Met tyrosine kinase, a receptor for a hepatocyte growth factor (HGF), plays a critical role in tumor growth, metastasis, and drug resistance. Mitochondria are highly dynamic and undergo fission and fusion to maintain a functional mitochondrial network. Dysregulated mitochondrial dynamics are responsible for the progression and metastasis of many cancers. Here, using structured illumination microscopy (SIM) and high spatial and temporal resolution live cell imaging, we identified mitochondrial trafficking of receptor tyrosine kinase Met. The contacts between activated Met kinase and mitochondria formed dramatically, and an intact HGF/Met axis was necessary for dysregulated mitochondrial fission and cancer cell movements. Mechanically, we found that Met directly phosphorylated outer mitochondrial membrane protein Fis1 at Tyr38 (Fis1 pY38). Fis1 pY38 promoted mitochondrial fission by recruiting the mitochondrial fission GTPase dynamin-related protein-1 (Drp1) to mitochondria. Fragmented mitochondria fueled actin filament remodeling and lamellipodia or invadopodia formation to facilitate cell metastasis in hepatocellular carcinoma (HCC) cells both in vitro and in vivo. These findings reveal a novel and noncanonical pathway of Met receptor tyrosine kinase in the regulation of mitochondrial activities, which may provide a therapeutic target for metastatic HCC. Various cytoplasmic effectors, including PI3K, Ras, PLC-γ, Shc, and SHP2, are recruited to the docking site and subsequently activated. Currently, at least 17 Met inhibitors, including JNJ-38877605, GEN-203, and ARQ-197, are undergoing clinical trials.6 However, no selective Met inhibitors, such as SU11274 and Tepotinib, have been proven despite the encouraging wave of recent drug approvals for HCC.10 Due to adverse side reactions and limited therapeutic effects, many clinical trials of selected Met inhibitors were hindered at Stage II or III,12 There still remains a need for a clearer understanding of the HGF/Met pathway to accelerate Met-targeting strategy development.Liver cancer is the second leading cause of cancer death worldwide because of the high rate of metastasis.14 Endosome-carried Met was supposed to be degraded along with the other contents of mature endosomes fusing with lysosomes.16 Following endocytosis, Met was found to signal either from the peripheral endosome to fully activate ERK1/214 and Rac1 or from the perinuclear endosome to activate STAT3.13 Recently, Met was reported to be located in the cell nucleus and phosphorylate PARP to facilitate BRCA inhibitor resistance.17 These findings suggest that Met may be redistributed to multiple organelles after internalization. Kang et al. reported that 38% of proteins whose expression was perturbed by Met inhibition were mitochondrial proteins,18 and a selective mitochondrial-targeting Met kinase inhibitor potently killed erlotinib-resistant lung cancer cells,19 suggesting that Met-regulated mitochondrial activity is strongly associated with cancer cell survival.The internalization of membrane docking Met after HGF stimulation and activation has been studied for nearly two decades.20 Altered mitochondrial dynamics have been linked to abnormal cell functions22 and many human diseases,26 including cancers.27 Studies have shown that excess mitochondrial fission29 and upregulation of the fission component Fis1-Drp1 are frequently involved in tumorigenesis and metastasis.32 How mitochondrial fission regulates cancer cell movements remains elusive. A few lines of evidence showed that the following fission, fragmented mitochondria were distributed to the leading edge of cancer cells to facilitate the formation of lamellipodia or invadopodia, which is a key step in cell migration and invasion.35Mitochondria are highly dynamic organelles that are continually undergoing fission and fusion.In the present study, using structured illumination microscopy (SIM) and high spatial and temporal resolution live cell imaging, we found that Met localized at mitochondrial dividing sites and catalyzed Fis1 Tyr38 phosphorylation to promote mitochondrial fission. Fragmented mitochondria thus facilitated the redistribution of mitochondria to the leading edge of HCC cells to fuel actin filament remodeling and lamellipodia or invadopodia formation. We