Proteases or peptidases are hydrolases that catalyze the breakdown of polypeptide chains into smaller peptide subunits. Proteases exist in all life forms, including archaea, bacteria, protozoa, insects, animals, and plants due to their vital functions in cellular processing and regulation. There are several classes of proteases in the MEROPS database based on their catalytic mechanisms. This review focuses on post-proline cleaving enzymes (PPCEs) from different peptidase families, as well as prolyl endoprotease/oligopeptidase (PEP/POP) from the serine peptidase family. To date, most PPCEs studied are of microbial and animal origins. Recently, there have been reports of plant PPCEs. The most common PEP/POP are members of the S9 family that comprise two conserved domains. The substrate-limiting β-propeller domain prevents unwanted digestion, while the α/β hydrolase catalyzes the reaction at the carboxyl-terminal of proline residues. PPCEs display preferences towards the Pro-X bonds for hydrolysis. This level of selectivity is substantial and has benefited the brewing industry, therapeutics for celiac disease by targeting proline-rich substrates, drug targets for human diseases, and proteomics analysis. Protein engineering via mutagenesis has been performed to improve heat resistance, pepsin-resistant capability, specificity, and protein turnover of PPCEs for pharmacological applications. This review aims to synthesize recent structure–function studies of PPCEs from different families of peptidases to provide insights into the molecular mechanism of prolyl cleaving activity. Despite the non-exhaustive list of PPCEs, this is the first comprehensive review to cover the biochemical properties, biological functions, and biotechnological applications of PPCEs from the diverse taxa. Peptidases, proteases, proteinases, or proteolytic enzymes break down peptide molecules, mostly by attacking peptide bonds via hydrolysis. Extensive protease studies found seven main catalytic types, which can be categorized by the nucleophiles for the proteolytic reaction with water molecules. The nucleophiles mainly comprise different amino acid residues, including aspartate, cysteine, glutamate, serine, and threonine, whilst another type contains metal ions (mostly zinc) at their active site, denoted as metalloproteases . The sevThis review focuses on post-proline cleaving enzymes (PPCEs) from different peptidase families, besides the well-known prolyl endoprotease/oligopeptidase (PEP/POP) from the serine peptidase family. The term PPCEs in this review is used to encompass other proteins with post-proline cleaving (PPC) activity. Based on the 108,005 cleavage site records of the MEROPS database, we used the “What peptidase can cleave this bond?” search function to compile a list of peptidase family showing specific post-proline cleavages and supported by literature searches. Many PPCEs can be found in the MEROPS peptidase database from 17 families with different substrates and proline-cleavage specificities . This noPost-proline proteolysis is prevalent given its importance in nature. PPCEs are involved in various biological functions in diverse taxa of organisms including microbes, fungi, animals, and plants. The biological functions described in the section below include microbial invasion through the protective barrier, pathophysiology of human diseases, and plant nutrient uptake/mobilization, growth, and development. Apart from proteolysis, PPCEs can affect the biological system through protein–protein interactions (PPIs), such as the acceleration of α-synuclein aggregation that can lead to Parkinson’s disease . Hence, The purpose of this review is to discuss the recent findings on the structure-function analysis of PPCEs to provide insights on the molecular mechanism of PPC activity for further investigations. The biochemical properties, such as substrate specificity and optimal conditions of enzymatic activity were summarized for both the native and recombinant PPCEs from the recent experimental findings. Lastly, the utilization of PPCEs for different biotechnological applications in the food/beer brewing industry, pharmaceuticals/therapeutics, and proteomics will be described with efforts in protein engineering. Sphingomonas capsulate (1.8 Å resolution) and Myxococcus xanthus (1.5 Å resolution) [Protein structures of PPCEs have been studied across different taxa including bacteria, fungi, animals, and plants . In thisolution) . Based on the sequence alignments and structural models, POP proteins from the MEROPS S9 family have two conserved domains . The C-tArabidopsis thaliana, Homo sapiens, and Sus scrofa (porcine) showed that each POP possesses a unique affinity toward different sizes of ligands despite high sequence and structural similarities [PEP/POP from different species have different β-propeller sizes, thus conferring specificity to different sizes of substrates targeting proline residues that are usually resistant to protein cleavage by other peptidases ,77. For larities . Besideslarities , whereaslarities . The difAspergillus niger PEP (AN-PEP) structure solved through an x-ray crystallography [There is a recent report of the lography . AN-PEP Another family of PPCEs is the prolyl aminopeptidase (PAP) from the peptidase family S33, which can be found in apricot seeds  and cabbOn the other hand, a protease from the carnivorous tropical pitcher plants known as neprosin with PPC activity was classified as an unknown peptidase family (U74). There is a recent neprosin structure–function analysis based on the AlphaFold2 modeling that suggests neprosin belongs to the glutamic peptidase family with two glutamic acid residues as the catalytic dyad . Unlike In comparison, all the PPCEs from family S9, S28, S33, and neprosin have a two-domains structure. The α/β hydrolase domain and neprosin domain act as the catalytic domain, whereas the second domain  appears to be involved in other functions such as substrate size limitation in S28 and S33 PEP, potential protein–protein interactions for S9 POP , and enzPPC activity has also been reported in a few members of zinc metallopeptidase families . Peptidyl-dipeptidase ANCE (M02.003), angiotensin-converting enzyme-2, ACE2 (M02.006), and neurolysin (M03.002) comprise two domains annotated as zinc metallopeptidase domain I and domain II ,66,83. AMetallopeptidase M12 BmooMPalpha-I  with a molecular mass of 22.6 kDa has conserved features of P-I class snake venom metallopeptidases, namely the five-stranded β-sheet, four long helices, and a short α-helix near N-terminal stabilized by three disulfide bridges . The zinSphingomonas capsulate PEP, as it failed to digest the 33-mer substrate (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF) effectively with a diameter larger than the 4 Å channel formed by the β-propeller domain. However, this theory cannot explain why S9 PEP from Myxococcus xanthus and Flavobacterium meningosepticum can digest the 33-mer substrate in the same study. Later, it is proposed that substrate binding with the β-propeller domain induces conformation changes, causing the domain to move and expose the active site. This theory is supported by the open state of Sphingomonas capsulate PEP structure, where an opening of 30 Å is observed between the α/β hydrolase and β-propeller domain [Typically, PEP/POP from the clan SC of family S9 comprises a catalytic triad with the following order, Ser-Asp-His with the catalytic Ser lies in between the Gly-X-Ser-X-Gly motif . Howeverr domain .Aeromonus punctata PEP and proposed an induced-fit mechanism as the conformational changes in the PEP can be induced by adding substrate and inhibitor [Pyrococcus furiosus POP (S09.002) shows that the conformational change is modulated by the “latching loop” mechanism and is essential for the catalysis mechanism of POP, especially for the loop containing catalytic histidine residue (H592) [Another study obtained an open state of nhibitor . The opee (H592) . The conFor peptidase family S28, it is suggested that the difference in the catalytic pocket located between α/β hydrolase and the SKS domain could confer substrate specificity based on the recent AN-PEP crystal structure . The catSerratia marcescens (S33.001) has a similar α/β hydrolase domain and another smaller helical domain made up of six helices, which are smaller than the SKS domain on S28. The smaller domain of prolyl aminopeptidase is believed to contribute to the exopeptidase specificity of S33 prolyl protease as the specificity hydrophobic pocket is formed on the helical domain [Serratia marcescens concur that residues Phe139, Tyr149, and Glu204 play an important role in the substrate recognition [Different from S9 and S28 families, the S33 family has exopeptidase properties whereby it cleaves the proline residues at the N-terminal of a peptide. Prolyl aminopeptidase (PAP) from l domain . Two mutognition ,87. Phe1ognition . Substraognition . The catOther than the serine peptidases, a glutamic peptidase was reported with PPC activity and a catalytic dyad comprising two catalytic glutamic acid residues . In silipNA (ZGPpNA) and other chromogenic substrates are commonly used [pNA (p-nitroanilide) or βNA (β-naphthylamide), which can be detected spectrophotometrically at a wavelength of 410 nm and 520 nm, respectively. Due to their specificity, these chromogenic substrates are often used in the functional characterization and activity measurement of PPCEs. Furthermore, proline-rich gliadin is also a common substrate to investigate the capability of certain PPCEs in gluten detoxification. The fragments of gliadin hydrolysates can be detected through ELISA, Western blot, or immunoblot with specific antibodies. To ascertain the protein cleavage sites, a liquid chromatography–mass spectrometry (LC-MS) approach can be taken to identify the digested peptide sequences. The native  and reconly used . PPCEs spNA peptide at the excitation (340 nm) and emission wavelengths (410 nm) [Specific amino acid preferences of targeted substrate sequences are curated in the MEROPS database with a collection of cleavage sites . The sub(410 nm) . In this(410 nm) . Meiothermus ruber H328, shows a stringent preference of residues next to proline but a greater flexibility in preferences of the second and third residues near proline [PPCEs can hydrolyze proteins with different preferences of amino acids adjacent to the proline residues. For example, the recombinant PEP from a Gram-negative thermophile,  proline . In a st proline . Both ca proline . TherefoEurygaster integriceps, Haliotis discus, Sus sucrofa, Porphyromonas gingivalis, and Nepenthes × ventrata  has a unique transpeptidation activity that catalyzes macrocyclization of a 25-mer peptide to form a monocyclic octapeptide [Aspergillus oryzae (S28.004) exhibits a similar PPC activity at an optimal pH of 2.5 with the capability of digesting much larger substrates such as intact casein [In addition to the PPC activity that cleaves at two conserved proline residues of α-amanitin pro-peptide, POP-B from apeptide . A PEP ft casein . Like S2t casein . Prolyl t casein . All PPCFlavobacterium meningosepticum and Serratia marcescens (S33.001), could be involved in pathogenicity by breaking down peptides rich in proline and hydroxyproline such as collagen [Mycobacterium tuberculosis that can stimulate the secretion of proinflammatory cytokines by peritoneal macrophage and lead to inflammatory response during M. tuberculosis infection [Galerina marginata (S09.077) from the same family is required for the maturation of α-amanitin, a toxin that is responsible for the fatal mushroom poisoning [Aspergillus sp. with different biochemical properties than the other paralogs from gene duplication, which functions together with other secreted aminopeptidases and carboxypeptidases to digest large peptides for