °C. Ice temperature was controlled using tubes circulating glycol coolant along the floor surface, and an ambient air temperature of 2.5 ± 2.0 °C was maintained throughout the experiment. In combination, these factors allowed a smooth, dull, ice walkway surface to be formed with minimal melting at the interface . The relative humidity in the WinterLab for dry ice conditions was about 45%. Starting with a dry ice surface, wet ice conditions were created by holding the ambient air temperature of the room at 8.0 ± 2.0 °C and the ice surface temperature at −1.5 ± 1.0 °C. The warmer ambient temperatures, in combination with the near-freezing ice temperature, helped to maintain a thin layer of water over the ice surface. The relative humidity in the WinterLab for wet ice conditions was about 36%.In the current protocol, the slope angle of the walkway is progressively increased by 2° until the first failure. An angle was considered the failure angle if the participant could not initiate gait or if both feet slipped simultaneously while traversing the slope. An angle was deemed as a maximum achievable angle (MAA) if the participant walked successfully on two out of three trials at this angle, and the participant failed on two out of three trials at the angle 1° higher than this angle. A trained observer inside WinterLab detected any slips that the participant encountered during their trials.Mean MAA score: uses the mean value of 8 MAAs, i.e., 4 participants × 2 directions, which represents the performance in-between an ascent and descent on each ice surface .mean) to be compared with min) to be compared with The mechanical test results for each footwear style on each ice surface were calculated in two ways: the mean of the COFs from the three modes  and all repeats as a “Mean values” scenario , and residual sum of squared (RSS) are considered to compare mechanical test results at KITE (KITE_COF) and MAA_COF values. The RSS is obtained as follows:Bland–Altman (B + A) analyses  were madResults from Phase 2 showed that for some winter boots, the ratings from the MAA and the mechanical tests conflict. Therefore, a second human-centred test  protocol was developed to check the number of slips that participants would experience while walking on a level icy surface in WinterLab. For this test, five healthy male participants were recruited. The demographic information of each participant is shown in A previously developed machine learning algorithm ,49 was uMonitoring the ice tray’s ice temperatures with thermistors at IRSST and KITE laboratories revealed that the actual ice surface temperature was colder than the setpoint temperature and colder than the temperature reading on the display of the STM 603ICE.Monitoring of the ice tray’s ice temperatures with thermistors also revealed that the ice surface temperature fluctuates as a function of the ice refrigeration cycle that corresponded to the stop-and-start cycle of the compressor allowing the STM 603ICE to maintain the setpoint temperature. This fluctuation showed slightly different patterns between the two labs: at KITE, it seemed that the ice took less time to warm up than it took to cool, whereas at IRSST it was the reverse, with the ice warming up more slowly but cooling more quickly .Specific temperature setpoints and restricted temperature ranges for testing on dry and wet ice surfaces were determined for each lab to ensure the ice temperatures measured by the thermistors were as similar as possible at the two labs and matched the WinterLab’s ice temperatures. The temperature setpoint −2 °C was found to be the best for producing a dry ice surface. At this setpoint, the display of the STM603ICE showed the temperature fluctuated between 0 °C and −2 °C, which corresponds to a fluctuation of the actual temperature of the ice between −2.5 °C and −6.5 °C at IRSST, and between −3 °C and −7 °C at KITE. In order to be as close as possible to the WinterLab’s dry ice condition, the COF values were measured when the displayed temperature was in the range of  at KITE . Mean MAA_COF, on the other hand, showed that the two boots had different slip-resistance qualities  highlighted in However, these results disagreed with the MAA method. For wet ice c,d, modemean and MAA_COFmean, and the correlation coefficient was 0.95  was 0.01, which shows a small deviation between KITE_COFwas 0.95 . On the analysis  confirmeThe results from Phase 2 indicated that the mechanical and MAA methods ranked the performance of the footwear differently. For example, when considering MAA mean scores on the wet ice condition, F5 had a higher MAA score compared to F1 , with pooled relative SDs of 9.0% for dry ice and 16.8% for wet ice. The inter-operator reproducibility (IRSST) was satisfactory since the trained operator did not have any significant effect on the measurements. In a shoe-on-quarry tile inter-laboratory study of the standardized test method ASTM F2913-19 involving 10 labs with one single model of shoe, the repeatability