defined a vital role of Met in the regulation of mitochondrial fission to promote metastasis of HCC cells in vitro and in vivo. Our data suggested a novel and noncanonical pathway of Met receptor tyrosine kinase in the regulation of mitochondrial activities, which may provide a therapeutic target for metastatic HCC.37 and it forces us to think about the possibility of Met localization at mitochondria. So far, none has been reported about the mitochondrial localization signal (MLS) of Met. To predict the probability of Met import to mitochondria, we analyzed the Met protein sequence by using the MitoProt database.38 The MitoProt software predicted MLS and a cleavage site after the first 22 amino acids at the N terminus with a probability score of 0.07. We further identified the localization of Met at mitochondria using conventional confocal microscopy as well as structured illumination super-resolution microscope (SIM). We performed confocal microscopy analysis in Huh7 cells expressing high levels of Met and found that Met had a propensity to colocalize with mitochondria in the absence of any stimuli  Huh7 cells (generated using CRISPR/Cas9-mediated gene editing and previously characterized),40 and found that the expression levels of KD mutant Met were significantly lower than WT Met in the isolated mitochondrial fraction  HeLa cells re-expressed WT or KD Met tagged with an enhanced green fluorescent protein (EGFP–Met–WT or EGFP–Met–KD) and mitochondria-targeting mCherry fluorescent protein (mCherry–Mito). Approximately, 13% of EGFP–Met–WT was tethered with mitochondria, and HGF treatment markedly triggered Met mitochondrial localization  and kinase-dead ion Fig. . Similarion Fig. , whereasion Fig. . Overall41 type of Met was mainly maintained in the cell membrane, with a small percentage located in mitochondria, in agreement with the observations above ,42 did not show mitochondrial localization and was diffusely distributed in the cytoplasm as well as vesicles  protein was expressed in the isolated mitochondrial fraction and the expression levels were significantly increased with HGF treatment Fig.  but decrove Fig. . Howeverles Fig. , indicat43 Dysregulation of Drp1 has been reported to contribute to tumor growth, metastasis, and chemoresistance.35 After HGF treatment, we assessed mitochondrial localization of Drp1 and found that HGF significantly stimulated Drp1 expression in isolated mitochondrial fractions  and WT, kinase dominated (KD), kinase-truncated (KT), or kinase-only Met tagged with EGFP. We subjected these cells to live SIM imaging to examine the actions of Met at mitochondria division sites. In WT cells treated with HGF, we noticed that mitochondrial fission sites were predominantly marked by Met protein before the event happened (white arrows) and the dynamic process of Met induced mitochondria division could be observed . We identified mitochondrial fission protein (Fis1) interacting directly with Met Fig. . Met intWe then tested whether Met kinase would catalyze the phosphorylation of Fis1 tyrosine residue with an in vitro kinase assay. We verified that the total phosphorylation of Fis1 was promoted by Met kinase but attenuated with Met kinase inhibition or phosphatase addition Fig. , suggest44 Therefore, we attempted to determine that whether Met-mediated phosphorylation of Y38 of Fis1 would affect Drp1 mitochondrial assembly and mediate mitochondrial fission.Fis1 was reported to recruit Drp1 to mitochondria and facilitate mitochondrial fission. Structurally, two regions of Fis1 have been previously implicated in Drp1 recruitment: an autoinhibitory N-terminal “arm”(NTE) and a concave surface formed by Fig evolutionarily conserved residues in the tetratricopeptide repeat (TPR) domain. Whereas NTE and TPR domains are insufficient to regulate Drp1 binding to Fis1, and there may be the mitochondrial outer membrane or a covalent modification, such as phosphorylation.Met−/− Huh7 cells re-expressed with KD-Met exhibited reduced mitochondrial expression of Drp1 compared to cells re-expressed with WT-Met  assay and the association was significantly enhanced with HGF stimulation Fig.  but atteFis1−/− cells, Y38E mutant Fis1 showed significantly enhanced association with Drp1 compared to WT Fis1, whereas the Y38F mutant exhibited