Monoamine oxidase A (MAO A) is a key enzyme for serotonin metabolism. Knockout MAO A in mice results in elevated serotonin, altered serum autoantibodies, and autistic-like behavior. There is a subset of patients with autism spectrum disorder (ASD) who exhibit hyperserotonemia. The link between the MAO A, hyperserotonemia, immunity, and ASD is still unknown. To address this question, we harness the high-density human proteome microarray to profile the serum autoantibodies in ASD patients with or without MAO A deficiency. We recruited 25 subjects, including 20 ASD patients, from National Yang Ming Chiao Tung University Hospital and determined their plasma serotonin levels, screened for MAO A gene mutations and identified one patient with a C374 G mutation, which abolished MAO A activity and showed the highest serotonin level (hsASD; 518.42 ng/ml) and severe ASD symptoms. In another family with twin brothers, one was diagnosed with mild ASD and exhibited an increased serotonin level (isASD; 31.48 ng/ml), while the other twin was a healthy control. These three subjects were used for serum autoantibody profiling using high-density human proteome microarrays. Comparing serum antibodies from hsASD with healthy control, we identified 354 up-regulated and 398 down-regulated autoantibodies in hsASD. By comparing isASD with healthy controls, we reported 235 up-regulated and 279 down-regulated autoantibodies in isASD. Interestingly, the up-regulated autoantibodies for hsASD were enriched in the brain region and exhibited distinct features from that of isASD. This study indicates that MAO A deficiency and serotonin levels significantly impact the immunological changes in ASD patients, which may shed some light on pathological mechanisms and provide potential biomarkers for translational research in ASD.

Bacterial proteome microarrays are high-throughput, adaptable tools that allow the simultaneous investigation of thousands of proteins from various bacterial species. These arrays are used to explore bacterial pathogenicity, pathogen-host interactions, and clinical diseases. Recent advancements have expanded their application to profiling human antibodies, identifying biomarkers for infectious and autoimmune diseases, and studying antimicrobial peptides (AMPs). This review highlights significant outcomes from recent studies, focusing on their diverse applications in biomedical research. Notable findings include the identification of novel antigens and diagnostic markers for gastrointestinal infections, autoimmune diseases, and mental health disorders. This technology promises to further elucidate the complex relationship between bacteria and their hosts, ultimately informing the development of new diagnostic, therapeutic, and preventive strategies.

Protein-lipid interactions are crucial for various cellular biological processes like intracellular signaling, membrane transport, and cytoskeletal dynamics. Therefore, studying these interactions is essential to understand and unravel their specific functions. Nevertheless, the interacting proteins of many lipids are poorly understood and still require systematic study. Liposomes are the most well-known and familiar biomimetic systems used to study protein-lipid interactions. Although liposomes have been widely used for studying protein-lipid interactions in classical methods such as the co-flotation assay (CFA), co-sedimentation assay (CSA), and flow cytometric assay (FCA), an overview of their current applications and developments in high-throughput methods is not yet available. Here, we summarize the liposome development in low and high-throughput methods to study protein-lipid interactions. Besides, a constructive comment for each platform is presented to stimulate the advancement of these technologies in the future.

Tyrosine phosphorylation is one of the most important posttranslational modifications in bacteria, linked to regulating growth, migration, virulence, secondary metabolites, biofilm formation, and capsule production. Only two tyrosine kinases (yccC (etk) and wzc) have been identified in Escherichia coli. The investigation by similarity has not revealed any novel BY-kinases in silico so far, most probably due to their sequence and structural variability. Here we developed a reverse-phase protein array from 4126 overexpressed E. coli clones, lysed, and printed on coated glass slides. These high-density E. coli lysate arrays (ECLAs) were quality controlled by the reproducibility and immobilization of total lysate proteins and specific overexpressed proteins. ECLAs were used to interrogate the relationship between protein overexpression and tyrosine phosphorylation in the total lysate. We identified 6 protein candidates, including etk and wzc, with elevated phosphotyrosine signals in the total lysates. Among them, we identified a novel kinase nrdD with autophosphorylation and transphosphorylation activities in the lysates. Moreover, the overexpression of nrdD induced biofilm formation. Since nrdD is a novel kinase, we used E. coli proteome microarrays (purified 4,126 E. coli proteins) to perform an in vitro kinase assay and identified 33 potential substrates. Together, this study established a new ECLA platform for interrogating posttranslational modifications and identified a novel kinase that is important in biofilm formation, which will shed some light on bacteria biochemistry and new ways to impede drug resistance.

Background and Objective:Proteome microarrays are one of the popular high-throughput screening methods for large-scale investigation of protein interactions in cells. These interactions can be measured on protein chips when coupled with fluorescence-labeled probes, helping indicate potential biomarkers or discover drugs. Several computational tools were developed to help analyze the protein chip results. However, existing tools fail to provide a user-friendly interface for biologists and present only one or two data analysis methods suitable for limited experimental designs, restricting the use cases.Methods:In order to facilitate the biomarker examination using protein chips, we implemented a user-friendly and comprehensive web tool called BAPCP (Biomarker Analysis tool for Protein Chip Platforms) in this research to deal with diverse chip data distributions.Results:BAPCP is well integrated with standard chip result files and includes 7 data normalization methods and 7 custom-designed quality control/differential analysis filters for biomarker extraction among experiment groups. Moreover, it can handle cost-efficient chip designs that repeat several blocks/samples within one single slide. Using experiments of the human coronavirus (HCoV) protein microarray and the E. coli proteome chip that helps study the immune response of Kawasaki disease as examples, we demonstrated that BAPCP can accelerate the time-consuming week-long manual biomarker identification process to merely 3 min.Conclusions:The developed BAPCP tool provides substantial analysis support for protein interaction studies and conforms to the necessity of expanding computer usage and exchanging information in bioscience and medicine. The web service of BAPCP is available at https://cosbi.ee.ncku.edu.tw/BAPCP/.

Abstarct Ectopic ATP synthase on the plasma membrane (eATP synthase) has been found in various cancer types and is a potential target for cancer therapy. However, whether it provides a functional role in tumor progression remains unclear. Here, quantitative proteomics reveals that cancer cells under starvation stress express higher eATP synthase and enhance the production of extracellular vesicles (EVs), which are vital regulators within the tumor microenvironment. Further results show that eATP synthase generates extracellular ATP to stimulate EV secretion by enhancing P2X7 receptor–triggered Ca2+ influx. Surprisingly, eATP synthase is also located on the surface of tumor-secreted EVs. The EVs-surface eATP synthase increases the uptake of tumor-secreted EVs in Jurkat T-cells via association with Fyn, a plasma membrane protein found in immune cells. The eATP synthase-coated EVs uptake subsequently represses the proliferation and cytokine secretion of Jurkat T-cells. This study clarifies the role of eATP synthase on EV secretion and its influence on immune cells.