SD for the heel slip mode was 0.020 (relative SD of 3.9%) on a wet quarry tile and 0.044 (4.6%) on a dry quarry tile, as well as a reproducibility SD of 0.046 (relative SD of 9.0%) on a wet quarry tile and 0.086 (8.9%) on a dry quarry tile . It shouReconsidering the choice of the experimental unit (mean of last 5 consecutive runs) may also be warranted. Although an a posteriori assessment showed that the variation in the last 5 consecutive runs was 10% or less in most, some cases did reveal variations exceeding 10% over successive runs, especially on wet ice. More in-depth study of these variations in successive runs would be valuable, especially as the COF values are heavily influenced by the ice refrigeration cycle, which can show differing rates of change over the cycle. It should be noted that this experimental unit is not suited to soles with crampons or grit, like boot F9 with Green Diamond technology. For that boot, the COF of the first test run would probably have been more representative of the sole slip resistance compared to the mean of the five measurements taken when the grit had gone through the same grooves several times.Regarding inter-laboratory reproducibility, the analyses showed that the measurements with wet ice were equivalent in both labs with respect to both similar COF values obtained and ranking of the boots (in which boots F9 and F10 stood out as distinctly better performers than the other models). For dry ice, the COF values recorded at the IRSST lab were systematically higher than those obtained at KITE, with a bias of 0.06. Still, the agreement between the two labs was relatively good and the linear correlation was high.There are a number of sources of variability that may explain the differences noted between the two labs. First, the refrigeration cycles showed different patterns, especially for dry ice. For this type of ice, the test windows had to be defined at a time when the ice was warming up at the IRSST (from −2 °C to −1 °C) and cooling down at the KITE (from −1 °C to −2 °C). The differences in patterns observed in the refrigeration cycles could be due to possibly different operations of the ice trays . They may also be due to the laboratory environment, where the temperature, relative humidity and speed of circulation of the ambient air can affect thermal exchange between the air and the ice surface and have an impact on the ice refrigeration cycle.Another source of variability can be found in the procedure for removing frost from the ice surface with a wet cloth. While meetings were held with operators from the two labs to standardize the procedure and use the same cloth, a better match between the procedures would be desirable . In future, the impacts of these variables on the results will be investigated by conducting separate studies; we will also include standard methods to measure the ice roughness to better quantify the ice surface morphology.The analysis of the data indicated that the mechanical test results were not in agreement with the MAA test results on dry ice. Especially, the MAA method found boots F4, F7 and F8 as among the lowest performers (below 0.12), while the mechanical method rated them as being among the best performers. It may therefore be possible that the mechanical method overestimates the performance of some boots in certain cases. However, it is worth noting that both methods found that boots F9 and F10 offered good slip resistance. There were several reasons that could explain these variabilities. Firstly, the test parameters used in the mechanical method did not reflect the differences in human gait patterns, as is the case with several mechanical methods ,52. SecoThe dry ice condition was defined by the absence of any water film visible on the surface. However, we note that determining that there is no water film on the ice surface can be challenging, and therefore, the dry ice condition should be more carefully defined . The mecOn the other hand, the mechanical test and MAA test results seemed to be in agreement in wet ice conditions. The COF values on wet ice surfaces were generally very low, except for F9 and F10. As shown in In addition, although efforts were made to obtain ice surfaces with the ice tray that were as similar as possible to the ice surfaces of the WinterLab, the temperatures at the ice surface, and especially the ambient air conditions, were still not the same. Moreover, the conditioning of the boots prior to the tests was not done at the same temperatures. Mechanical tests were done at 23 °C ambient temperature, while MAA testing was done at 2.5 °C ambient temperature for dry ice condition and 8.0 °C for wet ice condition. It is, therefore, possible that this parameter has an impact on the difference in the results between the two test methods. The decision not to condition the boots at a colder temperature, for the mechanical method developed in this study, was made because keeping the boot cold is quite a challenge in a laboratory where the