only a weak interaction, as showed by SE-FRET assay in HeLa cells and co-IP experiments in Huh7 cells  or nonphosphorylatable phenylalanine . When re-expressed in lls Fig. . Meanwhills Fig. , supportlls Fig. . It was 46 In contrast to the dynamic binding between phosphorylated Fis1 and Drp1 relying on Met kinase, interactions between Drp1 and Mff or MiD51/49 stayed consistent irrespective of the phosphorylation state of Fis1, indicating that Fis1 pY38 promoted Drp1 mitochondrial recruitment independent of other receptors   Fig. . Togethe34 However, in cells undergoing migration or invasion, extra ATP synthesized mainly in mitochondria is required for cytoskeletal rearrangement. Zhao et al. reported that in invasive breast cancer cells, mitochondrial fission regulates the invasion and migration of breast cancer cells by redistribution of mitochondria in the leading edge of cells to fuel lamellipodial formation.35 However, the mechanism under which mitochondrial fission is initiated and monitored under the condition of cell metastasis is not clearly understood. Our study elucidated a new mechanism by which Met, a tyrosine kinase receptor, directly regulates fission and promotes liver cancer metastasis.Reorganization of the cytoskeleton is crucial for the motility of tumor cells and requires the consumption of a large amount of ATP. In terms of energy metabolism, tumor cells are abnormal in their abundance of biosynthesis during proliferation, which requires a high level of aerobic glycolysis.50 Therefore, Met may play an important role in drug resistance and metastasis rather than EGFR. We found that Met could be directly located in mitochondria and regulate mitochondrial fission through kinase activity. HGF stimulation enhanced mitochondrial fission, while Met inhibitor treatment significantly suppressed mitochondrial fission. The invasion and migration ability is in line with Met kinase-regulated mitochondrial fission, as knocking out Fis1 resulted in hampered mitochondrial fission and cell invasion and migration, which would be prompted by HGF stimulation. Therefore, through the Met tyrosine kinase receptor, the signal carried by extracellular HGF is thus transmitted to mitochondria and promotes the metastasis of tumors by activating mitochondrial fission. Our work provides a novel perspective to better understand the tyrosine kinase receptor Met and may be helpful for targeting therapeutics.Met shares many structural and functional similarities with EGFR. Both are protein tyrosine kinase receptors and can promote cell proliferation and tissue renewal under physiological conditions. Overexpression and activation of Met have been reported frequently in advanced non-small cell lung cancers with resistance to EGFR inhibitors or antibodies.51 It is generally accepted that Fis1 participates in the recruitment of Drp1 to mitochondria either directly or through the regulation of other structural proteins, such as MFF and Mid49/51.52 It has been reported that overexpression of Fis1 promotes the occurrence of mitochondrial fission in either normal or cancer cells.30 Interestingly, in some cases, Fis1 is not necessary for mitochondrial fission in eukaryotic cells, and the cells with Fis1 knockout can replicate and proliferate without obvious changes in mitochondrial morphology.53 According to these findings, we infer that under static conditions, the role of Fis1 in mediating mitochondrial fission may be substituted by other components; however, under certain circumstances, such as tumor cells undergoing migration or invasion with high energy requirements, Fis1 can be activated and strongly promotes mitochondrial fission for special energy demands. Our studies demonstrate that Met phosphorylates Y38 of Fis1 in vitro and in vivo, and the phosphorylation of Fis1 activates its ability to recruit Drp1 and facilitate mitochondrial fission. HCC cells with Y38E (phosphomimetic) mutant Fis1 exhibited enhanced migration and invasion in vitro and in vivo. It has been reported that the Fis1 N-terminal region (1–20 amino acids) can form an autoinhibitory structure, which blocks Drp1 recruitment. Y38 phosphorylation could abolish Fis1 N-terminal negative structure so that Drp1 binding increased with Fis1.44 By IHC staining of liver cancer tissues, we also