uptake [Many PPCEs that have been purified and characterized are from microbes. The strawberry mottle virus glutamic peptidase unit (G03.001) can cleave at P↓AFP sites of the coat protein domain in the polyprotein and is hypothesized to be involved in the regulatory step of controlling viral RNA encapsidation . Prolinecollagen . Rv0457cnfection . Besidesoisoning . There ir uptake . Trypanosoma cruzi and T. brucei are involved in the pathophysiology of Chagas disease and human African trypanosomiasis, respectively [Trypanosoma spp. attached to the extracellular matrix (ECM) releases POPs that degrade collagen. The entry of Trypanosoma spp. with degraded collagen peptides signal host leukocytes to release matrix metalloproteases (MMPs) that further break down the ECM barrier. Ultimately, this allows the parasite to break through protective barriers such as ECM or the blood–brain barrier in causing diseases. These examples demonstrate that microbial and protozoa PPCEs have diverse biological roles, many of which are important in pathogenicity.On the other hand, the POP zoomastigote (S09.033) from the unicellular parasitic flagellate protozoa ectively . The TryPPCEs have been associated with diverse physiological roles in animals, especially humans. In mice, POP (S09.001) was highly expressed in the brain, kidney, testis, and thymus, with the kidney having the least enzymatic activity . MoreovePPCEs appear to play an important role in memory and learning as well as cognitive and behavioral development. For example, alterations of a POP and dipeptidyl-peptidase IV  levels in different brain parts were observed in mice of different genders administered with DPP-IV inhibitor . DPP-IV Other than the localization of POP in the brain, the distribution of POP in the human brain resembled that of inositol 1,4,5-triphosphate (IP3) receptors, which supports the theory that POP plays a role in movement regulation, cognition, and IP3 signaling . High POAltered POP activities were found in patients suffering from cognitive diseases and other health implications. Increased POP activity is associated with inflammation  as well ace2 gene knock-out experiments, disruption in mice causes severely impaired heart function whereas the disruption of ace2 homolog in Drosophila embryo resulted in a serious defect in the heart morphogenesis [On the other hand, peptidyl-dipeptidase ANCE (M02.003) is involved in the renin-angiotensin-aldosterone system (RAAS) that regulates blood pressure by hydrolyzing angiotensin I into angiotensin II (vasoconstrictor) and its physiological substrate bradykinin, which is a vasodilator . Moreoveogenesis . Additioogenesis , as showogenesis . Lastly, neurolysin with PPC activity is widely distributed in the mammalian tissues and can be found in different subcellular locations, in cytosolic, membrane-bound forms, or with mitochondrial targeting sequences . It is aDaucus carota) from the screening of over 40 vegetables and fruits, including spinach, carrot, pumpkin, soybean, green onion, and parsley for PEP activity [The first plant PEP was isolated from carrot  were found to be differentially expressed in two closely related Coffea arabica varieties, cultivars “Tall Mokka” and “Typica” [Vigna radiata suggests that DPP-II breakdown protein storage in seed into free amino acid during the germination period [Arabidopsis thaliana resulted in a faster flowering process and the production of more siliques [POP genes , Triticum durum (UAS-428), and Triticum dicoccum (DDK-1025) for proteolytic degradation and shown to have 99.95% less gluten content than the control pasta. The sensory evaluation test showed that the quality of the treated wheat flour is comparable to the control pasta, despite having a brownish color [PPCEs have many uses in industry. For example, PEP enzymes are useful for the gluten-free food processing industry for people with gluten intolerance, such as celiac disease. Celiac disease occurs in individuals with HLA-DQ2 or HLA-DQ8 genetic predisposition, where the ingestion of gluten can trigger a T-cell immune response in the small intestine . Gluten sh color . Hence, Furthermore, to test the efficiency of AN-PEP gluten degradation in a randomized placebo-controlled crossover study, 18 celiac disease patients were given a porridge containing 0.5 g gluten together with two tablets either containing a high or low dose of AN-PEP, or a placebo. In a meal physiological setting, samples were then taken from the stomach and duodenal content to calculate the gluten content. The results showed over a 50% reduction in gluten content in both stomach and duodenum as compared to the placebo. Furthermore, gluten degradation was shown to be more apparent in the stomach compared to before it entered the duodenum, showing AN-PEP as a potent protease in degrading gluten content when provided in a meal setting .Another study showed that AN-PEP degrades gluten in a dynamic system (TIM) that closely mimics the human gastrointestinal tract . Gluten Sphingomonas capsulate and Myxococcus xanthus as oral supplements for gluten degradation, but these proteases failed to detoxify gluten after irreversibly inactivated by pepsin and acidic pH in the stomach [Nepenthes × ventrata has demonstrated the capability to digest gliadin at a higher potency than AN-PEP [There are also efforts to utilize POP from  stomach . Instead stomach . On the n AN-PEP . This shLactobacillus helveticus WSU19 with PPC activity can effectively debitter aged cheddar cheese. The oligopeptidase O2 was experimentally proven to cleave three sites after Pro196, Pro200, and Pro206 for the complete digestion of the bitter β-casein (f193–209) {Chen, 2003 #17}.Meanwhile, PEP can reduce the hordein content in beer that may interact with polyphenol causing cloudy precipitation (chill-haze) during storage . One altA study also showed that barley treated with AN-PEP for five days during germination can reduce beer hordein content by 46% compared to the control when tested using an R5 antibody-based competitive enzyme-linked immunosorbent assay . In theoBerberis vulgaris, flavonoid baicalein from Scutellaria baicalensis Georgi, and alkaloid californidine from Eschscholzia californica [Due to the important functions of certain PEP/POP in human cognitive diseases (described in 3.2), understanding the mechanism of PPCEs is useful for the design of drugs or enzyme inhibitors as therapeutics. PEP inhibitors can be categorized broadly into natural and synthetic inhibitors. Natural PEP inhibitors can be found in traditional medicinal herbs, such as isoquinoline alkaloid berberine from ifornica . Berberiifornica . The curifornica . For exaifornica . Notablyifornica , colocalifornica , the inhifornica , and intifornica .Aside from PEP/POP, metallopeptidase M3 neurolysin (M03.002) with the ability to cleave multiple types of neuropeptides hinted at its potential as a single therapeutic target for multiple diseases. Hence, neurolysin and POP are currently being studied systematically as potential therapeutic targets to cure human brain diseases . MoreoveTrypanosoma brucei and T. cruzi are currently in progress to combat Chagas diseases and human African trypanosomiasis {Bastos, 2005 #58}. A recent study has shown that Tc80 POP from T. cruzi can be used as an antigen for the development of an effective vaccine against T. cruzi infection by reducing the parasite burden and tissue damage induced in the vaccinated mice [Other than human PPCEs, rational drug designs that target the POP of ted mice . The assPPCEs with prolyl cleaving activity is different from other peptidases such as trypsin that is commonly used in the MS-based proteomics . ConventPPCEs can aid in the generation of peptide fragments with a better grouping of PTM. This is due to the nature of PPCEs, which cleave after proline can disrupt protein secondary structures and are usually unmodified. However, not all PPCEs are suitable for the task due to their substrate size limitation and specificity. For instance, POPs are unable to cleave peptides larger than 30 residues. AN-PEP has a cleavage specificity outside proline and alanine while having broader substrate recognition. The activity of AN-PEP for MS is comparable to that of conventionally used pepsin with an equivalent number of peptides generated by AN-PEP at a lower concentration . MoreoveN. × ventrata was used to digest 1251 HeLa cell proteins for a proteomics study [Neprosin with PPC activity was applied in the MS analysis of histone modifications due to its selective prolyl endoprotease nature as compared to the conventional trypsin-based methods to study the information of the histones H3 and H4 . Neprosics study , which sStenotrophomonas maltophilia prolyl endopeptidase (SmPEP) improves the enzymatic activity on synthetic peptides. The introduction of mutants on these specific residues weakens the inter-domain interaction of the enzymes to reduce the restriction of bigger substrates to enter the active site [Aspergillus oryzae [Studies to improve the activity of PEP enzymes have been performed through protein engineering. For example, PEP mutagenesis on residues F263A and E184G on the β-propeller domain of the novel ive site . The incs oryzae . Mutagens oryzae .Flavobacterium meningosepticum (Fm-POP) that was expressed in the wheat endosperm showed the ability to reduce gluten content under gastrointestinal conditions with a higher ability to tolerate high temperatures over 37 °C up to 90 °C [The amino acid mutation (Glu289Gly) of a human recombinant PEP prolongs the half-life of the enzyme in phosphate buffer at 37 °C to be used for in vivo Antibody-Directed Enzyme Prodrug Therapy (ADEPT) . The proto 90 °C . Gluten to 90 °C . Hence, Sphingomonas capsulate showed the improvement in enzyme activity when simulated under gastric conditions [Moreover, the co-administration of cysteine endoprotease EP-B2 with improved mutagenesis variants of PEP from nditions . The cysnditions . The PEPnditions . This ennditions . HoweverThis review explores the structure–function studies of PPCEs from different taxa and highlights many of their biological functions. PPCEs from family S9, S28, S33, and neprosin shared a common two-domain structure but with different catalytic and non-catalytic domains in the control of substrate binding and post-proline cleaving activity. Meanwhile, the zinc metallopeptidase families show diverse structures, such as that of M2 family with two metallopeptidase domains sandwiching the active channel with a bound zinc ion, which is different from the M12 family with N-terminal and C-terminal domains. PPCEs found in diverse organisms are useful in various ways given their capability to degrade proline-rich proteins. Moreover, enzyme characterization studies for both the native and recombinant PPCEs from various species suggest a promising potential for PPCEs to be applied according to their optimum conditions for various purposes. This review provides examples of extensive PPCE biotechnological applications in the beer-brewing industry, as potential therapeutic agents for individuals intolerant to gluten, as drug targets for various diseases, and contribute to proteomics analysis. Furthermore, inhibitors of PPCEs can be applied to type 2 diabetes treatment, memory learning enhancement, and prodrug cancer treatment. Many investigations have been conducted to find the ideal enzyme that can degrade gluten in small doses under gastric conditions, mostly focusing on microbial enzymes. More studies of PPCEs are needed in plants to understand their physiological roles for beneficial agricultural applications, such as nutrient mobilization and stress resistance. Hopefully, further protein engineering and structure-function studies with the aid of ab initio structural prediction will uncover more useful PPCEs for new biotechnological applications.