ambient air temperature is 23 °C and the temperature of the boot sole is increasing at a rate of approximately 1 °C per minute from 0 °C to 10 °C, and because conditioning the footwear to the colder temperatures would not allow running as many repetitions in one day as conditioning at the laboratory ambient temperature. For the further development of the mechanical method, however, it could be useful to rerun some tests by conditioning the boots in colder temperatures.The mean value scenario was a reasonable approach to compare the mechanical and MAA results because it took into account different ways of slipping  to evaluate the overall performance of the boots. However, the minimum values scenario seems to be the most appropriate when providing information on footwear performance to the consumer, as done by the KITE for the MAA method . One reaThe low number of participants for MAA testing is a limitation of the study. The criteria used in sample size estimation for the MAA method meant that a 95% confidence interval (CI) for the mean maximum achievable angle for each type of footwear should be no more than two degrees in total width. In other words, we determine the sample size and number of repetitions that will yield a 95% CI of x ± 1.0°. An internal study was conducted at KITE in 2014, with 15 male and 15 female participants, to establish the required sample size. The results revealed that a sample size of 4 with a repetition of 1 yielded a 95% CI of x ± 0.95°, and a sample size of 8 with a repetition of 3 yielded a 95% CI of x ± 0.48°. Therefore, a sample size of 4 is considered as a good compromise between cost efficiency and accuracy. Future work will include a study to consider more participants and more types of footwear to estimate the sample size to develop a more accurate model.Finally, it may be worthwhile to investigate the performance of the boots under real conditions in the field to get a better assessment of the value of the mechanical method in the laboratory and under a variety of real winter conditions.The comparison between the two boots, based on the three parameters, indicated that F1 was less slip-resistant than F5. This outcome was consistent with the results from the MAA test method, but not with those from the mechanical test method. The fact that the MAA and level walking tests were done under the same environmental conditions (wet ice inside the WinterLab) was certainly a factor in the similarity of the results. It should be noted, however, that even when walking conditions were different , participants experienced the same performances with the two boots.Even though there was high variability between participants, the phase 3 test protocol, based on the number of slips and slip distance (instead of COF), showed good potential for footwear slipperiness comparison. However, the detection of slip events and slip distances was very challenging. The small sample size was one of the limitations of this phase. Other limitations include a relatively short walking distance for the testing protocol in both phases 2 and 3, where only young participants were tested. More research will be needed to improve this method. Future work should include a protocol similar to that of phase 3, but taking place outdoors in real-world icy conditions, while the numbers of slip events are tracked by a wearable slip-detector (currently in development). In addition, it may be useful to test other conditions that produced conflicting outcomes, such as on dry ice with boots F1 and F4 , or boots F8 and F10 . An improved level-walking test could provide further information on the performance of the footwear, which could be used as an additional method to validate the MAA test method. In future, we will investigate different factors such as participants’ demographic, walking speed, and step length in this proposed human-centred protocol to provide a more rigorous statistical analysis.Overall, this work provided a better understanding of the limitations of the mechanical test method using the SATRA ice tray for measuring slip resistance. Our findings indicate that the mechanical method must be further refined to make its results more comparable to human-centred methods, which are more likely to reflect real-world slip resistance performance.The mechanical slip resistance testing results obtained in phase 1 from our two labs showed a high linear correlation (>0.94) and good agreement on both ice surfaces. However, they revealed a bias (~0.06) between the two labs on the dry ice condition. Comparing these mechanical test results to the human-centred test results in phase 2 showed a better correlation in the wet ice condition (R = 0.95) compared to the dry ice condition (R = 0.34). Finally, the rating of the footwear slip resistance based on the number of slips counted in the alternate human-centred testing methods in phase 3 was consistent with the rating by the human-centred test method (phase 2), but not the mechanical method (phase 1).