confirmed that p-Fis1 was a good predictor of recurrence and overall survival of patients with HCC, and high expression of p-Fis1, as well as HGF, was positively associated with poor prognosis. Therefore, our work provides closer insight into the indeterminate role of Fis1 protein, illuminating that Fis1 is particularly activated in tumor cells that are migrating and invading with large energy demands.Fis1 is an important recruiting protein on the outer membrane of mitochondria. In prokaryotic cells, Fis1 assembles with Drp1 on the outer mitochondrial membrane to form a Drp1 ring and initiates mitochondrial fission by ring contraction and scission. In eukaryotic cells, the component of the mitochondrial fission complex is much more intricate and involves more members and regulatory mechanisms.55 Cytoplasmic signaling cascades mediated by RAC1/CDC42 and PAK elicit cytoskeletal changes for cell motility.57 Signals through the RAP1 and RAC1/CDC42 pathways reach the plasma membrane and control cadherin and integrin adhesion molecules and thereby affect cell migration.58 Our study further improved the downstream regulation network of Met in tumor metastasis. As wound healing assay showed, HGF induced enhanced cell migration was almost blocked by Fis1 knockout  to construct mCherry-Met and EGFP-Met. Met-KD cDNA was subcloned into pEGFP-C1 Vector to construct EGFP-Met-KD. Using EGFP-Met expression vector as a template, depletion mutants including KT EGFP-Met and kinase-only EGFP-Met were developed. Human full-length Met cDNA  was subcloned into pcDNA3.1 Vector . Human full-length Fis1 cDNA  was subcloned from FIS1 (Myc-DDK-tagged)  and inserted into pcDNA3.1 Vector, PCDH-EF1-MCS-IRES Vector , or pGEX6P1 Vector . Using pcDNA3.1-Fis1-Flag as a template, several deletion mutants including Flag-Fis1-△α1, Flag-Fis1-△TPR1, Flag-Fis1-△TPR2, Flag-Fis1-(TPR2 + CT) were developed. Using PCDH-Fis1-Flag as a template, mutant plasmids including PCDH-Fis1-Flag-Y38E, PCDH-Fis1-Flag-Y38F, PCDH-Fis1-Flag-Y87E, PCDH-Fis1-Flag-Y87F, PCDH-Fis1-Flag-Y38/87E, PCDH-Fis1-Flag-Y38/87F were developed by performing site-directed mutagenesis. Human full-length Fis1 cDNA, Fis1-Y38F cDNA, Fis1-Y87F cDNA, Fis1-Y38/87F cDNA were subcloned into pGEX6P1 Vector with GST encoding sequence replaced by 6× His encoding sequence. Human full-length Fis1 cDNA, Fis1-Y38E cDNA, and Fis1-Y38F cDNA were subcloned into pECFP-C1 Vector . mCh-Drp1 (#49152) was obtained from addgene.34 Recombinant human HGF (CYT-244) was obtained from Prospec. Met inhibitors crizotinib , tivantinib , SU11274 (S1080), and mitochondrial fission inhibitor mdivi-1 (S7162) were purchased from Selleck Chemicals.To construct pLYS1-mCherry-Mito, mCherry cDNA was subcloned from pmCherry-C1 Vector  and inserted into pLYS1-FLAG-MitoGFP-HA HeLa cells expressing mCherry-Met with mitochondria stably marked by EGFP or Huh7 cells expressing WT or mutant EGFP-Met with mitochondria stably marked with mCherry were plated on 20-mm glass-bottomed dishes (NEST) at a density of 10,000 cells per dish. Images of cells were acquired using Olympus live cell imaging system using 100× oil objectives (for representative time-lapse images) at excitation wavelengths of 488 and 559 nm for EGFP and mCherry, respectively. CV1000 (YOKOGAWA) was used for imaging obtain. For mitochondrial fission events observation, images of cells were snapped at the interval of 15 s for 200 times with 7 slices ranging 3 µm.2 at 1 frame every 2–3 s. Dual-color videos were acquired as consecutive green–red images and tricolor videos were acquired as consecutive green–red–blue images.All confocal images were acquired on Olympus FV1000 laser scanning confocal microscope with GaAsp detectors using a PlanApo λ 100 × 1.45 NA oil immersion objective (Olympus). Live cells were imaged in a temperature-controlled chamber (37 °C) at 5% CO2 in a TokaiHit stage top incubator.SIM super-resolution images were taken on a Nikon N-SIM system with a 100× oil immersion objective lens, 1.49 NA (Nikon). Images were captured using Nikon NIS-Elements and reconstructed using slice reconstruction in NIS elements. Images for live-cell imaging (live N-SIM) were taken at a single Z-plane, while images of fixed cells for 3D N-SIM were taken using Z-stacks with step sizes of 0.12 µm. Cells