How to promote employees’ knowledge-sharing behaviors has become a focus of managers and researchers. Based on the theory of relative deprivation, this study explored the mechanism of organizational procedural justice on employees’ intra-team knowledge sharing, as well as the mediating role of relative deprivation and the moderating role of group identification. A path analysis was conducted on 416 valid questionnaire data, and the results revealed that: (1) Procedural justice has a positive effect on intra-team knowledge sharing; (2) Both group relative deprivation and individual relative deprivation play a mediating role between procedural justice and intra-team knowledge sharing, but they have opposite effects. Procedural justice reduces both group relative deprivation and individual relative deprivation, but individual relative deprivation decreases employees’ intra-team knowledge sharing, while group relative deprivation increases it. (3) Group identification has an enhancing moderating effect on the relationship between group relative deprivation and intra-team knowledge sharing, while the moderating effect on the relationship between individual relative deprivation and intra-team knowledge sharing is not significant. Therefore, enterprises should make procedures such as performance appraisal and salary allocation justify and transparent to reduce individual relative deprivation, but should moderately trigger group relative deprivation flexibly according to the situation, while enhancing employee group identification through cultural construction. In a knowledge-based economy, the ability to compete among organizations is increasingly dependent on intangible assets such as employee expertise and experience skills within the organization . EmployeKnowledge sharing is a fundamental behavior to create and apply knowledge in organizations , which iIn addition, relative deprivation theory states that relative deprivation is a link between the external environment and individual behavior, and individuals assess the external environment which they are in through social comparison, which leads to a certain degree of relative deprivation that affects their behavior . ResearcAccording to social identity theory, group identity is a part of an individual self-concept , and indIn summary, based on relative deprivation theory and social identity theory, this study established a model with relative deprivation as a mediating variable and group identification as a moderating variable to explore the influence of procedural justice on employees’ intra-team knowledge sharing, to enrich the understanding of the influence mechanism of procedural justice on employees’ knowledge-sharing behavior, and provide a reference for organizations to improve corporate allocation procedures, enhance employees’ group identification, and promote employees’ intra-team knowledge sharing.Procedural justice refers to employees’ perceived fairness about the processes and methods used in reward and other decisions , and it Hypothesis 1: Procedural justice has a positive impact on intra-team knowledge-sharing behavior.Relative deprivation is the subjective cognition and emotional experience that individuals or groups perceive that they are at a disadvantage through horizontal or vertical comparison with the reference group, which leads to negative emotions such as anger and resentment . It is aHypothesis 2: Procedural justice has a negative effect on individual relative deprivation.Hypothesis 3: Procedural justice has a negative effect on group relative deprivation.Because individual and group deprivation often occur simultaneously , there aTherefore, this study argues that employees will be more inclined to reduce knowledge-sharing behaviors within their teams when they experience a sense of individual relative deprivation.Hypothesis 4: Individual relative deprivation plays a mediating role between procedural justice and intra-team knowledge sharing. Specifically, through the mediation of individual relative deprivation, procedural justice has a positive impact on intra-team knowledge sharing.Group relative deprivation is often associated with group-based responses and can predict collective action support . IndividHypothesis 5: Group relative deprivation mediates the relationship between procedural justice and intra-team knowledge sharing. Specifically, through the mediation of group relative deprivation, procedural justice has a negative impact on intra-team knowledge sharing.Group identification means that an individual recognizes that he belongs to a specific social group, and also recognizes the emotional and value significance brought to him as a member of the group . It enhaHypothesis 6: Group identification has a weakening effect on the relationship between individual relative deprivation and intra-team knowledge sharing. That is, the higher the individual’s group identification, the smaller the negative effect of individual relative deprivation on intra-team knowledge sharing.Research confirmed that the higher the group member’s identification with the group, the more one can experience the negative emotions caused by relative deprivation . IndividHypothesis 7: Group identification plays an enhancing role in the relationship between group relative deprivation and intra-team knowledge sharing. That is, the higher the individual group identification, the greater the positive effect of group relative deprivation on intra-team knowledge sharing.The participants were mainly knowledge-based employees in institutions, administrative organs, and various private enterprises in Hangzhou and other Chinese cities. Suitable participants were first selected relying on interpersonal resources, and then, the initial participants selected appropriate research objects in their organizations. Six hundred on-site paper questionnaires were distributed, 455 questionnaires were returned, and 416 of which were valid. Males accounted for 42.5% and females 57.5%; the average age was 41.03 years old, and the standard deviation was 8.149; 90.4% of subjects have a college or bachelor’s degree, and 6.7% have a master’s or doctoral degree.The details of the sample data are shown in Procedural justice was measured by the procedural justice dimension of the organizational justice scale developed by Individual relative deprivation was measured by a scale developed by Group relative deprivation was measured by a scale developed by Intra-team knowledge sharing was measured by scale developed by Group identification was measured by scale developed by All scales were scored on a five-point Likert scale, with 1–5 indicating “strongly disagree” to “strongly agree,” respectively. Control variables included gender, age, education, and length of service in the organization.Spss25.0 was used for reliability test, common method bias test, correlation analysis and hierarchical regression analysis to test the reliability of the scales and the relationships between variables, with a p value smaller than 0.05 considered a statistically significant difference. And to further estimate the model, this study adopted the Bootstrap method with 5,000 samples in SPSS 25.0.First, the reliability of scales was analyzed by SPSS25.0, and the Cronbach’s alpha of procedural justice, individual relative deprivation, group relative deprivation, knowledge sharing, and group identification scales was 0.872, 0.818, 0.872, 0.867, and 0.890, which showed scales selected had good reliability.2/dfX = 5.914, CFI = 0.797, TLI = 0.766, SRMR = 0.071, RMESA = 0.109) of the five-factor model was significantly better than that of the other models.Confirmatory factor analysis was performed on all items of variables using MPLUS7.0 to test the discriminant validity between variables. This study compared the fit of models with one to five factors. The four factors model took individual relative deprivation and group relative deprivation as one factor; the three factors model took individual relative deprivation, group relative deprivation, and group identification as one factor; the two factors model took individual relative deprivation, group relative deprivation, group identification, and knowledge sharing as one factor. The fitting index , but the model could not fit adding the method factor. Both tests showed that there was no serious common method bias in this data.Given that all data were filled in by participants at one time, Harman’s single-factor test was used, and it showed the percentage of variance explained by the first factor was 27.27%, which was lower than 40%. Furthermore, the method factor was added as a global factor on the basis of the five-factor model. The five-factor model fitted well , and significantly negatively correlated with individual relative deprivation  and group relative deprivation ; individual relative deprivation is significantly negatively correlated with intra-team knowledge sharing ; and group relative deprivation is significant positive correlated with intra-team knowledge sharing . The hypotheses were initially tested.β = 0.155, p < 0.001). Hypothesis 1 was verified.As shown in p < 0.001), so hypothesis 2 was verified. In M3, only the control variables are added to the regression equation. To explore the relationship between procedural justice and group relative deprivation, based on M3, procedural justice was added to obtain M4. In M4, procedural justice has a significant negative effect on group relative deprivation , so hypothesis 3 was verified.In M1, only the control variables are added to the regression equation. To explore the relationship between procedural justice and individual relative deprivation, based on M1, procedural justice was added to obtain M2. In M2, procedural justice has a significant negative effect on individual relative deprivation , and group relative deprivation has a significant positive effect on intra-team knowledge sharing . It indicated that individual deprivation and group deprivation mediate the relationship between organizational procedural justice and intra-team knowledge sharing. Hypotheses 4 and 5 were verified.In addition, based on M6, individual relative deprivation and group relative deprivation were added to obtain M7. In M7, individual relative deprivation has a significant negative effect on intra-team knowledge sharing . It indicates that group identification moderates the relationship between individual relative deprivation and intra-team knowledge sharing, and group deprivation and intra-team knowledge sharing. Hypotheses 6 and 7 were verified.To explore the moderating effect of group identification,the interaction term of individual relative deprivation and group identification and group relative deprivation and group identification was constructed.On the basis of M7, the interaction term was added to obtain M8. In M8, both interaction terms have significant positive effects on intra-team knowledge sharing  and individual relative deprivation has a significant mediation between procedural justice and intra-team knowledge sharing . Hypothesis 4 was again tested. Also, the mediation group relative deprivation between procedural justice and intra-team knowledge sharing is significant . Hypothesis 5 was again verified. Thus, it showed that procedural justice not only directly and positively affects intra-team knowledge sharing, but also enhances it by reducing individual relative deprivation while also reduces it by reducing group deprivation.To further estimate the model, this study adopted the Bootstrap method with 5,000 samples in SPSS 25.0. According to different mediation paths, two model 14 tests are performed and results showed that procedural justice has a significant direct positive effect on intra-team knowledge sharing (β = 0.075 p < 0.009), indicating that group identification plays a moderating role between group relative deprivation and intra-team knowledge sharing, but it does not moderate the relationship between individual relative deprivation and intra-team knowledge sharing. Hypothesis 7 was verified and hypothesis 6 was not verified.In addition, the interaction between group relative deprivation and group identification has a significant positive effect on intra-team knowledge sharing  and the Social Science Foundation of Jiangxi Province (no. 22JY23).