Reactive oxygen species (ROS), released as byproducts of mitochondrial metabolism or as products of NADPH oxidases and other processes, can directly oxidize the active-site cysteine (Cys) residue of protein tyrosine phosphatases (PTPs) in a mammalian cell. Robust degradation of irreversibly oxidized PTPs is essential for preventing accumulation of these permanently inactive enzymes. However, the mechanism underlying the degradation of these proteins was unknown. In this study, we found that the active-site Cys215 of endogenous PTP1B is sulfonated in H9c2 cardiomyocytes under physiological conditions. The sulfonation of Cys215 led PTP1B to exhibit a conformational change, and drive the subsequent ubiquitination and degradation of this protein. We then discovered that Cullin1, an E3 ligase, interacts with the Cys215-sulfonated PTP1B. The func-tional impairment of Cullin1 prevented PTP1B from oxidation-dependent ubiquitination and degradation in H9c2 cells. Moreover, delivery of the terminally oxidized PTP1B resulted in proteotoxicity-caused injury in the affected cells. In conclusion, we elucidate how sulfonation of the active-site Cys215 can direct turnover of endogenous PTP1B through the engagement of ubiquitin-proteasome system. These data highlight a novel mechanism that maintains PTP homeostasis in cardiomyocytes with constitutive ROS production.