used for live-cell imaging were maintained in a temperature-controlled chamber (37 °C) at 5% COn = 48 cells were analyzed per condition for Fis1 (WT), Fis1 (Y38E), and Fis1 (Y38F), and the FRET intensity was normalized to average SE-FRET values for Fis1 (WT).HeLa cells were plated on 20-mm glass-bottomed dishes (NEST) at a density of 10,000 cells per dish. The following day, cells were transfected using lipofectamine with FRET pairs . Cells were fixed in 4% (vol/vol) paraformaldehyde for 10 min and incubated in phosphate-buffered saline. Images of fixed HeLa cells were acquired using Olympus confocal microscope using 100× objectives (for representative time-lapse images) at excitation wavelengths of 453 nm, and 561 nm for ECFP and mCherry, respectively. FV1000 (Olympus) was used for FRET analysis to calculate SE-FRET and to unbiasedly generate regions of interest by tracing individual cells in the red fluorescence view. A total of 35 The expected probability that a Met dots would be at the site of a mito-fission event by random chance was calculated as the density of Met dots in the cytosol from n = 25 living cells, using ImageJ ). All contacts analyzed for the duration were those that had already formed at the beginning of the video. The minimum duration of contact in HeLa cells was quantified as the time before contact termination and dissociation (Met dots and mitochondria detaching from one another) over a 5 min (300 s) video. Any contacts that lasted throughout the entire 5-min video and were still in contact by the end of the video were categorized as 300 s in bar graphs and as >5 min in histograms for the minimum duration of Met mitochondrial contacts. The percentage of Met in contacts with mitochondria was quantified as the percentage of vesicles that formed contacts (defined above) with mitochondria divided by the total number of vesicles in the region of interest. The duration of contact in cells was quantified from videos of ≥300 s. Mitochondrial morphology was analyzed using ImageJ (NIH), MiNA-Master system. The rate of mito-fission was defined by calculating the number of fission events per cell from videos of n = 25 living cells and ≥300 s.The mitochondrial skeleton structure was reconstructed using Imaris software. Mitochondrial localization of Met was defined as those Met dots overlapping with mitochondria over a quarter of its area. Mitochondrial contacts of Met imaged in living cells were categorized as those that showed mitochondria and Met dots in close proximity (<0.1 µm) for >10 s in time-lapse images. Mito-fission events were defined as those that showed the clear division of a single mitochondrion into two distinct daughter mitochondria that moved independently of one another after division.t test was used to compare two groups of independent samples. Repeated measure ANOVA analysis was used to evaluate the statistical significance of dose curve response. Correlations were analyzed using the Pearson chi-square test. A p value < 0.05 was considered statistically significant.Unless otherwise noted, each experiment was repeated at least three times. All error bars represent the standard error of the mean. Student’s Supplementary Movie S1Supplementary Movie S2Supplementary Movie S3Supplementary Movie S4Supplementary Movie S5Supplementary Movie S6Supplementary Movie S7Supplementary Materials


Immunoglobulin G4-related disease (IgG4-RD) is a fibro-inflammatory disease characterized by organ enlargement and elevated serum IgG4 levels. In 2003, IgG4-RD was proposed as a distinct form of IgG4-related systemic disease based on a histopathological study involving patients with autoimmune pancreatitis. IgG4-RD occurs mainly in older men and can affect almost any organ simultaneously or metachronously. Pathophysiologically, IgG4-RD occurs when an autoantigen triggers an immune response characterized by Th2 predominance with increased production of cytokines, such as interleukin 4 (IL-4), IL-5, IL-10, IL-13, and tumor growth factor-β (TGF-β), in the affected organ. IL-10 and TGF-β produced by the increased number of regulatory T cells induce a switch from B cells to IgG4-producing plasma cells and fibrosis, respectively. The characteristic histological features consist of dense infiltration of lymphocytes and IgG4-positive plasma