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Neurodegenerative diseases are currently incurable. Numerous experimental data accumulated over the past fifty years have brought us closer to understanding the molecular and cell mechanisms responsible for their development. However, these data are not enough for a complete understanding of the genesis of these diseases, nor to suggest treatment methods. It turns out that many cellular pathologies developing during neurodegeneration coincide from disease to disease. These observations give hope to finding a common intracellular target(s) and to offering a universal method of treatment. In this review, we attempt to analyze data on similar cellular disorders among neurodegenerative diseases in general, and polyglutamine neurodegenerative diseases in particular, focusing on the interaction of various proteins involved in the development of neurodegenerative diseases with various cellular organelles. The main purposes of this review are: (1) to outline the spectrum of common intracellular pathologies and to answer the question of whether it is possible to find potential universal target(s) for therapeutic intervention; (2) to identify specific intracellular pathologies and to speculate about a possible general approach for their treatment. Neurodegenerative diseases are an extensive group of human pathologies of various genesis, among which both hereditary diseases and pathologies developed de novo are described. These diseases develop over decades, so they are more common in older people. In many countries of the world in recent decades, especially in countries with developed economies, in parallel with the increase in average life expectancy there has been an increase in the number of neurodegenerative diseases. They have become one of the main problems that worsen the quality of life in old age. Despite the difference in clinical manifestations, the development of neurodegenerative diseases may be based on similar molecular and/or cellular mechanisms. Considerable attention has been paid to the elucidation of these mechanisms in the last fifty years, and significant progress has been made in understanding the causes of their occurrence at the molecular, cellular and organic levels. However, this knowledge is still insufficient not only for the prevention, but also for the treatment of neurodegenerative diseases. In addition, if we do not talk about hereditary or genetically determined pathologies, there is also a problem of diagnosis and early detection of sporadically developing neurodegenerations.The most common and actively researched neurodegenerative diseases are Alzheimer’s (AD) and Parkinson’s (PD) diseases. There is a wide range of less common pathologies, including hereditary causes. The classification of these diseases can be based on various principles: by clinical manifestations , by anatomical features (atrophy of various brain parts), as well as by the nature of molecular disorders and cellular pathologies underlying the disease development ,2. ClassAt the moment, it is known that the basis of neurodegenerations are proteinopathies, disorders in the structure and function of various proteins that lead to their aggregation and toxic effects on cells. The most common neurodegenerative proteinopathies are amyloidosis (amyloid extracellular plaques in AD), tauopathy (various dementias), α-synucleopathy (Lowy bodies in PD), prionopathy, and TDP-43 proteinopathy ). Among hereditary neuropathologies, a group of polyglutamine diseases can be distinguished caused by the repetition of the cytosine-adenine-guanine (CAG) trinucleotide in the coding segment of certain genes, which leads to the appearance of mutant proteins. Huntington’s disease (HD) is one of the best studied polyglutamine diseases .In addition, it is known that with the development of some neurodegenerative diseases, the accumulation of several different proteins is observed. Simultaneous accumulation of tau protein and amyloid-β (Aβ) protein in AD , Aβ and The greatest difficulty for the timely clinical diagnosis of neurodegenerative diseases is their sporadic forms. They are associated with a variety of genetic factors and environmental influences. The causes of their occurrence remain largely unknown. In general, the development of sporadic forms of neurodegenerative diseases leads to the same cellular pathologies as in hereditary forms, including the accumulation of toxic proteins. Modern scientific research is aimed at identifying specific biomarkers that would select potential patients in the early stages of the disease before the first symptoms appear. Determining the risk group is of great importance, because now methods have been developed to slow down the development of the disease, and the earlier such therapy begins, the better for the patient. For instance, in a number of publications devoted to this problem, an increased level of methylation of risk genes associated with the development of neurodegenerative diseases is discussed ,10,11.In all neurodegenerative diseases, similar cellular pathologies are observed, among which are listed the accumulation of protein aggregates, violation of protein proteolysis, mitochondrial disorders, transport disorders (including neurotransmitters), changes in protein expression, and others. A question naturally arises: which processes accompanying neurodegeneration are the consequence of the cellular pathology development, and which are the cause? Neurodegenerations often develop according to a similar scenario at the tissue and organizational level. However, from a practical point of view, it is important to understand whether it is possible to identify intracellular disorders responsible for the development of several neurodegenerative diseases. This issue is often the subject of discussion during scientific conferences and round tables, and the purpose of such discussions is to try to identify, at least theoretically, intracellular targets for creating a general targeted therapy. Identifying possible targets may provide a key to the neurodegeneration’s treatment. Nevertheless, the fundamental question remains open: whether it is possible to find a common approach to the treatment of all (or at least a group of) neurodegenerative diseases, or an individual approach is required for each disorder? In this review we will try to answer this question by considering both widespread intracellular pathologies characteristic of many neurodegenerative diseases and unique ones peculiar only to a certain disease.Most neurodegenerative diseases are proteinopathies that occur as a result of disorders in the structure and function of certain proteins. These defective proteins disorganize the normal function of intracellular organelles, which ultimately leads to toxic damage to nerve cells.AD is one of the most common and severe neurodegenerative diseases. It is considered to be proven that AD is amyloidosis by nature; the disease is characterized by the formation of extracellular amyloid plaques in the nervous tissue. It is believed that their accumulation leads to the development of neurodegenerative disorders in patients, however (and we cannot fail to note this), these long-established ideas can be revised .It has been shown that Aβ is formed by the cleavage of Aβ precursor protein (APP) by β- and γ-secretase. A number of β-amyloid peptides with a length of 39 to 43 amino acid residues are formed, the hydrophobic nature of which promotes self-aggregation and neurotoxicity ,14. A seAPP is an expressed transmembrane protein  thatAβ oligomers formed during APP cleavage, can form pore-like structures with channel activity that leads to damage to synapses ,27. In aMany studies in the field of understanding the functions of Aβ appear almost daily. Nevertheless, we still have little idea of how to look for approaches to the qualitative therapy of neurodegenerative diseases associated with amyloid accumulation. Perhaps it is after the discovery of native APP functions that an understanding of the occurrence mechanisms, timely diagnosis and prevention of these diseases will appear.The cause of some dementias is considered to be tauopathy. Tau protein was first isolated from cells together with tubulin. It turned out that this protein can stimulate the assembly of microtubules . NormallThe tau protein, originally defined as MAP (microtubule-associated protein), has over time been discovered to possess many alternative functions. It has been shown to bind to the p150 subunit of the dynactin complex, which provides the interaction of dynein with cargo . Some stThe wide range of tau protein interactions with other proteins and cellular structures presents ample opportunities for research activities that may lead to the discovery of new treatments for neurodegenerative processes.α-Synucleinopathies are the cause of dementia and, in particular, PD. In its native state, α-synuclein is a soluble protein consisting of 140 amino acid residues. Due to the central hydrophobic region, α-synuclein has a high tendency for aggregation. It forms an intermediate ring structure called a protofibrill, which eventually transforms into insoluble polymers or fibrils . These i0-F1-ATPsynthase also affects ATP production [Despite many studies, the exact function of α-synuclein remains unclear. Initially, this protein was detected in presynaptic endings , howeveroduction .From all of the above, it follows that α-synuclein performs a number of important functions in the cell and affects various cellular organelles. However, researchers have yet to determine its main role in normal cellular metabolism.Sc) is the most studied mammalian prion. It is formed from the normal cellular prion protein (PrPC) by either spontaneous conversion or dominant mutations in the PRNP gene [Sc [Sc.Prions (PrP) are unique pathogenic proteins that have the property of self-replication when acquiring an alternative conformation. Scrapie prion protein  is a highly conserved heterogeneous nuclear ribonucleoprotein that controls the transcription, splicing and RNA stability of certain genes (reviewed in ). This pCurrently, a number of neurodegenerative diseases have been described that have hereditary genetically determined causes, for example, polyglutamine diseases. This group of polyglutamine diseases, caused by the expansion of trinucleotide cytosine–adenine–guanine (CAG), repeats in the coding segment of certain genes. Such expansion leads to the appearance of mRNA with abnormally long repetitive CAG triplets and respective proteins with polyglutamine tracts in the cells. Today, a total of nine polyglutamine disorders (spinocerebellar ataxias (SCA) types 1, 2, 3, 6, 7, 17 ,105,106;HTT gene and the consequence of this mutation and defective huntingtin (HTT) expression, is the death of neurons, mostly striatal neurons. HD is the most studied among the polyglutamine neurodegenerative diseases. Its development is associated with a mutation in the It has been shown that HTT is associated with numerous partner proteins, interacts with many intracellular structures and is involved in many cellular processes; therefore, HD is a very perspective model for the investigation.HTT gene. At the same time, there is no striatum atrophy characteristic of HD, although such patients have a family history of HD cases with typical symptoms for this disease [Modern studies have been conducted, describing ALS patients with a pathological increase in CAG repeats in the  disease ,109. Mea disease . On the  disease . These dHTT gene expresses in all tissues and organs in humans and mice, but HTT expression level is the highest in neural tissue [HTT is a major cellular protein. As one of the research teams aptly