Full text Figures and data Side by side Abstract eLife assessment Introduction Results Discussion Methods Data availability References Peer review Author response Article and author information Abstract The pathogenesis of antibodies in severe alcoholic hepatitis (SAH) remains unknown. We analyzed immunoglobulins (Ig) in explanted livers from SAH patients (n=45) undergoing liver transplantation and tissues from corresponding healthy donors (HD, n=10) and found massive deposition of IgG and IgA isotype antibodies associated with complement fragment C3d and C4d staining in ballooned hepatocytes in SAH livers. Ig extracted from SAH livers, but not patient serum exhibited hepatocyte killing efficacy. Employing human and Escherichia coli K12 proteome arrays, we profiled the antibodies extracted from explanted SAH, livers with other diseases, and HD livers. Compared with their counterparts extracted from livers with other diseases and HD, antibodies of IgG and IgA isotypes were highly accumulated in SAH and recognized a unique set of human proteins and E. coli antigens. Further, both Ig- and E. coli-captured Ig from SAH livers recognized common autoantigens enriched in several cellular components including cytosol and cytoplasm (IgG and IgA), nucleus, mitochondrion, and focal adhesion (IgG). Except IgM from primary biliary cholangitis livers, no common autoantigen was recognized by Ig- and E. coli-captured Ig from livers with other diseases. These findings demonstrate the presence of cross-reacting anti-bacterial IgG and IgA autoantibodies in SAH livers. eLife assessment This important study tested the hypothesis that liver-derived but not serum-derived antibodies that are cross-reactive to E.coli and to host proteins can play a role in the hepatic damage found in severe alcoholic hepatitis (SAH). Using a solid methodology that includes state-of-the-art microscopy, proteome arrays, and gene ontology assays, it provides strong evidence that liver-derived IgG and IgA with cytotoxic properties and reactivity to both gut-derived E. coli and autoantigens accumulated in hepatocytes of SAH patients but not of healthy controls. The study would benefit from a broader analysis of gut microbiota proteome and further characterization of B cells infiltrating the liver tissue including their numbers/field and their origin (infiltrating versus resident cells). The work opens new avenues of understanding for the pathogenesis of severe alcoholic hepatitis and is of great interest to researchers and clinicians in the field. https://doi.org/10.7554/eLife.86678.2.sa0 About eLife assessments Introduction Severe alcoholic hepatitis (SAH) is a distinct clinical syndrome that can develop suddenly and quickly lead to liver failure. It carries a particularly poor prognosis with a 28-day mortality ranging from 30% to 50% (Dugum et al., 2015; Thursz and Morgan, 2016; Sehrawat et al., 2020). Unfortunately, there is little to offer medically to such critically ill patients beyond supportive care with steroids, which improves survival in only a minority. Studies have established connections between alcohol abuse, disruption of gut microbial homeostasis, and alcoholic liver disease (ALD). It has been speculated for over four decades that antibodies targeting intestinal microbes might play a role in pathogenesis of ALD (Bjorneboe et al., 1972; Simjee et al., 1975; Kanagasundaram et al., 1977; Kater et al., 1979; Trevisan et al., 1983; Koskinas et al., 1992). For example, the presence of IgA and IgG on the cell membrane of hepatocytes was detected by direct immunofluorescence in patients with ALD, and the percentage of IgG-positive hepatocytes correlated with transaminase levels, independently of the histological findings (Trevisan et al., 1983). Although a number of studies demonstrated liver IgA deposition in ALD in the 1980s, other reports concluded that IgA deposition in the liver was not specific for ALD but might reflect the reduced metabolism of the damaged livers (van de Wiel et al., 1987; Amano et al., 1988) or the clearance of excess IgA from the circulation (Nagura et al., 1989). A recent study (Moro-Sibilot et al., 2016) confirmed that human livers contained IgA-secreting cells originating from Peyer's patches and directed against intestinal antigens. Interestingly, livers from mice with ethanol-induced injury contain increased numbers of IgA-secreting cells and have IgA deposits in sinusoids (Moro-Sibilot et al., 2016). The primary aim of this study was to determine if there was antibody deposition in SAH livers and whether antibodies extracted from SAH livers exhibited hepatocyte killing efficacy. The second aim was to determine if antibodies deposited in the liver were cross-reactive antibodies against both bacterial antigens and human proteins and whether the cross-reactive antibodies were presented uniquely in SAH livers. Results Immunoglobulins in ballooned hepatocytes in SAH patients To determine whether antibodies deposit in SAH livers, we collected explanted liver tissues from SAH patients during liver transplantation at Johns Hopkins. Liver tissue sections with H&E staining from SAH patients showed histologic features of SAH including macrovesicular steatosis, neutrophilic lobular inflammation, ballooning hepatocyte degeneration, Mallory-Denk bodies, and portal and pericellular fibrosis (Figure 1A). Immunohistochemistry (IHC) staining by using anti-human immunoglobulin (Ig) antibodies demonstrated massive IgA and IgG deposition in ballooned hepatocytes in SAH livers, while none of the hepatocytes were stained with anti-human Ig antibodies in liver tissue sections from healthy donors (HD) except for positive staining in some hepatic sinusoid cells (Figure 1B and C). To further confirm the deposition of Ig in SAH livers, the presence of Ig in liver tissue homogenates form SAH (n=7) or HD (n=7) was assessed by western blot analysis and ELISA assays. Western blot analysis demonstrated that the levels of IgA and IgG were dramatically increased in all SAH livers as compared with the donor livers (Figure 1D and E). The IgM but not the IgE level was also significantly increased in SAH livers. The increase of IgA, IgG, and IgM levels in SAH liver tissue homogenates was further confirmed by ELISA. IgA and IgG isotypes were major Ig in SAH livers (Figure 1F). Further analysis of IgG subclasses demonstrated that the IgG subclass levels – predominantly IgG1 – were significantly higher in SAH livers than that in HD (Figure 1G). On the basis of these findings, we performed IHC staining for human IgG in SAH livers from 45 patients with liver transplantation and 10 donor livers in a clinical pathology lab at Johns Hopkins in a double-blind manner. The IgG+ hepatocytes in scanned slides of stained tissues sections were analyzed by using HALO Image Analysis Software. Positive cells were reported as percentage stained surface area of total annotated area by digital analysis (Figure 1—figure supplement 1). Few IgG+ hepatocytes were identified in donor livers. In contrast, on average ~40% of the hepatocytes (ranging from 4% to 80%) were IgG positive in the SAH livers (Figure 1H). These findings demonstrated the deposition of Ig antibodies in ballooned hepatocytes in SAH livers. Figure 1 with 1 supplement see all Download asset Open asset Immunoglobulin (Ig) deposition in ballooned hepatocytes of explanted livers from severe alcoholic hepatitis (SAH) patients. (A) Liver tissue sections with H&E staining showed histologic features of SAH. (b, c) Immunohistochemistry staining by using anti-human IgA (B) or IgG (C) antibodies demonstrated IgA and IgG deposition in ballooned hepatocytes in SAH livers. Representative tissue sections from 45 SAH or 10 healthy donor (HD) livers. (D–E) Ig levels in liver tissue homogenates from SAH or HD (n=7/group) were quantified by western blot analysis (D). Western blot analysis demonstrated that the levels of IgA, IgG, and IgM were significantly increased in SAH livers as compared with the HD livers (E). (F–G) Ig isotypes (f) and IgG subclass levels (G) were quantified by ELISA (n=7/group). (H) IgG-positive hepatocytes in tissue sections from 45 SAH patients and 10 HD were quantified by immunohistochemistry staining and using HALO Image Analysis Software. Ig deposition is associated with activation of complement in ballooned hepatocytes and Ig extracted from SAH livers exhibits hepatocyte killing efficacy in vitro IgG, especially IgG1, plays a critical role in the classical complement activation pathway. To determine if Ig in ballooning hepatocytes induces activation of complement, C3d and C4d were analyzed in SAH livers. IHC staining showed the presence of both C3d and C4d in ballooning hepatocytes in SAH livers but not in the donor livers (Figure 2A and B). Double staining for IgG and complement fragments C3d or C4d showed IgG co-stained with C3d or C4d in ballooning hepatocytes (Figure 2C and D). These results indicated that IgG deposition in hepatocytes was associated with activation of complement. Furthermore, complement activation including the presence of C3d and C4d in SAH liver was confirmed by western blot analysis (Figure 2E and F). Finally, we asked whether antibodies (Ig) extracted from SAH livers exhibit hepatocyte killing efficacy in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay. Compared to isotype control human Ig from HD, no increased hepatocyte killing was observed when peripheral blood mononuclear cells (PBMCs) (effector cells) from HD were added into cultured human hepatocytes (target cells) in the presence of serum Ig from SAH patients. However, the hepatocyte killing efficacy was significantly increased when the same levels of Ig extracted from SAH livers were added into the hepatocytes/PBMCs co-culture system (Figure 2G). These results demonstrated that Ig antibodies deposited in hepatocytes of SAH livers could induce activation of complement but more importantly, exhibited antibody-dependent cellular cytotoxicity of hepatocytes. Therefore, deposition of Ig antibodies may contribute to the hepatocyte ballooning degeneration and necrotic damage in SAH. Figure 2 Download asset Open asset Immunoglobulin (Ig) deposition is associated with activation of complement in ballooned hepatocytes and Ig extracted from severe alcoholic hepatitis (SAH) livers exhibits hepatocyte killing efficacy in vitro. To determine if immunoglobulin in ballooning hepatocytes induces activation of complement, complement fragments C3d and C4d were analyzed in SAH livers. (A–B) Immunohistochemistry staining showed the presence of both C3d (A) and C4d (B) in ballooning hepatocytes in SAH livers but not in the donor livers. (C) Double staining for IgG and complement fragments C3d or C4d showed IgG co-stained with C3d or C4d in ballooning hepatocytes. Representative tissue sections from seven samples per group. (E–F) C3d and C4d levels in SAH livers were quantified by western blot analysis (n=7). (G) Ig extracted from SAH livers but not serum exhibit hepatocyte killing efficacy in antibody-dependent cell-mediated cytotoxicity (ADCC) assay. Representative data from three independent experiments. To further define the Ig and C4d deposition on the membrane of ballooned hepatocytes, we performed multiplex analyses by co-staining liver tissue sections with multiple cell markers for hepatocytes (Heppar1), Kupffer cells (CD68), hepatic sinusoid endothelial cells (CD32), and hepatocyte membrane (β-catenin). Images of confocal microscopy showed the presence of both IgG and IgA in hepatic sinusoid endothelial cells but not hepatocytes in the donor livers, while the majority of ballooned hepatocytes co-stained with IgG and IgA in SAH livers (new Figure 3A). Further, co-staining with β-catenin detected heavy IgG and IgA deposition on hepatocyte membrane (Figure 3B). Interestingly, the IgG and IgA on hepatocyte membrane were co-stained with C4d as well (Figure 3C). Figure 3 Download asset Open asset To determine if immunoglobulin (Ig) recognize cell surface antigens of ballooned hepatocytes, multiplex immunofluorescence staining was performed in liver tissue sections. (A) Images of confocal microscopy showed the presence of both IgG (red) and IgA (green) in ballooning hepatocytes (blue) in severe alcoholic hepatitis (SAH) livers (lower panels), while only hepatic sinusoid endothelial cells (CD32+, purple) stained with IgG and IgA in donor livers (upper panels). (B) Co-staining with β-catenin (blue) demonstrated IgG (red) and IgA (green) deposition on membrane of ballooning hepatocytes in SAH livers. (C) Triple staining for IgG (red), IgA (green), and C4d (blue) showed both IgG and IgA co-stained with C4d on the surface of hepatocyte. Representative tissue sections from six samples per group. Human proteome array-identified autoantigens were recognized by Ig extracted from SAH livers We used the human proteome microarray (HuProt), comprising 21,240 individual purified human proteins, to perform antibody profiling assays (Hu et al., 2017). Each liver specimen was treated to release tissue-deposited Ig (Figure 4A). After neutralization, the extracted antibodies from each liver sample were separately probed to the HuProt arrays, using isotype-specific secondary antibodies to obtain the IgG, IgA, IgM, and IgE autoimmune signatures of the same liver sample (Figure 4B). Many positive human proteins were recognized by each of the four Ig isotypes in all five SAH samples. Each antibody profiling assay was performed in duplicate and only those reproducible signals were scored. A substantial fraction of autoantigens was shared by the antibodies in all five SAH livers, regardless of the Ig isotype (Figure 4C, Figure 4—figure supplement 1). The total numbers of the shared autoantigens recognized by the IgG, IgA, IgM, and IgE isotypes were 346, 319, 194, and 10 (Figure 4—figure supplement 1), respectively, suggesting that the shared autoantibodies of the IgG and IgA isotypes were the most prevalent, while the counterparts of IgE isotype were the scarcest. With tissues of five donor livers, the numbers of positive human proteins were much lower (Figure 4C ). Although 95 shared IgG autoantigens were identified by the donor liver samples, 79 (83.2%) of them were also shared by the SAH livers (Figure 4C). More importantly, a large fraction (i.e., 267 proteins) of the SAH-shared autoantigens were not found in the donor livers (Figure 4C). Figure 4 with 5 supplements see all Download asset Open asset Human and Escherichia coli proteome arrays that identify a group of unique antibodies in the severe alcoholic hepatitis (SAH) liver recognize both human and bacterial antigens. (A) Each liver tissue piece was ground up and treated under low pH to release tissue-deposited Ig. After neutralization, the extracted Ig from each liver sample were separately probed to the HuProt or E. coli protein arrays, followed by incubation with the isotype-specific secondary antibodies to obtain the Ig isotype immune signatures of the same liver sample. (B) Representative images of HuProt arrays. (C) Venn diagram analysis to identify shared autoantigens of each liver disease. 