cells, storiform fibrosis, and obliterative phlebitis. IgG4-RD is diagnosed based on a combination of clinical, serological, radiological, and histopathological findings. Differentiating IgG4-RD from malignant tumors or similar inflammatory diseases in the affected organs is important. The 2019 America College of Rheumatology/European League against Rheumatism classification criteria for IgG4-RD have high diagnostic sensitivity and specificity. IgG4-RD generally responds well to treatment with steroids, and a swift response is reassuring and provides further diagnostic confirmation. However, relapses are common during tapering or after cessation of steroids. In Japan, low-dose steroid maintenance therapy is usually given to prevent a relapse. B-cell depletion with rituximab is effective in patients resistant to or dependent on steroids. Most patients with IgG4-RD who receive steroid therapy show good short-term clinical, morphological, and functional outcomes. However, long-term outcomes, such as relapse, fibrosis development, and associated malignancies, have not been clearly defined. Therefore, novel treatment strategies, including rituximab, need to be tested in international randomized controlled clinical trials. It can essentially involve any organ or site simultaneously or metachronously (2). LPSP is characterized by dense infiltration of lymphocytes and plasma cells with fibrosis in the pancreas. In 1995, the concept of autoimmune pancreatitis (AIP) was proposed based on a case of chronic pancreatitis with hypergammaglobulinemia, which improved with steroid therapy (3). Hamano et al. reported significant elevation of serum IgG4 levels in patients with AIP in 2001 (4) and infiltration of IgG4-bearing plasma cells in the pancreas and ureter of three patients with AIP and retroperitoneal fibrosis in 2002 (5). Kamisawa et al. (6) detected histopathologically dense fibrosis with abundant infiltration of IgG4-positive plasma cells in the pancreas and other involved organs, such as the peripancreatic tissue, cervical lymph nodes, biliary tract, and salivary gland in seven patients with AIP, and pointed out a close relationship between AIP and multifocal fibrosclerosis (7), an uncommon fibroproliferative systemic disorder with multiple manifestations, including retroperitoneal fibrosis, sclerosing cholangitis, fibrotic orbital pseudotumor, and salivary gland fibrosis. Furthermore, they found abundant IgG4-positive plasma cell infiltration in various organs and sites, such as the liver, stomach, and colon, in patients with AIP (8). On the basis of these findings, in 2003, the authors proposed “IgG4-related systemic disease” as a new clinicopathological systemic entity (9).A peculiar histological entity of lymphoplasmacytic sclerosing pancreatitis (LPSP) was proposed in 1991 based on a histopathological analysis of a resected specimen of mass-forming pancreatitis that was initially thought to be pancreatic cancer (10), and the condition was called Mikulicz’s disease. However, Mikulicz’s disease has been classified in the West as an atypical type of Sjögren’s syndrome. Yamamoto et al. reported elevated serum IgG4 levels in patients with Mikulicz’s disease in 2004 (11). In 2005 (12), they reported an infiltration of numerous IgG4-positive plasma cells in the lacrimal and salivary glands of patients with Mikulicz’s disease and pointed out some clinical differences between Mikulicz’s disease and Sjögren’s syndrome, including the sex ratio and the degree of keratoconjunctivitis sicca and steroid responsiveness. On the basis of these findings, they proposed that Mikulicz’s disease is systemic and renamed it systemic IgG4 plasmacytic syndrome in 2006 (13). In 2010, these various entities were unified under the single term, IgG4-RD, by Japanese research teams (14).In 1892, Mikulicz et al. described a patient with symmetrical enlargement of the lachrymal, parotid, and submandibular glands with massive infiltration of mononuclear cells (1).No simple diagnostic test currently exists for IgG4-RD, which comprises many conditions previously thought to be isolated, single-organ diseases with no known underlying systemic disease. Further, IgG4-RD often presents with findings that mimic malignancy both clinically and radiologically. Therefore, diagnosing