said, huntingtin is ‘here, there, everywhere’ . The HTTl tissue . Autosoml tissue ,113,114.Glued dinactin subunit and the kinesin family protein KIF5C [Despite intensive investigations, the functions of both mutant (mHTT) and wild-type HTT remain not fully understood. HTT can play an active role in cell physiology, being involved in a wide range of intracellular processes, such as cell transport, endocytosis, protein degradation and other cellular and molecular processes. HTT, as well as mHTT, interacts with different cell organelles directly or through their associated proteins . It has in KIF5C ,120. HTTin KIF5C ,121,122.in KIF5C ,124. HTTin KIF5C ,126). Thin KIF5C ,128, thein KIF5C  and the in KIF5C ,131,132.According to many studies, the number of HTT partner proteins can range from 100 to 350 ,133,134.It was suggested that HTT interacting proteins are genetic modifiers in neurodegenerative disorders . These aExperimental data indicate the involvement of many cellular processes in the neurodegenerative diseases development. These processes can be both unique to one disease and characteristic of many neurodegenerations. There are many discussions in the literature about phenomena such as the accumulation of protein aggregates or toxic protein fragments, violation of protein proteolysis, mRNA toxicity, mitochondrial disorders, nuclear violations, DNA damage, changes in protein expression and disruption of cellular transport. An attempt to compare and generalize these phenomena is given in The common manifestations of neurodegenerative diseases also include activation of microglia, cytokines, reactive astrogliosis and the launch of a broad inflammatory immune response ,137. TheSummarizing numerous experimental data , we tried to determine which intracellular structures and cytophysiological processes mainly undergo pathological changes during the development of the most common neurodegenerative diseases .It transpired that in various neurodegenerative diseases on the subcellular structural level, in addition to the obvious general problems with the corresponding proteins , mitochondria most often suffer from .Mitochondrial disorders are the most frequent in neurodegenerative diseases and are observed in both hereditary and sporadic forms. Excessive formation of free radicals, decreased ATP production, and damage to mitochondrial DNA are the most common mitochondrial disfunctions. It is these injuries that often become targets for the development of therapeutic approaches , and such approaches are universal for neurodegenerative diseases. Thus, drugs aimed at maintaining mitochondrial function, for example, mitochondrial-targeted antioxidants, can be an essential part of the treatment of all neurodegenerative proteinopathy disorders.The biological aspects of the influence of proteins involved in neurodegeneration remains a subject of debate. It is known that in AD Aβ inhibits some components of the respiratory chain, thus causing mitochondrial depolarization, and also causes an increase in calcium levels, which leads to oxidative stress ,174,175.The transcription of some neurotransmitters genes is disrupted in polyglutamine diseases, and for the HTT protein, the direct effect upon gene transcription is evident. Additionally, in neurodegenerative diseases there is a violation of the transcription factors’ transport from the cytoplasm to the nucleus (reviewed in ). Most nRecent experimental data have confirmed the effectiveness of alternative splicing modulators in eliminating aberrant mHtt-induced splicing. The potential to use these modulators as a method of successful HD therapy looks very promising .Proteomic analysis in AD, PD and polyglutamine diseases demonstrates changes in the expression of proteins involved in energy metabolism, synaptic function, axon growth, chaperone activity and protein degradation (well described in review ). Such sModern medicine commonly uses exclusively symptomatic therapy, which is not able to affect the dynamics of the pathological process. Therapeutic approaches are designed to eliminate the manifestations of neurological disorders and somatic disorders that have arisen as a result of the disease development. Treatment methods include drugs that affect the neurotransmitter component  inhibitors, MAO-B inhibitors, anticholinergics, L-Dopa, dopamine agonists and MAO-B inhibitors in PD; dopamine antagonists in HD); various channel blockers in ALS and ataxia (neuroprotectors); antipsychotics; antioxidants; and sedatives and muscle relaxants for the relief of choreic symptoms in HD. The therapy used is not specific to certain forms of neurodegenerative diseases and nor have a specific point of application to the regulation mechanisms of a certain protein, as such a violation of the conformation or structure causes the disease development.As it follows from the analysis of modern research data , mitochoExamples of such approaches include trials of mitochondrial-targeted antioxidants, such as SkQ1 and MitoQ, against neurodegenerative diseases (well described in review ). TheoreIn recent years, innovative immunological, genetic engineering and molecular biological approaches to the treatment of neurodegenerative diseases have been developed in the laboratory. One of the promising ways to combat the progressive development of neuron death is the vaccination of patients against neurodegeneration proteins  , as wellDespite the theoretical validity of the innovative approaches described above, the numerous methods of treatment of neurodegenerative diseases being developed have a very significant drawback. The basis of their therapeutic concept is a more or less unique approach to the elimination of one specific molecular pathology. At the same time, the accumulated experimental data already indicate that the occurrence and progression of neurodegeneration is always associated with many different internal and external factors. Many neurodegenerative diseases may occur as a result of damage to not one, but several genes, as we see in the example of HD and ALS. In such cases, studies aimed at identifying the specific functions and unique properties of all involved neurodegeneration-associated proteins can help in the development of innovative therapies. Thus, it is undesirable to exclude an individual approach in the treatment of each disease and its subtype, but it must be combined with the general principles of therapy . Only in this case can targeted therapy give significant results in improving the condition of patients.As follows from the attempts described above to create effective strategies for the treatment of neurodegenerative diseases, their inefficiency is often associated with the problem of late detection due to the lack of early diagnostic methods. When the process of neuronal death has already started and has led to the development of atrophic disorders in the nervous system, the most modern molecular approaches come to a dead end and do not bring tangible relief to patients. Thus, the efforts of scientists should be directed to the search for biomarkers that would allow the determination of the risks of neurodegeneration in patients before the symptoms of the disease. This task is not solved at the moment, but work in this direction is underway and researchers offer a wide variety of approaches. For example, to detect AD in the early stages, the use positron emission tomography, which detects even a small accumulation of Aβ , has beeAlternative approaches to early diagnosis are also described in the literature, for example, muscle action potential measurement methods. A simple physical method of muscle action potential measurement in ALS was proposed for practical application as a biomarker for longitudinal follow-up and clinical trials in ALS .A very urgent problem of modern medicine is the study of mechanisms, and the search for new ways to compensate for damage and protect neurons in chronic neurodegenerative diseases of the central nervous system, since they are currently incurable. The methods of symptomatic treatment used in clinics make it possible to alleviate the condition of patients to some extent and slow down the development of neurodegenerative processes, but they cannot bring the disease under control or noticeably prolong the patient’s life.Despite the huge amount of research being conducted, many fundamental questions have remained unanswered until now. In particular, the native functions of many proteins participating in the neurodegenerative process and the trigger mechanisms provoking the onset of the disease are not characterized at the molecular level. That is why modern researchers’ interest in the mechanisms of the development of neurodegenerative processes is often aimed at studying the native functions of proteins, whose mutant forms are involved in the pathogenesis of neurodegenerative diseases. It is possible that here lies the key to the diagnosis and prevention of neurodegeneration in the early stages.The compilation of the available experimental data on morphological and functional pathologies of intracellular structures characteristic of the most common neurodegenerative diseases allows us to conclude that the most vulnerable cellular organelles are mitochondria . Thus, tObviously, it is impossible to offer a universal approach to the treatment of all neurodegenerative diseases. It is necessary to search for individual approaches to protect the structure and functions of specific proteins involved in the development of neurodegeneration at the cellular level. In this regard, promising are the studies conducted on the HD model, allowed to detect HTT-interacting proteins that are genetic modifiers in neurodegenerative disorders . Non-funGenetic engineering approaches aimed at editing mutant genes , strategThus, a combination of therapy aimed at maintaining mitochondrial function and any of the above innovative approaches aimed at editing mutant genes, at suppressing (by means of neurodegeneration suppressor proteins), or reducing the level of mutant proteins in the cell (for diseases that are not the result of a genetic mutation) , current


Exploring new linkage chemistry for covalent organic frameworks (COFs) provides a strong driving force to promote the development of this emerging class of crystalline porous organic materials. Herein we report a strategy to synthesize COFs with azo linkage, one of the most important functional unit in materials science but having not yet been exploited as a linkage of COFs. This strategy is developed on the basis of in situ linker exchange, by which imine-linked COFs are completely transformed into azo-linked COFs (Azo-COFs). Moreover, distinct properties of Azo-COFs from their corresponding imine-linked precursors are observed, indicating unique property of Azo-COFs. This strategy provides a useful approach to develop new linkage chemistry for COFs. It also has established a synthetic method for azo-linked COFs, which not only enriches the family of COFs but also offers a platform to explore properties and applications of this class of crystalline porous conjugated polymers. Exploring new linkage chemistry for covalent organic frameworks (COFs) provides a strong driving force to promote the development of this class porous materials. Here, the authors report a strategy to synthesize COFs with azo linkages based on an in situ linker exchange strategy which transforms imine-linked COFs into their azo-linked counterparts, and explore the unique properties of azo-linked COFs. Benefiting from their unique structural features, COFs have achieved versatile applications in many fields such as gas storage, separation, catalysis, optoelectronics, sensing, drug delivery, electrochemical applications, etc6. During the development history of COFs, exploring new linkage chemistry has always been one of research focuses, for the emergence of a new type of linkage not only enriches the structural diversity of COFs but more importantly could bring in distinctive properties and applications. Over