346 autoantigens were shared by the IgG antibodies in all five SAH livers (left panel), 95 autoantigens were shared by the IgG antibodies in all five healthy donor (HD) livers (middle panel), and a large fraction (i.e., 267 proteins) of the SAH-shared IgG autoantigens was not found in the HD livers (right panel), suggesting existence of an SAH-specific autoimmune signature. (D) A seven-way Venn diagram analysis showed that 45 autoantigens were commonly recognized by IgG isotype autoantibodies in liver tissue homogenates extracted from different liver diseases, while 188 unique IgG autoantigens were recognized by tissue homogenates from the SAH livers. (E) Representative images of E. coli protein arrays. (F) Venn diagram analysis to identify shared E. coli antigens recognized by each liver disease. 435 E. coli antigens were commonly recognized by IgG antibodies in the five SAH livers (left panel), while only 1 E. coli antigen was commonly recognized by IgG antibodies in the five HD livers (middle panel). 434 out of 435 E. coli antigens were uniquely recognized by IgG antibodies in SAH livers but not HD livers (right panel). (G) A seven-way Venn diagram analysis showed that unique IgG bacterial antigens were only identified by using liver tissue homogenates from SAH or AC. Figure 4—source data 1 The number of unique autoantigens recognized by antibodies extracted from the diseased liver tissues (HuProt arrays). https://cdn.elifesciences.org/articles/86678/elife-86678-fig4-data1-v1.docx Download elife-86678-fig4-data1-v1.docx Figure 4—source data 2 The number of unique E. coli antigens recognized by antibodies extracted from the diseased liver tissues (E. coli proteome arrays). https://cdn.elifesciences.org/articles/86678/elife-86678-fig4-data2-v1.docx Download elife-86678-fig4-data2-v1.docx We applied the above approach to a group of five livers explanted from patients with AC, AIH, primary biliary cholangitis (PBC), NASH, HCV, and HBV infection. SAH still exhibited the highest number of shared IgG autoantigens, while HBV showed the highest number of shared IgM, IgA and IgE autoantigens, and PBC showed the highest number of shared IgM autoantigens (Figure 4—figure supplement 2). The numbers of shared IgG, IgA, and IgM autoantigens were much higher in SAH livers (n=859) as compared with AC (n=349), HBV (n=735), other liver diseases (n<428), or HD livers (n=448). Although the shared IgE autoantigens were the lowest in all seven liver diseases, each disease showed a distinct autoantibody signature (Figure 4—figure supplement 2). We compared the shared autoantigens recognized by SAH Ig to their counterparts from the other six liver diseases. By Venn diagram analysis 45, 41, 68, and 8 autoantigens were commonly recognized by IgG, IgA, IgM, and IgE isotype autoantibodies in liver tissue extracted from different liver diseases (Figure 4—figure supplement 3). 188 unique IgG autoantigens, 45 unique IgA autoantigens, and 7 unique IgM autoantigens were recognized by tissue homogenates from the SAH livers, whereas the second highest in this category was HBV livers in which 1 unique IgG autoantigen, 88 unique IgA autoantigens, and 25 unique IgM autoantigens were identified (Figure 4D, Figure 4—figure supplement 3). The third in this category was PBC in which 7 unique IgG autoantigens, 2 unique IgA autoantigens, and 81 unique IgM autoantigens were identified. The tissue homogenates from HCV livers recognized 32 unique IgG autoantigens, 4 unique IgA autoantigens, and 3 unique IgE autoantigens, while only 4 unique IgA autoantigens were recognized by tissue homogenates from AC livers (Figure 4—figure supplement 3), showing disease-distinct autoantibodies in diseased livers regardless of their etiology. A large number of unique autoantigens were recognized by Ig recovered from SAH livers (Figure 4—source data 1), indicating Ig (especially IgG) deposited to the SAH livers (Figure 1) might play an important role in pathogenesis. Ig from SAH or AC livers recognize a unique set of bacterial antigens Antibodies secreted into the gut mostly target bacteria and bacterial products (Kato et al., 2014). Ig deposited in SAH livers might be from the gut, and these Ig may be antibodies targeting intestinal bacterial antigens. To test this, we employed a bacterial proteome array, comprising 4256 purified E. coli proteins encoded by a commensal strain K12, to do antibody profiling assays (Chen et al., 2009; Figure 4E). Using the same liver tissues and approach described above, we obtained the immune signatures of the 40 livers from 7 diseases and 5 HD in duplicate. Many more bacterial than human antigens were recognized by Ig from alcoholic livers (AC and SAH) compared with HD livers (Figure 4—figure supplement 4). The differences in shared bacterial antigens between alcoholic livers and HD livers were more pronounced across all four Ig isotypes. For instance, only one bacterial antigen was commonly recognized by IgG antibodies from HD livers, but 435 shared bacterial antigens (about 10.2% of 4256 purified E. coli proteins) were recognized by IgG antibodies in the five SAH livers (Figure 4F), and 466 shared bacterial antigens were recognized by IgG antibodies in the five AC livers (Figure 4—figure supplement 4). The numbers of the commonly shared bacterial antigens identified by the IgG antibodies extracted from the other 5 liver diseases were much lower, ranging from 7 to 63 (Figure 4—figure supplement 4). In addition, the numbers of IgA-, IgM-, or IgE-recognized bacterial antigens by liver tissue homogenates from SAH or AC were much higher than that in other liver diseases, except PBC showing a higher number of IgM antigens than AC (Figure 4—figure supplement 4). This observation suggests that chronic or acute ALD is associated with specific anti-bacterial antibody signatures. A large number of shared bacterial antigens were recognized by liver tissue homogenates from each liver disease. We found that 1, 6, 24, and 5 E. coli antigens were commonly recognized by IgG, IgA, IgM, and IgE isotype antibodies in liver tissue homogenates extracted from different liver diseases (Figure 4F, Figure 4—figure supplement 5). Unique IgG or IgA bacterial antigens were only identified by using liver tissue from SAH or AC (Figure 4G, Figure 4—figure supplement 5). More unique bacterial antigens were recognized by the SAH than AC IgA (110 vs 54), IgM (54 vs 1), and IgE (45 vs 1), but less so by the SAH IgG (53 vs 83) (Figure 4—source data 2). Notably, 69 unique IgM bacterial antigens were recognized by PBC liver tissue. Common anti-E. coli protein antibodies are present in diseased livers regardless of etiology, but unique anti-E. coli IgG and IgA antibodies exist only in alcoholic livers predominantly in SAH. The prevalent anti-bacterial immunoactivity of the Ig in SAH livers suggested that Ig deposited in SAH livers might be derived from leaky gut and these anti-bacterial antibodies cross-react with human liver proteins. We used proteins from E. coli (strain K12) immobilized on magnetic beads to capture Ig pooled from the SAH livers (Figure 5A) (E. coli-captured Ig) which were then released and incubated on the HuProt arrays to determine whether these bacterium-recognizing antibodies could also cross-react with human proteins (examples shown in Figure 5B). At a stringent cutoff value (e.g., SD=10) 694, 796, 451, and 42 human proteins were reproducibly identified by E. coli protein-captured IgG, IgA, IgM, and IgE antibodies from the five SAH livers, respectively (Figure 5C and antibody-recognized protein sets in Supplementary file 1). Strikingly, many of these E. coli binding Ig recognized proteins (74.06%, 76.46%, 48.39%, and 49.41%, respectively) were also found to be the autoantigens recognized by Ig recovered directly from the five SAH livers. These results demonstrated that there exist a large number of cross-reacting antibodies that recognize both bacterial and human proteins in the SAH livers. Figure 5 Download asset Open asset E. coli antigens-captured immunoglobulins (Ig) from severe alcoholic hepatitis (SAH) livers recognize human protein antigens. (A) To determine if anti-bacterial antibodies cross-react with human proteins in the liver, total proteins from E. coli (strain K12) were extracted and immobilized on NHS-activated magnetic beads to capture Ig pooled from the five SAH livers. E. coli protein-captured antibodies (E. coli-Ig) were then released and incubated on the HuProt arrays. (B) Representative images of E. coli-Ig on HuProt arrays. (C) 937, 1041, 932, and 85 human proteins were reproducibly identified by E. coli protein-captured IgG, IgA, IgM, and IgE antibodies from the five SAH livers. Venn diagram analysis showed many of these proteins (694/937, 796/1041, 451/932, and 42/95, respectively) were also found to be the autoantigens recognized by Ig recovered directly from the five SAH livers. Gene ontology (GO) enrichment analysis of proteome arrays identified autoantigen-enriched unique common cellular components recognized by both Ig- and E. coli-captured Ig in SAH livers. To determine if autoantigens recognized by Ig from diseased livers were specifically presented in cellular components, we performed GO enrichment analysis on antibody-recognized protein sets. Proteins recognized by IgG antibodies from SAH livers were significantly over-represented in eight cellular components including cytosol, cytoplasm, nucleus, mitochondrion, focal adhesion extracellular exosome, mitochondrial intermembrane space, and ruffle membrane, while autoantigens recognized by IgA antibodies from SAH livers were over-represented in cytosol and cytoplasm (Figure 6A). These proteins are involved in a number of biological processes including nucleobase-containing small molecule interconversion, protein phosphorylation, signal transduction, regulation of cell proliferation, mitochondrial ribosome reassembly, and mitochondrion organization (Figure 6B and C). With the exception that cellular components were identified by IgM antibodies from PBC livers (Figure 6—source data 1), no cellular component was recognized by Ig from other diseased livers. Interestingly, E. coli-captured IgG and IgA from SAH livers recognized five out of eight cellular components which were recognized by Ig directly extracted from SAH livers (Figure 6D). This was observed only in SAH livers (Figure 6—source data 2). These results further demonstrated the presence of unique cross-reacting anti-E. coli autoantibodies in the SAH livers. Figure 6 Download asset Open asset Gene ontology (GO) enrichment analysis of proteome arrays identifies autoantigen-enriched common cellular components recognized by both immunoglobulin (Ig)- and E. coli-captured Ig in severe alcoholic hepatitis (SAH) livers. (A) Cellular components recognized by IgG and IgA antibodies in SAH livers. (B–C) Biological processes are involved by IgG autoantigens (B) and IgA autoantigens (C). (D) Common cellular components recognized by both Ig- and E. coli antigens-captured Ig in SAH livers. Figure 6—source data 1 Cellular components recognized by IgM and E. coli enriched IgM extracted from PBC liver tissues. https://cdn.elifesciences.org/articles/86678/elife-86678-fig6-data1-v1.docx Download elife-86678-fig6-data1-v1.docx Figure 6—source data 2 Cellular components recognized by IgG or IgA antibodies extracted from the diseased liver tissues (Observed vs. Expected). https://cdn.elifesciences.org/articles/86678/elife-86678-fig6-data2-v1.docx Download elife-86678-fig6-data2-v1.docx The infiltration of B and plasma cells in SAH livers is associated with increased Ig gene expression Alcohol-derived leaky gut may promote translocation of gut bacterial products and Peyer's patches IgA-secreting plasma cells to the liver (Moro-Sibilot et al., 2016). To determine if the migration of bacteria and/or bacterial products from the bowel to liver occurred in SAH, we performed IHC staining for the gram-negative bacterial (E. coli) product livers. Both LPS and LTA were in liver tissue from SAH patients cf. controls (Figure 7A), especially in the inflammatory areas. The increase of LPS levels in SAH liver tissues was confirmed by using Pierce Chromogenic Endotoxin Quant Kit (Figure 7B). IHC staining for CD20+ and CD138+ cells revealed that most of these cells were localized in the inflammatory areas of SAH livers, with none in HD livers (Figure 7C). These results suggest that increased gut bacterial antigens in the SAH liver are associated with increased B cells and plasma cells. Figure 7 Download asset Open asset Increased bacterial antigens, B and plasma cell infiltration, and immunoglobulin gene expression in severe alcoholic hepatitis (SAH) livers. (A) Immunohistochemistry staining for the gram-negative bacterial product lipopolysaccharides (LPS) and gram-positive bacteria antigen lipoteichoic acid (LTA) in SAH livers. (B) LPS levels in SAH liver tissues were quantified by using Pierce Chromogenic Endotoxin Quant Kit (n=7/group). (C) Immunohistochemistry staining for CD20+ B cells and CD138+ plasma cells in SAH livers. Representative images from 20 SAH or 10 healthy donor (HD) livers (A–C). (D) Double immunofluorescence staining with CD20 (green) and LTA (red) showed colocalization of B cells and bacteria antigens in the inflammatory areas. Representative tissue sections from six samples per group. (E) RNA-sequencing analysis identified differentially expressed genes representing immunoglobulin fragments in SAH livers. Purple dots represented 91 differentially expressed immunoglobulin genes (log2FC >1 and adjusted p-value <0.01) in the comparison of SAH patients between normal donors. Among them, 87 were upregulated and 4 downregulated. Gray dots were the remaining immunoglobulin genes that did not meet the thresholds. n=7 per group. (F) Schematic representation of mechanism of cross-reacting anti-bacterial autoantibody-mediated hepatocyte damage in SAH. Based on our findings that Ig extracted from SAH livers but not present in the serum exhibited hepatocyte killing efficacy (Figure 2G), we suspected that Ig antibodies deposited in hepatocytes might not be produced systemically but by plasma cells infiltrating the liver. To determine if infiltration of B and plasma cells in the SAH liver is associated with expression of genes coding for certain Ig or fragments of Ig, we analyzed RNA-seq data generated from SAH and HD livers (GEO GSE143318). Specifically, we exam

This study aimed to optimize the per-O-acetylation, deacetylation, and benzylidene protection of carbohydrates and investigate the scope of saccharides via alum-catalysis. Initially, optimization was based on the previously reported PTA-catalyzed acetylation of glucose, but alum-catalysis required heating conditions. The study expanded to investigate the per-O-acetylation of different saccharides, including galactose and mannose, which produced stereoisomeric mixtures. The study proposed a plausible reaction mechanism for the isomerization observed in galactose and xylose after acetylation. The mechanism involved chelation of the active alum-catalyst with the O-6 position and beta-type O-1 position of galactopyranose to form a transition state complex via ring opening. This study investigated the generality of the de-O-acetylation method on a variety of carbohydrates using per-O-acetyl saccharide substrates bearing different functional groups. The direct protection of benzylidene in addition to acetylation/de-O-acetylation with a focus on optimizing reaction conditions for the synthesis of carbohydrate derivatives. Overall, the study offers insights into optimizing de-acetylation, per-O-acetylation, and benzylidene protection investigating the scope of saccharides via alum-catalysis.

The pathogenesis of antibodies in severe alcoholic hepatitis (SAH) remains unknown. We analyzed immunoglobulins (Ig) in explanted livers from SAH patients (n=45) undergoing liver transplantation and tissues from corresponding healthy donors (HD, n=10) and found massive deposition of IgG and IgA isotype antibodies associated with complement fragment C3d and C4d staining in ballooned hepatocytes in SAH livers. Ig extracted from SAH livers, but not patient serum exhibited hepatocyte killing efficacy. Employing human and Escherichia coli K12 proteome arrays, we profiled the antibodies extracted from explanted SAH, livers with other diseases, and HD livers. Compared with their counterparts extracted from livers with other diseases and HD, antibodies of IgG and IgA isotypes were highly accumulated in SAH and recognized a unique set of human proteins and E. coli antigens. Further, both Ig- and E. coli-captured Ig from SAH livers recognized common autoantigens enriched in several cellular components including cytosol and cytoplasm (IgG and IgA), nucleus, mitochondrion, and focal adhesion (IgG). Except IgM from primary biliary cholangitis livers, no common autoantigen was recognized by Ig- and E. coli-captured Ig from livers with other diseases. These findings demonstrate the presence of cross-reacting anti-bacterial IgG and IgA autoantibodies in SAH livers.