the past dozen years, COFs with –B–O–8, –C=N–10, –B–N–11, –C=C–13, –C–N–20, –C–O–23, –Si–O–24, and –C–C–26 linkages have been achieved. However, it remains a formidable challenge in developing new linkages for COFs to further explore novel connectivity and functionality.Covalent organic frameworks (COFs) are an emerging class of crystalline porous organic materials assembled by covalently linking building blocks in sequence29, narrow bandgap30, Lewis base property31, and redox activity32. In this context, construction of azo-linked COFs (Azo-COFs) undoubtedly will lead to a novel type of crystalline porous polymers with fascinating properties. Indeed, recent theoretical calculations have revealed that Azo-COFs have great potentials in photocatalysis33. However, thus far –N=N– has not yet been reported as a linkage to construct COFs, albeit a few COFs carrying azo moieties were prepared through polycondensation of judiciously selected monomers with pre-installed –N=N– units38. In those examples the azo units do not function as linkages, but as part of building blocks. This might be attributed to lack of an appropriate reaction or condensation condition to implement azo bond into crystalline frameworks. In this contribution, a synthetic method for Azo-COFs has been established, by which –N=N– linkage has been developed for COFs. Moreover, distinct photocatalytic property between an Azo-COF and its corresponding imine-linked COF progenitor is also revealed.Azo unit (–N=N–), as a vital functional group in chemistry and materials science, has aroused wide interest because of its intriguing features such as photo-isomerization40. However, only amorphous products were produced even though various condensation conditions were screened  and COFs with different linkages43. However, so far it has been limited to the linkages already established. Whether it could be employed to explore new linkage chemistry for COFs is unclear. We envisioned that the original frameworks could serve as templates to guide the growth of new frameworks in the building block replacement process. As a result, the crystallization problem encountered in the polycondensation of monomers might be overcome. Based on this concept, we changed the synthetic method of Azo-COFs from direct polycondensation of monomers to the linker-exchange strategy. To this end, the experiment was designed to establish azo bond by using 1,4-dinitrosobenzene (DNB) to replace the linker corresponding to terephthalaldehyde (TPA) in imine-linked COFs.To achieve the synthesis of Azo-COFs, we firstly tried Mills reaction which has been widely used to construct azo bond via the reaction of amine and nitrosocompound45 and Im-COF-246) with DNB under solvothermal conditions, azo-linked COFs, termed Azo-COF-1 and Azo-COF-2, respectively, were successfully obtained  disappears in the spectrum of Azo-COF-1. Correspondingly, two peaks belonging to –N=N– moiety (1404 and 1452 cm−1) are observed in the same spectrum, suggesting successful construction of azo-linked structure47. Similar results are also found for the transformation from Im-COF-2 to Azo-COF-2  spectroscopy Fig. . The banF-2 Fig. , as illu13C cross-polarization magic angle spinning (CP-MAS) NMR spectra of the COFs indicate that the carbon signals of –C=N– linkages appear at 157 and 156 ppm for Im-COF-1 and Im-COF-2, respectively  and emergence of a peak corresponding to azo bond (1432 cm−1) after the reaction, suggesting conversion from imine to azo compound. For the transformation from Im-COF-1 to Azo-COF-1, the comparison between their spectra reveals disappearance of imine linkage (1628 cm−1) and presence of azo linkage (1441 cm−1) in the spectrum of the latter , and appearance of azo peak (1443 cm−1) were also observed . The comparisons between the PXRD patterns of the Im-COFs and Azo-COFs show high similarity in both cases Fig. g, h, inds shifted from 398.6 eV of Im-COF-1 to 399.7 eV of Azo-COF-1  provides another evidence for the transformation from imine-linked COFs to azo-linked COFs  was employed to reveal morphology of the COFs before and after the transformation. For Im-COF-1 and Azo-COF-1, similar morphology was identified by their SEM images, which showed amalgamation of flakes. However, Azo-COF-1 exhibited a smaller size domain of flakes compared with its maternal Im-COF-1. A remarkable difference in morphology was observed for Im-COF-2 and Azo-COF-2, wherein the former had a granular-aggregation morphology and the latter displayed a ribbon-like structure (Supplementary Section d6 disappeared to form a clear and transparent solution upon the addition of deuterium chloride (D2O solution), indicating a complete hydrolysis. The peaks corresponding to protons of TPA (10.06 and 8.05 ppm) and 4,4′,4″,4‴-tetraaniline (ETTA) (7.20 and 7.10 ppm) were identified after the acidic treatment tetraaniline (PTTA) in the solvent. As a result, its signals are very weak (7.80 and 7.63 ppm)  Fig. . The inc30. To check such property in Azo-COFs, UV–vis–NIR diffuse reflectance spectra (DRS) were collected for the COFs and model compound 2  was identified as the primary active species in the photocatalytic process  with dissolved O2, it could be anticipated that the CB level of Azo-COF-1 matched better with E0 (O2/·O2− = −4.17 eV vs. Vacuum) than that of Im-COF-1. This assumption was verified by the density functional theory (DFT) calculations, which indicated that Azo-COF-1 had a lower CB level than Im-COF-1 and thus favored the charge transfer process  spectra, the emission of Azo-COF-1 was significantly quenched in comparison with that of Im-COF-1, which preliminarily confirmed an efficient suppression of the electron–hole recombination in Azo-COF-1 Fig. . To gainirs Fig. . Furtheress Fig. . Since tess Fig. . Moreoveess Fig. . To furtess Fig. , 48.Fig.In summary, an approach to explore new linkage chemistry for COFs have been developed via linker exchange. Based on this approach, a synthetic method for azo-linked COFs has been realized, which leads to a complete transformation from reversible –C=N– to irreversible –N=N– in the COFs. The template effect of precursor COFs provides guidance for the construction of target COFs, through which the crystallization problem could be overcome. Benefiting from the unique property of azo linkage, the Azo-COFs exhibit distinct properties from their corresponding imine-linked counterparts. The azo-linked COFs represent a type of crystalline π-conjugated polymers, which not only enrich the linkage chemistry of COFs, but also provide a platform to explore their application as a class of semiconductors. Moreover, this work provides a useful tool to explore new types of COF linkages inaccessible by the traditional direct polycondensation of monomers, through which development of more novel COF linkages could be expected.13C-labeled Im-COF-1 was synthesized according to the same procedure using 3C-labeled TPA.The monomers 4,4′,4″,4‴-tetraaniline  and terephthalaldehyde  were added into a mixture of dioxane and 6 M HOAc  in a glass ampoule. After three freeze-pump-thaw cycles, the glass ampoule was sealed under vacuum and allowed to stand at 120 °C for 96 h. After cooling to room temperature, the resulting solid was collected by filtration, followed by immersion in dry dioxane to give Im-COF-1 as a yellow powder after filtration . 13C-labeled Im-COF-2 was synthesized according to the same procedure using 3C-labeled TPA.4,4′,4″,4‴-tetraaniline , terephthalaldehyde , and a mixture of dioxane and mesitylene  were added into a glass ampoule in sequence. Upon being sonicated for 10 min, aqueous acetic acid  was added as a catalyst. After three freeze-pump-thaw cycles, the glass ampoule was sealed under vacuum and heated at 120 °C for 72 h to afford Im-COF-2 as a yellow powder . The purification process was similar to that of Im-COF-1. 2O/glacial acetic acid  in a glass ampoule, followed by standing at 120 °C for 3 days. After cooling to ambient temperature, the mixture was filtered, and washed with aqueous NaOH solution (1 M), water, acetone, DMF, and THF in sequence. The as-obtained solid was subject to Soxhlet extraction using CHCl3 and THF, each for 12 h, respectively, from which Azo-COF-1 was obtained as a red powder . Note: The excessive yield is attributed to adsorbed guest molecules  which are hard to remove due to low solubility of DNB and strong binding ability of –N=N– units to water and acid derived from their Lewis base property. The adsorbed DNB was evidenced by XPS. An incomplete conversion was observed when the equivalence of DNB was less than 3 equiv.Im-COF-1 (10 mg) and DNB  were added into a mixture of dioxane/H2O/trifluoroacetic acid  in a glass ampoule. The following procedure was similar to that described for the synthesis of Azo-COF-1. The Azo-COF-2 was obtained as a brown powder .Im-COF-2 (10 mg) and DNB  were added into a mixture of dioxane/HNote: The excessive yield is attributed to adsorbed guest molecules  which are hard to remove due to low solubility of DNB and strong binding ability of –N=N– units to water and acid derived from their Lewis base property. The adsorbed DNB was evidenced by XPS. An incomplete conversion was observed when the equivalence of DNB was less than 4 equiv.d6 (1 mL), and DCl  was added. The mixture was stirred at 60 °C for 6 h and the resulting mixture was subjected to 1H NMR analysis.The hydrolysis experiments were performed under acidic condition following the procedure below. To a vial charged with a mixture of the COF sample (10 mg), DMSO-e versus time, where Ce and C are the concentrations of Rh B at the adsorption–desorption equilibrium and t minutes of the photocatalytic reaction, respectively.In a typical photocatalytic procedure, the photocatalyst  was dispersed in an aqueous solution of Rh B , followed by magnetically stirring in dark. After reaching adsorption-desorption equilibrium, the system was exposed to a 300 W xenon lamp with an optical cutoff filter (λ ≥ 420 nm). For the collection of UV–vis spectra, 1 mL of aliquot was extracted and centrifuged to remove the photocatalyst at the given intervals. After that, the resulting supernatant was diluted with distilled water (2.0 mL), and then analyzed with a UV–vis spectrophotometer. For comparison, the photocatalytic activity of Im-COF-1 was also assessed following the similar experimental procedure. The performance of photocatalytic degradation of Rh B catalyzed by the COFs was evaluated by plot of C/C2SO4 aqueous solution as the electrolyte. Xenon light was used as the light source. The bias voltage was set as 0.2 V.Photocurrent response measurement was performed in a three-electrode electrochemical quartz glass cell. Prior to the test, the photocatalysts (5 mg) were dispersed in 5% nafion (50 uL) and EtOH (0.45 mL) to afford a suspension, which was then dip-coated on the ITO surface and allowed to dry for 24 h at room temperature. Photocurrent measurements were conducted on a CHI660D electrochemical workstation in a standard three-electrode system with the photocatalyst composites as the working electrode, Pt foil as the counter electrode, saturated calomel electrode as the reference electrode, and 0.1 M NaThe TRPL decay curves were collected on an Edinburgh FLS1000 spectrophotometer with EPL 375 nm as the source. The TRPL decay curves could be described as the Eq. 3 package. The generalized gradient approximation with Perdew–Burke–Ernzerhof was applied for exchange-correlation energy. DFT semi-core pseudopotential core treatment was implemented for relativistic effects, with C and O calculated in the all-electron method. The double numerical plus polarization (DNP) basis set was used with a “fine” orbital cutoff. Energy, Force, and displacement convergence were 10−5 Ha, 0.002 Ha Å−1 and 0.005 Å, respectively. All self-consistent field calculations were performed with convergence criterion of 1 × 10−6 Ha with smearing.The HOMO, LUMO, and density of states were calculated by spin-polarized DFT method using the DmolSupplementary Information