Monoamine oxidase A (MAO A) is the critical enzyme to degrade serotonin in the brain and the knockout mouse exhibits hyperserotonemia and abnormalities that are observed in autism spectrum disorder (ASD). Thus, the MAO A knockout mouse is a valuable model for studying neurological and behavioral impairments in ASD. Based on the immune dysfunction hypothesis, dysregulated humoral immunity may cause neurological impairments. To address this hypothesis, we use high-density proteome microarray to profile the serum antibodies in both wild-type and MAO A knockout mice. The distingue autoantibody signatures were observed in the MAO A knockout and wild-type controls and showed 165 up-regulated and 232 down-regulated autoantibodies. The up-regulated autoantibodies were prone to target brain tissues while down-regulated ones were enriched in sex organs. The identified autoantibodies help bridge the gap between ASD mouse models and humoral immunity, not only yielding insights into the pathological mechanisms but also providing potential biomarkers for translational research in ASD.

Cell-penetrating peptides (CPPs) have distinct properties to translocate across cell envelope. The key property of CPPs to translocation with attached molecules has been utilized as vehicles for the delivery of several potential drug candidates that illustrate the significant effect in in-vitro experiment but fail in in-vivo experiment due to selectively permeable nature of cell envelop. Penetratin, a well-known CPP identified from the third α-helix of Antennapedia homeodomain of Drosophila, has been widely used and studied for the delivery of bioactive molecules to treat cancers, stroke, and infections caused by pathogenic organisms. Few studies have demonstrated that penetratin directly possesses antimicrobial activities against bacterial and fungal pathogens; however, the mechanism is unknown. In this study, we have utilized the power of high-throughput Saccharomyces cerevisiae proteome microarrays to screen all the potential protein targets of penetratin. Saccharomyces cerevisiae proteome microarrays assays of penetratin followed by statistical analysis depicted 123 Saccharomyces cerevisiae proteins as the protein targets of penetratin out of ~5800 Saccharomyces cerevisiae proteins. To understand the target patterns of penetratin, enrichment analyses were conducted using 123 protein targets. In biological process: ribonucleoprotein complex biogenesis, nucleic acid metabolic process, actin filament-based process, transcription, DNA-templated, and negative regulation of gene expression are a few significantly enriched terms. Cytoplasm, nucleus, and cell-organelles are enriched terms for cellular component. Protein-protein interactions network depicted ribonucleoprotein complex biogenesis, cortical cytoskeleton, and histone binding, which represent the major enriched terms for the 123 protein targets of penetratin. We also compared the protein targets of penetratin and intracellular protein targets of antifungal AMPs (Lfcin B, Histatin-5, and Sub-5). The comparison results showed few unique proteins between penetratin and AMPs. Nucleic acid metabolic process and cellular component disassembly were the common enrichment terms for penetratin and three AMPs. Penetratin shows unique enrichment items that are related to DNA biological process. Moreover, motif enrichment analysis depicted different enriched motifs in the protein targets of penetratin, LfcinB, Histatin-5, and Sub-5.