Automated breast ultrasound (ABUS) is a useful choice in breast disease diagnosis. The axillary lymph node (ALN) status is crucial for predicting the clinical classification and deciding on the treatment of early-stage breast cancer (EBC) and could be the primary indicator of locoregional recurrence. We aimed to establish a prediction model using ABUS features of primary breast cancer to predict ALN status. A total of 469 lesions were divided into the axillary lymph node metastasis (ALNM) group and the no ALNM (NALNM) group. Univariate analysis and multivariate analysis were used to analyze the difference of clinical factors and ABUS features between the two groups, and a predictive model of ALNM was established. Pathological results were as the gold standard.P < 0.05). The four risk factors were used to build the predictive model, and it achieved an area under the receiver operating characteristic (ROC) curve (AUC) of 0.791 . The accuracy, sensitivity and specificity of the prediction model were 72.5%, 69.1% and 75.26%. The positive predictive value (PPV) and negative predictive value (NPV) were 66.08% and 79.93%, respectively. Distance to skin, MD, margin, shape, internal echo pattern, orientation, posterior features, and hyperechoic halo showed significant differences between stage I and stage II (P < 0.001).Ki-67, maximum diameter (MD), posterior feature shadowing or enhancement and hyperechoic halo were significant risk factors for ALNM in multivariate logistic regression analysis (ABUS features and Ki-67 can meaningfully predict ALNM in EBC and the prediction model may facilitate a more effective therapeutic schedule.The online version contains supplementary material available at 10.1186/s12885-022-10034-3. The axillary lymph node (ALN) status is crucial for predicting the clinical classification and decisions on treatment of early-stage breast cancer (EBC) and could be the primary indicator of locoregional recurrence , 2. LympAutomated breast ultrasound (ABUS), as a noninvasive and effective imaging modality, has been increasingly widely used on account of its automated volumetric scanning of the breast lesions with high frequency broadband transducers , 12. ABUThe retrospective study was approved by the Institutional Review Board of Harbin medical university cancer hospital. The need for informed consent was waived because of the retrospective nature of the cohort study.Patients with breast cancer lesions, diagnosed by surgery or biopsy specimen, between May 2019 and January 2021 were included in this study. All patients consecutively underwent ABUS before surgery or biopsy. Clinical information of patients was recorded, and ABUS examinations were performed by skilled technologists. The patient collection process is shown in Fig. The Invenia™ Automated Breast Ultrasound System  with an automated 6–14 MHz linear broadband transducer (covering volumes of 15.4 × 17.0 × 5.0 cm) was used in the study. The patients were placed similar to those with conventional US. Thus, anteroposterior, medial, and lateral orientation items of volume data were obtained, and, if required, superior and inferior orientations were performed additionally. After acquisition, all the images were sent to the workstation for review by the two experienced radiologists (with 5 years and 4 years of work experience in ABUS). ABUS imaging analysis is based on the same features as conventional ultrasound (CUS), simultaneously adding 3D analysis by generating a coronal view , 18, 19.The ER, PR, Ki-67 and P53 statuses of all patients were determined by immunohistochemical analysis, and HER-2 were determined by immunohistochemistry or fluorescent in situ hybridization (FISH). The definitions of P53, ER, PR, HER-2 status was as follows: Ki-67 status , P53 status , ER and PR status , and HER-2 status . FISH was performed to make a final determination when HER-2 status was 2 + . If the gene-to-chromosome ratio was more than 2.0, HER-2 was considered gene amplification –23. The According to the pathological results, all lesions were divided into the ALNM group and NALNM group. The ABUS features and each clinical factor between the two groups were compared by univariate analysis. The correlation between each variable and ALNM was studied by univariate analysis and multivariate analysis. According to the obtained significant results, a prediction model of ALNM was established. The specificity and sensitivity of the significant variables were drawn in ROC space, and AUCs with 95% confidence intervals (CIs) were calculated. In addition, the correlation between ABUS features and cancer staging  was studP < 0.05 was considered significant (to avoid eliminating significant variables). The variables found to be significant in the univariate analysis were included in the multivariate analysis. Logistic regression was used for ALNM prediction model development with the variables identified as significant in the univariate analysis (P < 0.05). The Youden Index was used to select the cutoff value of the predicted probability. P < 0.05 indicates that the differences were statistically significant.SAS9.4 software was used for statistical analysis. The mean ± standard deviation was used to describe measurement data conforming to the normal distribution. T test was used for comparison between groups. If data did not conform to the normal distribution, the median and quartile  were used for statistical description. Rank sum test were used for comparison between the groups. The count data were described by count and percentage, and the Chi-square test was used for comparisons between the groups. Univariate logistic regression analysis was performed and covariates with a P < 0.05). Lesions with ALNM were significantly larger than those with NALNM [2.587 ± 0.852 cm vs. 2.086 ± 0.893 cm] (P < 0.001). Lesions with shorter distances to the skin [0.628 ± 0.462 vs. 0.747 ± 0.591] and larger MDs [2.587 ± 0.852 cm vs. 2.086 ± 0.893 cm] were more prone to occur in stage II (P < 0.001).In total, there were 469 female patients in the study with 469 EBC lesions. The average age of the patients was 53.2 ± 10.05 years  and mean lesion MD was 2.276 ± 0.90 cm  as measured by ABUS. Among these lesions, 291  had NALNM, and 178  had ALNM. A total of 165 lesions  were classified as stage I, and 304  were classified as stage II. Body Mass Index (BMI) of all the patients was 23.717 ± 3.107. The comparison of clinical factors and ABUS features between EBC lesions with and without ALNM is shown in Tables P < 0.05). Lumina A was a protective factor (P < 0.001) against ALNM relative to the triple negative (TN) subtype. Echo pattern with heterogeneous/complex cystic and solid pattern was protective factor for ALNM relative to the hypoechoic pattern (P = 0.023)   Table . Based oThe p denotes the probability of ALNM with a cutoff value of 0.4424. The e denotes the natural logarithm with a value of 2.71828. The accuracy, sensitivity and specificity of the model were 72.5%, 69.1% and 75.26%, respectively. The positive predictive value (PPV) and negative predictive value (NPV) were 66.08% and 79.93%, respectively.The ROC curve was drawn and AUC was calculated Fig. . It showP < 0.001). As shown in Table P < 0.05). Lesions with circumscribed/angular or indistinct margins, irregular shape, micro calcifications, combined pattern or negative retraction phenomenon may have higher Ki-67 status. This result may provide helpful information in for the prediction of Ki-67 status. The typical cases are demonstrated in Fig. In this study, we further analyzed the correlation among MD, ABUS features and Ki-67 status. The correlation between Ki-67 and MD was statistically significant; that is, the larger the MD of breast cancer lesions was, the higher the value of Ki-67 . Breast cancer cells can migrate to the ALN via the lymphatic plexuses and network in the breast parenchyma and interstitium. The inconsistent edge of the tumors may promote tumor cell invasion into the adjacent tissues at different growth rates. This can contribute to the increase in tumor size and ALNM [Some studies reported that size larger than 2 cm was significantly related to ALNM. Lesions in this study with ALNM were significantly larger than those of NALNM . Ki-67 positive was a significant predictor of ALNM in multivariate regression analysis. As reported in previous studies, assessment of the Ki-67 index represents an easy and reliable method for evaluating cell proliferative activity in breast cancer. The rapid proliferation and invasion of tumor cells will cause larger size, irregular shape, uncircumscribed margin, heterogeneous or positive hyperechoic halo in ultrasound features of EBC [Ki-67 protein expression has been confirmed to be correlated with cell proliferation and the active phases of the cell cycle. Generally, high levels of Ki-67 expression are strongly associated with more proliferation and poor prognosis , 29, ands of EBC , 31. ConRapid proliferation, high content of collagen fibers in the interstitial tissue and invasion into the adjacent tissues also contribute to the ultrasound features of irregular shape, uncircumscribed margin and shadowing . The posA characteristic manifestation of ABUS in the coronal plane is the convergence sign of the “retraction phenomenon”. ABUS has been proven to improve early detection in dense breasts and diagnostic accuracy because of the retraction phenomenon. In addition, the retraction pattern is generally more severe in luminal A than in luminal B, HER-2-enriched and TN IDCs . The retMarital status, pregnancy and fertility status, obesity, menopause status, smoking and alcohol habit have been reported to be correlated with breast cancer. Late menopause, smoking, alcohol use and obesity can increase breast cancer risk –44. EverThe sensitivity of our prediction model was 69.1%, which was higher than the results that the sensitivity value ranged from 26.4% to 75.9% of CUS. The specificity of our study was 75.26%, which was consistent with the results ranged from 55.6% to 97.3% of CUS , 9, 46. Our prediction model showed moderate predictive efficacy with an AUC of 0.791. This result is similar to those of recent studies, which have investigated the potential value of CUS features of breast lesions in predicting ALNM with reported AUCs ranging from 0.731 to 0.848 . There wOur study has several limitations: First, due to the retrospective analysis and single-center study, our results may be biased. Second, ABUS cannot observe the Color Doppler information and ALN features, so in this study we did not investigate the relationship among color Doppler flow, ALN features and ALNM. Third, only a few patients had CUS data of tumors, so our model failed to predict ALNS with CUS.ABUS has been increasingly widely used in clinical workflows for convenient operation, excellent repeatability and adjunct screening of women with dense breasts.Although the results of the predictive model established based on ABUS cannot fundamentally change the decision-making of SLNB and the method of surgery of EBC, it can provide more clinical references. In the future, multimode methods, such as ABUS combined with CUS, contrast-enhanced ultrasound, ultrasound elastography evaluation or artificial intelligence, should be necessarily used to improve diagnostic performance.Additional file 1:Figure S1. (A) ABUS image of 48-year-old woman in stage II (MD = 2.1 cm) breast cancer with ALNM. The coronal plane shows proper nipple position (yellow point). ABUS detected a big hypoechoic lesion (arrows) on outer quadrant in left breast with irregular shape, angular margin, microcalcification, posterior features shadowing, positive hyperechoic halo and retraction phenomenon.  ER (+), PR (+), HER2 (1+), Ki-67 = 15%; . The model predicted that the lesion possibly has ALNM (p = 0.83). MS is Lumina B. ABUS = Automated breast ultrasound, ALNM = axillary lymph node metastasis, MD = maximum diameter, MS = molecular subtype.Additional file 2:Table S1. Relationship between Ki-67 status and ABUS features.