Schizophrenia (SZ) is influenced by genetic and environmental factors, and associated with chronic neuroinflammation. If the symptoms express after adolescence, environmental impacts are more substantial, and the disease is defined as adult-onset schizophrenia (AOS). Effects of environmental factors on antibody responses such as Escherichia coli (E. coli) to immunoglobulin G (IgG) and immunoglobulin M (IgM) might increase the severity of symptoms in SZ via the gut-brain axis. The purpose of this study is to reveal antibody profiles of SZ against bacterial protein antigens. We analyzed the IgG and IgM antibodies using E. coli proteome microarrays from 80 SZ patients and 40 healthy controls (HC). Using support vector machine to select panels of proteins differentiating between groups and conducted enrichment analysis for those proteins. We identified that the groL, pldA, yjjU, livG, and ftsE can classify IgGs in AOS vs HC achieved accuracy of 0.7. The protein yjjU, livG and ftsE can form the best combination panel to classify IgG in AOS vs HC with accuracy of 0.8. The enrichment results are highly related to ABC (ATP binding cassette) transporter in the protein domain and cellular component. We further found that the human ATP binding cassette subfamily b member 1 (ABCB1) autoantibody level in AOS is significantly higher than in HC. The findings suggest that AOS had different immunoglobulin production compared to early-onset schizophrenia (EOS) and HC. We also identified potential antibody biomarkers of AOS and found their antigens are enriched in ABC transporter related domains, including human ABCB1 protein.

Antimicrobial peptides (AMPs) are intensively studied in terms of alternative drugs. Sub5 is a synthetic 12-mer AMP with substitutions of five amino acids of bactenecin 2A (Bac2A), a linearized bactenecin variant of bovine. Sub5 is highly effective against fungi with an ability to translocate cell membrane, but its targets are unknown. Systematic analysis of Sub5 targets will facilitate our understanding on its mechanism of action. In this study, we used high-throughput Saccharomyces cerevisiae proteome microarrays to explore the potential protein targets of Sub5. The screening results showed 128 potential protein targets of Sub5. Bioinformatics analysis of protein targets of Sub5 revealed significant gene ontology (GO) enrichment in actin related process of “actin filament-based process”, “actin filament organization”, “actin cortical patch organization”, regulation of “actin filament bundle assembly”. Moreover, the other enriched categories in GO enrichment mostly contained actin associate proteins. In total, 11 actin-associated proteins were identified in the protein targets of Sub5. Protein family (PFAM) enrichment analysis shows protein domain enriched in actin binding, i.e., “Cytoskeletal-regulatory complex EF hand (helix E-loop-helix F motif)”. Being consistent with GO analysis, Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) analysis of the protein targets of Sub5 showed actin network with involvement of 15 protein targets. Along with actin-network, STRING analysis showed protein–protein interaction network in ribonucleoprotein, transcription and translation, chromosome, histone, and ubiquitin related, DNA repair, and chaperone. Multiple Expression motifs for Motif Elicitation (MEME) suite provided a consensus binding motif of [ED][ED]EEE[ED][ED][ED][ED][ED], in total of 75 protein targets of Sub5. This motif was present in 9 out of 15 actin-related proteins identified among protein targets of Sub5.

Treatment of peracetylated glycosides and β-isopropyl glycosides with halogen in the presence of TESH and BHQ has been found to result in the halogenation and the anomerization, respectively. Peracetylatedglycosides treaded with I2/TESH or Br2/TESH leading tothe formation of corresponding glycosyl halides, and b-isopropyl glycosidesreacted with Br2/BHQ resulting in the formation of a-glycosides. The anomerizationof glycosidic bond was considered to be catalyzed by in situ formation of hydrogenbromide from the mixing of Br2/BHQ.

With their wide repertoire of mechanisms, antimicrobial peptides (AMPs) are promising alternatives to fight against varied pathogenic microorganisms (bacteria, fungi, viruses, parasites, etc.). AMPs, novel components of the innate immune defense system, are secreted by all organisms. The aquatic environment represents a huge population and an enormous source of varied AMPs. Polyphemusin-I, a marine AMP isolated from hemocytes of an American horseshoe crab, possesses high antimicrobial activities. Studies on polyphemusin-I have verified the intracellular mechanisms of action, however, its intracellular targets are not yet explored. In this study, we employed Escherichia coli proteome microarrays to systematically screen the entire intracellular protein targets of polyphemusin-I. A total of 97 protein targets of polyphemusin-I were statistically analyzed from the quadruplicate Escherichia coli proteome microarrays assays. Among these identified protein targets, 56 proteins had cellular location inside the cell (i.e., cytoplasm), one in the plasma membrane, one in the periplasm and the rest 39 proteins had no specified cellular location. The bioinformatics analysis of these identified protein targets of polyphemusin-I in gene ontology (GO) enrichment category of molecular function revealed significant enrichment in nucleic acid related GO terms i.e., "RNA binding", "nucleotide binding", "nuclease activities", "uracil DNA N-glycosylase activities" and others. Moreover, enrichment in GO category of biological process also depicted enrichment in nucleic acid related GO terms, such as "nucleic acid phosphodiester bond hydrolysis", "deoxyribonucleotide metabolism", and others. In accordance to GO enrichment analysis, protein families (PFAM) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analysis also showed significant enrichment in nucleic acid terms. These enrichment results suggest that polyphemusin-I targets nucleic acid-associated proteins. Furthermore, to provide a comprehensive study, we compared the identified protein targets of polyphemusin-I with previously identified protein targets of four AMPs (P-Der, Lfcin B, PR-39, and Bac 7) using Escherichia coli proteome microarrays. The comparison study of five AMPs (polyhemusin-I, P-Der, Lfcin B, PR-39, and Bac 7) showed only nine common protein targets in all the five AMPs, whereas a total of 39 and 43 common protein targets were identified among the two marine AMPs (polyphemusin-I and P-Der) and three terrestrial AMPs (Lfcin B, PR-39 and Bac7), respectively. To further reveal the target pattern of marine and terrestrial AMPs, the enrichment results obtained from common protein targets of marine AMPs with terrestrial AMPs were compared. The comparison result indicated that AMPs have unique mechanism of action among marine or terrestrial AMPs. Hence, in this study, we have not only identified the intracellular protein targets of polyphemusin-I, but also revealed the protein target differences between marine AMPs and terrestrial AMPs.

Phosphoinositides (PIs) are a family of eight lipids consisting of phosphatidylinositol (PtdIns) and its seven phosphorylated forms. PIs have important regulatory functions in the cell including lipid signaling, protein transport, and membrane trafficking. Yeast has been recognized as a eukaryotic model system to study lipid-protein interactions. Hundreds of yeast PI-binding proteins have been identified, but this research knowledge remains scattered. Besides, the complete PI-binding spectrum and potential PI-binding domains have not been interlinked. No comprehensive databases are available to support the lipid-protein interaction research on phosphoinositides. Here we constructed the first knowledgebase of Yeast Phosphoinositide-Binding Proteins (YPIBP), a repository consisting of 679 PI-binding proteins collected from high-throughput proteome-array and lipid-array studies, QuickGO, and a rigorous literature mining. The YPIBP also contains protein domain information in categories of lipid-binding domains, lipid-related domains and other domains. The YPIBP provides search and browse modes along with two enrichment analyses (PI-binding enrichment analysis and domain enrichment analysis). An interactive visualization is given to summarize the PI-domain-protein interactome. Finally, three case studies were given to demonstrate the utility of YPIBP. The YPIBP knowledgebase consolidates the present knowledge and provides new insights of the PI-binding proteins by bringing comprehensive and in-depth interaction network of the PI-binding proteins. YPIBP is available at http://cosbi7.ee.ncku.edu.tw/YPIBP/.

Microbial proteases play pivotal roles in many aspects of bacterial physiological processes. Because a protease exerts its biological function by proteolytically regulating its substrates, the identification and characterization of the physiological substrates of a protease advance our understanding of the biological roles of the protease. Prc (also named Tsp) is anEscherichia coliperiplasmic protease thought to be indispensable forE. colito survive under low osmolality at 42 degrees C. The accumulation of the Prc substrate MepS due to Prc deficiency contributes to the conditional growth defect. Because preventing MepS accumulation only partially restored the growth of Prc-deficientE. coli, we hypothesized that other unidentified Prc substrates intracellularly accumulate due to Prc deficiency and contribute to the conditional growth defect. To identify previously undiscovered substrates, 85E. coliproteins able to physically interact with Prc were identified usingE. coliproteome arrays. Ten proteins were shown to be cleavable by Prcin vitro. Among these candidates, MltG was able to interact with Prc inE. coli. Prc regulated the intracellular level of MltG, indicating that MltG is a physiological substrate of Prc. Prc deficiency induced the accumulation of MltG in the bacteria. Blocking MltG accumulation by deletingmltGpartially restored the growth of Prc-deficientE. coli. In addition, Prc-deficientE. coliwith blocked MltG and MepS expression exhibited higher growth levels than those with only the MltG or MepS expression blocked under low osmolality at 42 degrees C, suggesting that these accumulated substrates additively contributed to the conditional growth defect. MltG is a lytic transglycosylase involved in the biogenesis of peptidoglycan (PG). In addition to MltG, the previously identified physiological Prc substrates MepS and PBP3 are involved in PG biogenesis, suggesting a potential role of Prc in regulating PG biogenesis.

We used yeast proteome microarrays (∼5800 purified proteins) to conduct a high-throughput and systematic screening of PI5P-interacting proteins with PI5P-tagged fluorescent liposomal nanovesicles. Lissamine rhodamine B-dipalmitoyl phosphatidylethanol was incorporated into the liposome bilayer to provide the nanovesicles with fluorescence without any encapsulants, which not only made the liposome fabrication much easier without the need for purification but also improved the chip-probing quality. A special chip assay was washed very gently without the traditional spin-dry step. Forty-five PI5P-interacting proteins were identified in triplicate with this special chip assay. Subsequently, we used flow cytometry to validate these interactions, and a total of 41 PI5P-interacting proteins were confirmed. Enrichment analysis revealed that these proteins have significant functions associated with ribosome biogenesis, rRNA processing, ribosome binding, GTP binding, and hydrolase activity. Their component enrichment is located in the nucleolus. The InterPro domain analysis indicated that PI5P-interacting proteins are enriched in the P-loop containing nucleoside triphosphate hydrolases domain (P-loop). Additionally, using the MEME program, we identified a consensus motif (IVGPAGTGKSTLF) that contains the Walker A sequence, a well-known nucleotide-binding motif. Furthermore, using a quartz crystal microbalance, both the consensus motif and Walker A motif showed strong affinities to PI5P-containing liposomes but not to PI5P-deprived liposomes or PI-containing liposomes. Additionally, the glycine (G6) and lysine (K7) residues of the Walker A motif (-GPAGTG6K7S-) were found to be critical to the PI5P-binding ability. This study not only identified an additional set of PI5P-interacting proteins but also revealed the strong PI5P-binding affinity (Kd = 1.81 × 10-7 M) of the Walker A motif beyond the motif's nucleotide-binding characteristic.

The polyetherimide diaphragm, sodium copper chlorophyllin (SCC), and copper ion coating composite used on earphones were observed to improve the high-frequency (10k–14k Hz) performance. This reinforcement phenomenon was expected to make the sound experience brighter and more diverse. By SEM observation, the mixed coating of SCC/Cu2+ on the polyethylenimine (PEI) diaphragm exhibited a planar blocky structure and was tightly bonded to the surface of the PEI polymer without the aid of colloids. The endothermic process of SCC and metal ion complexation was analyzed by isothermal titration calorimetry. The association ratios of SCC/Cu2+ and SCC/Ni2+ were 4/1 and 6/1, respectively, and the SCC/Cu2+ association yielded a stronger binding constant and more free energy. It was expected that the SCC/Cu2+(4/1) mixed liquid would be immobilized on the PEI polymer by multivalent interaction, including hydrogen-bonding networks between carboxyl groups of SCC and amine groups of PEI, and cross-linking of bridging copper ions. We used dimethylethylenediamine (DME) monomer instead of PEI polymer to analyze this multivalent interaction and observed a two-stage exothermic association of SCC/Cu2+(4/1) and DME with a total Gibbs free energy of 15.15 kcal/mol. We observed that the binding energy could be used to explain that the SCC/Cu2+ mixed formulation could be fixed on the surface of the PEI polymer and could enhance the strength of the PEI film. Compared with graphene films, which can continuously improve the performance of high and ultrasonic frequencies, this study was devoted to and was initiated for the purpose of applying porphyrin compounds to improve music performance.