CYP1A2 (rs 762551) and ADORA2A (rs 5751876) genes in a cohort of Brazilian athletes. One-hundred and fifty participants were genotyped for CYP1A2 and ADORA2A. After the recruitment and selection phase, 71  completed the CaffEQ-BR questionnaires and a self-report online questionnaire concerning sociodemographic data, general health status, and frequency of caffeine consumption. The order of completion of the CaffEQ-BR questionnaires was counterbalanced. The concordance between the full and brief versions of the CaffEQ-BR was analyzed using the intraclass correlation coefficient (ICC). To determine the discriminatory capacity of the questionnaires for genotype, the receiver operating characteristic (ROC) curve was applied for sensitivity and specificity (significance level of 5%). Mean caffeine intake was 244 ± 161 mg·day−1. The frequency of AA genotypes for CYP1A2 was 47.9% (n = 34) and 52.1% (n = 37) for C-allele carriers (AC and CC). The frequencies of TT genotypes for ADORA2A were 22.7% (n = 15) and 77.3% (n = 51) for C-allele carriers (TC and CC). All CaffEQ-BR factors, for the full and brief versions, were ICCs > 0.75, except for factor 6 , and presented ROC curve values from 0.464 to 0.624 and 0.443 to 0.575 for CYP1A2 and ADORA2A. Overall, the CaffEQ-BR (full and brief versions) did not show discriminatory capacity for CYP1A2 and ADORA2A gene polymorphisms. In conclusion, the CaffEQ-BR was not able to differentiate genotypes for the CYP1A2 or ADORA2A genes in this group of Brazilian athletes.This study investigated the ability of the Brazilian Caffeine Expectancy Questionnaire (CaffEQ-BR), full and brief versions, to differentiate genetic profiles regarding the polymorphisms of the  In addiass (BM) ,5,6. To ive dose ,8. It isive dose . Furtherive dose .CYP1A2 , which is related to the hepatic metabolization of caffeine [ADORA2A gene  is related to the sensitivity and responsiveness to caffeine [Genetic variants can affect caffeine metabolism , potenticaffeine . C allelcaffeine . In addicaffeine . This efCYP1A2 and ADORA2A genotypes affect the responsiveness and sensitivity of caffeine in the CNS is of great scientific and clinical relevance [CYP1A2 and ADORA2A genotypes of any individuals undergoing caffeine consumption, be it in research or in the real world, since these genes may alter responses. Nonetheless, genotyping is a time consuming and expensive process that is not easily available, meaning alternative, more cost-effective methods are desirable.Understanding how the elevance , includielevance ,14,20,21elevance ,23. In celevance ,24. ThusCYP1A2 genotype [ADORA2A gene [CYP1A2) and sensitivity  may lead to different response profiles to the CaffEQ-BR questionnaires.Therefore, assessing the expectations about caffeine’s effects is important due to its impacts on mood and sports performance. The Brazilian caffeine expectancy questionnaires (full and brief versions) (CaffEQ-BR) were developed to investigate expectations relating to caffeine ingestion in the adult Brazilian population, specifically regarding factors related to dependence, energy/work improvement, social/mood enhancement, physical performance, anxiety, and sleep disturbances ,26. Undegenotype , whereasA2A gene . Thus, gCYP1A2 and ADORA2A genes. The study hypothesis was that the CaffEQ-BR instrument could differentiate the polymorphisms of the CYP1A2 and ADORA2A genes. If this holds true, these questionnaires may be a cost-efficient method capable of identifying different genotypes that could be implemented in clinical research and practice to guide caffeine recommendations.The present study aimed to examine the ability of the CaffEQ-BR questionnaires (full and brief versions) to differentiate the polymorphisms of the CYP1A2 and ADORA2A polymorphisms who completed the full and brief versions of the CaffEQ-BR [This was a cross-sectional study involving 71 participants genotyped for affEQ-BR ,26. The n = 100) and Barreto et al. (n = 50) (data not yet published). Inclusion criteria were: (i) Brazilian adult (19–59 years old), resident in Brazil, regular consumer of caffeine (≥3x a week) from various sources, and agreement to complete the full and brief versions of CaffEQ-BR; (ii) had CYP1A2 −163C > A and ADORA2A 1976T > C genotypes determined. Participants were recruited among individuals who participated in the study by Spineli et al.  (n = 1002max: 44.0 ± 2.7 mL·kg−1·min−1) [2max: 51.1 ± 5.2 and 42.3 ± 8.12 mL·kg−1·min−1, respectively). It is important to emphasize that the athletes in the Spineli et al. study [In the Spineli et al. study , the par1·min−1) ; and in 1·min−1)   and T homozygote (TT) ADORA2A, the minimum sample would be 40 participants. Of the 150 individuals invited, 71 participated in the research.The distribution across genotypes generally includes fewer homozygous participants carrying the CC genotype for ectively ,27. ThusParticipants completed the full and brief versions of the CaffEQ-BR questionnaire. We have previously shown the full   and brieCYP1A2 gene extraction procedure followed the protocol proposed by Cornelis et al. [ADORA2A, that by Deckert et al. [CYP1A2 gene extraction procedure followed the protocol proposed by Salinero et al. [ADORA2A, that by Muñoz et al. [CYP1A2 (rs762551) was successful in all participants. However, five participants were not successful in determining the polymorphism of the ADORA2A gene (rs5751876). The researchers and participants were blinded to their genetic polymorphisms until all statistical analyses had been completed. For the samples of Spineli et al. , the CYPs et al. , and fort et al. . For theo et al. , and forz et al. . GenotypCPY1A2 and ADORA2A gene polymorphisms was evaluated by the receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) ranged from 0 to 1, an AUC = 0.5 indicates that CaffEQ-BR has no discrimination capability, and AUC = 0 or 1 corresponds to perfect discrimination. In addition, AUC 0.0–0.5 or 0.5–1.0 indicates that lower/higher CaffEQ-BR values indicate evidence of a positive state [CYP1A2 and TT homozygotes for ADORA2A, and three-tier clusters using AA, AC, and CC genotypes for CYP1A2 and TT, CT, and CC genotypes for ADORA2A. The ICC and AUC estimates are presented with their respective 95% confidence intervals and were evaluated by IBM SPSS Software version 22 .Descriptives analysis are presented through frequencies and percentages for categorical variables and mean and standard deviation for numerical variables (CaffEQ-BR scores). The CaffEQ-BR concordance between full and brief versions was verified using the intraclass correlation coefficient (ICC). The type of ICC adopted was the absolute agreement considering the average agreement of the two applications. The ICC calculation was based on a two-way mixed model. According to Cicchetti , an exceve state . The ana−1). The sample profile consisted of 90.1% males, 25 ± 8 y, body mass index 23.7 ± 3.9 kg/m2 , 33.8% CYP1A2 gene was 47.9% (n = 34), and that of C allele carriers (AC and CC genotypes) was 52.1% (n = 37). For the ADORA2A gene, 22.7% (n = 15) were TT homozygotes and 77.3% (n = 51) were C allele carriers (TC and CC genotypes).The frequency of AA homozygotes for the All factors showed excellent agreement  obtained a better discriminatory capacity than with three-tier clusters  (data not presented).The analyses performed with two-tier clusters  showed discriminatory ability for the ADORA2A genotypes in the brief questionnaire (AUC = 0.293). In ADORA2A genotypes.CYP1A2 and ADORA2A genes. Data showed that the full and brief versions of CaffEQ-BR were not able to differentiate the CYP1A2 or ADORA2A genotypes in these adult Brazilian athletes. Of all factors, only factor 6  in the brief questionnaire showed any discriminatory capacity for the genotype TT for the ADORA2A gene. However, the sample size was limited to two individuals, which precluded generalizing this result to an external population. Specifically, factor 6 in the brief CaffEQ-BR is composed of three questions related to the expected anxiety and negative physical effects of caffeine: “I don’t like the way I feel after drinking caffeine/coffee” (portuguese: Não gosto de como me sinto depois de tomar cafeína /café). “When I drink caffeine/coffee I get nervous” (portuguese: Quando bebo cafeína /café fico nervoso). “Caffeine/coffee makes me irritable” (portuguese: Cafeína /café me deixa irritado). Anxiety and sleep disturbance (factors 6 and 7) have been associated with the ADORA2A gene [ADORA2A polymorphisms.This is the first study that evaluated if an instrument (full and brief CaffEQ-BR questionnaires) can differentiate the polymorphisms of the A2A gene ; therefoCYP1A2, suggesting differences in caffeine metabolism do not lead to different expectations regarding caffeine effects. The importance of caffeine metabolism for exercise performance following supplementation is still unclear and inconsistent [CYP1A2 genotype on caffeine’s ergogenic effect [CYP1A2 genotypes. Differences in caffeine metabolism may not sufficiently alter variables that would modify caffeine expectancy, as the questionnaires here may provide questions that are too vague and generalized to detect differences in CYP1A2 genotypes. It cannot be ruled out that other questionnaires related to how symptoms develop and persist over time following caffeine ingestion may provide a more accurate method of determining an individual’s caffeine metabolism genotype.The CaffEQ-BR questionnaires were not capable of discriminating between genotypes for nsistent ,14. Mostc effect ,35,40,41c effect ,17,42. NADORA2A gene seem more consistent, especially regarding the negative effects of caffeine in more sensitive individuals (TT homozygotes), such as increased anxiety and sleep disturbance at higher doses (>6 mg·day−1) [−1), are likely TT homozygotes for the ADORA2A gene [ADORA2A genotypes.Studies that aim to find differences in the polymorphisms of the g·day−1) . In the g·day−1) ,26 and ig·day−1) ,43,44, tg·day−1)  showed tA2A gene ,45. ThusIndividuals with high dependency scores present strong correlation and high scores in CaffEQ factors 1, 2, 3, 4, and 5 . However, individuals who reported a desire to reduce or eliminate caffeine consumption from their routines had high scores on factors 6 and 7  . In the ADORA2A needs to be calibrated to have high sensitivity and specificity for those individuals who experience negative effects from caffeine. The present results suggest that future studies should include more individuals with low/irregular caffeine intake, as these may be, to a large extent, of the TT genotype for the ADORA2A gene. In all versions of the CaffEQ, the questionnaire presents more factors to support people who experience beneficial effects or a possible dependence on caffeine than negative effects, such as anxiety and sleep disturbance ,26,43,44CYP1A2 and ADORA2A genes. Furthermore, a good alternative could be to carry out an exploratory factor analysis, followed by a confirmatory analysis based on a biobank of individuals genotyped for CYP1A2 and ADORA2A polymorphisms in an attempt to assess the ability to differentiate the genotypes according to the responses to the caffeine expectancy questionnaire. A recent caffeine expectancy questionnaire (CaffCo) stressed the methodological caution needed to balance the number of positive/negative caffeine effects factors in the development of this kind of questionnaire . The CafCYP1A2 and ADORA2A genotypes. Nonetheless, regarding the practical applications, the CaffEQ-BR remains a useful tool to understand the expectation of caffeine intake to assess any potential risk of bias in clinical trials in sports science due to the possible ergogenic effects of placebo associated with the expected effects of caffeine in the placebo/control group [The current data suggest that the CaffEQ-BR questionnaire cannot be used to differentiate individuals for the ol group ,48, sincol group ,14.CYP1A2 and ADORA2A genotypes; importantly, we also showed good agreement between both versions of the questionnaire, confirming our previous work [CYP1A2 and TT for ADORA2A) and the small number of female participants. However, it is unclear if the questionnaire could lead to different discriminatory capacities for genotypes between men and women. Nevertheless, we recommended that future studies balance the sample profile in terms of gender. The research was conducted with the participants previously enrolled in the study of Spineli et al. [The strengths of the present study are that we explored the capacity of both full and brief versions of the CaffEQ-BR questionnaires to discriminate between ous work . Some poi et al.  and BarrCYP1A2; and TT, CT, and CC for ADORA2A), due to high sample segmentation and analysis with small groups. To achieve a more expressive number of genotypes of CC and TT for the CYP1A2 and ADORA2A genes (respectively), future studies should enroll larger samples due to the small presence of these homozygous individuals in the general population. We also encourage further studies to evaluate other applications of the CaffEQ-BR with more specific purposes, such as its application to the effect of placebo-controlled caffeine supplementation in clinical trials, as performed by Shabir et al. [The analyses were even less discriminatory when performed with three-tier clusters  was not able to differentiate genotypes for the