IgG4-RD remains a major clinical challenge. To provide the appropriate therapy, it is critically important to differentiate IgG4-RD from malignant tumors and similar inflammatory diseases in the affected organ in a timely and accurate manner (15) estimated that 13,436 patients had AIP and that the overall prevalence of cases was 10.1 per 100,000 population. The estimated number of newly diagnosed cases was 3,984, with an annual incidence of 3.1 cases per 100,000 population. Both numbers had more than doubled over the figures in the 2011 survey. This apparent increase may be attributable to more widespread awareness of the disease concept and refinements in the diagnostic criteria. The male-to-female sex ratio was 2.94, and the mean age at diagnosis was 64.8 years. IgG4-RD occurs mainly in older men.As IgG4-RD is relatively rare and not well known, its epidemiology is difficult to ascertain. A nationwide epidemiological survey of AIP in Japan in 2016 (17).Multiple pathogenic factors have been proposed as playing a key role in IgG4-RD development, but the pathogenic mechanism of the disease remains unclear. Initially, autoantigens trigger an immune response characterized by Th2 predominance and increased production of cytokines, such as interleukin 4 (IL-4), IL-5, IL-10, and IL-13, as well as tumor growth factor-β (TGF-β) in the affected organ. IL-10 and TGF-β produced by the increased number of regulatory T cells induce the switch from B cells to IgG4-producing plasma cells and fibrosis, respectively. IgG4 itself does not seem to be a driver of the pathogenesis (19).Abnormal innate immunity has also been demonstrated in some patients with IgG4-related disease. Watanabe et al. reported that activation of nucleotide-binding oligomerization domain-2 and Toll-like receptor ligands on monocytes or basophils from patients with AIP enhanced the IgG4 response via B-cell activating factor and IL-13 (20).Shiokawa et al. reported that laminin-511 is a target antigen in AIP. An autoantibody against laminin-511 was identified in 26 (51%) of 51 patients with AIP, and immunization of mice with human laminin-511 induced antibodies and pancreatic injury fulfilling the pathological criteria for human AIP Together with storiform fibrosis and obliterative phlebitis, dense infiltration of lymphocytes and plasma cells that are immunohistochemically positive for anti-IgG4 antibody are the characteristic histopathological features of IgG4-RD . Storifo(21).At present, the characteristic histology and immunohistochemistry of IgG4-RD represent the gold standard of diagnosis. However, biopsies often provide an insufficient amount of tissue for diagnostic confirmation. In addition, significant infiltration of IgG4-positive plasma cells in biopsied specimens is not specific to IgG4-RD, and this has been reported in other conditions commonly known to mimic IgG4-RD, including malignancies. Semiquantitative analysis of IgG4 immunostaining may help in distinguishing IgG4-RD from similar conditions. A frequently used cutoff value for IgG4-positive plasma cell infiltration is >10 cells per high power field, but this varies by tissue. Evaluating the ratio of IgG4-positive/total IgG-positive cells, typically with a minimum ratio of 40%, is also reportedly useful (21). Samples from previously biopsied or resected specimens may be useful if they are reviewed along with the IgG4-immunostaining of paraffin-embedded specimens.Although the findings of storiform fibrosis and obliterative phlebitis can enhance diagnostic specificity, correlation with clinicopathological findings is essential for a definitive diagnosis (22) are as follows: cellular infiltrates suspicious for malignancy that have not been investigated sufficiently, markers consistent with inflammatory myofibroblastic tumor, prominent neutrophilic infiltration, necrotizing vasculitis, prominent necrosis, primary granulomatous inflammation, and pathologic features of a macrophage/histiocytic disorder.The pathological exclusion criteria of the American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) classification criteria for IgG4-RD  (22) are(23).IgG4-RD is a systematic disease affecting various organs with organomegaly or hypertrophy. The clinical symptoms of IgG4-RD depend on the individual pattern of organ involvement and the severity of disease activity. Patients with IgG4-RD sometimes have a history of allergic disease or atopic features (24). Severe constitutional symptoms are rare, but organomegaly or hypertrophy can occasionally lead to serious complications, such as obstruction or compression, including obstructive jaundice, visual disturbance, and hydronephrosis in AIP or IgG4-related sclerosing cholangitis (IgG4-SC), IgG4-related dacryoadenitis, and IgG4-related retroperitoneal fibrosis, respectively. In addition, there are previous reports of persistent inflammation in the affected organs resulting in fibrosis and permanent organ dysfunction or failure (25). The organs most frequently involved are the pancreas and biliary tract, followed by the salivary glands, kidneys, and lachrymal glands (26).Most cases follow a subacute or chronic course, whereas others show spontaneous improvement (27) independent of predominant organ involvement have also been proposed. The 2019 ACR/EULAR classification criteria for IgG4-RD ((22).Diagnosis relies on a combination of various clinical, laboratory, radiological, and histopathological findings. Specific diagnostic criteria have been developed for several organs, and comprehensive diagnostic criteria for IgG4-RD  IgG4-RD  consist (28). In serological testing, polyclonal hypergammaglobulinemia, elevated immunoglobulin E levels, the presence of antinuclear antigen, and the presence of rheumatoid factor were found in 61%, 58%, 30%, and 20% of patients with IgG4-RD, respectively (28). Hypocomplementemia was found in 53% of patients with IgG4-related kidney disease (29).Serum IgG4 levels are frequently elevated in patients with IgG4-RD. In a previous study, elevated serum IgG4 levels (≥ 135 mg/dL) were reported in 84% (1586/1883) of patients with IgG4-RD (mean: 769 mg/dL) (22).ACR/EULAR serological exclusion criteria have been developed for leukopenia and thrombocytopenia with no explanation, peripheral eosinophilia, and positive disease-specific autoantibodies 18F-fluorodeoxyglucose positron emission tomography (PET) are useful in diagnosing IgG4-RD. Diffuse or focal swelling of organs or soft tissue masses appears with soft tissue attenuation, well-defined margins, and homogeneous, late-stage enhancement on enhanced CT images of IgG4-RD (30).Computed tomography (CT), magnetic resonance imaging (MRI), and (32).IgG4-RD responds well to steroids, so a swift response to steroids can help in confirming the presence of IgG4-RD in patients with collateral evidence. A steroid trial can be performed if enough biopsy specimens cannot be obtained and differentiating IgG4-RD from malignancy is difficult. However, a careful, diagnostic steroid trial should only be conducted after a negative work-up for malignancy that includes a histopathological approach or in cases where imaging modalities can be used to evaluate the effects of steroid therapy because symptomatic and hematological improvement can occur nonspecifically in response to steroids, even in cases of malignancy (30). The common clinical presentation of this disease involves painless obstructive jaundice (49%) and abdominal pain (26%), with about one-third of patients being asymptomatic (15). The International Consensus Diagnostic Criteria (32) recommend a combination of serum IgG4 level, characteristic imaging, other organ involvement, histology, and steroid responsiveness. Characteristic imaging findings of AIP show diffuse enlargement of the pancreas with delayed enhancement on enhanced CT ((30).In AIP, the pancreatic duct epithelium is well preserved despite the surrounding inflammation anced CT  accompananced CT . A capsu(33), whereas IgG4-SC is associated with 49% of AIP cases (15). In patients with IgG4-SC, jaundice is the most common presenting symptom, followed by pruritus and abdominal pain. However, 37% of cases are diagnosed in the absence of any symptoms (33). Histologically, transmural wall thickening with fibrosis and lymphoplasmacytic infiltration is observed ((31).AIP is the most commonly reported comorbidity (84%) in IgG4-SC observed  (31).(34).In IgG4-SC, stenosis of the lower bile duct occurs most frequently. When the hilar and/or intrahepatic bile ducts are involved, differentiating IgG4-SC from primary sclerosing cholangitis (PSC) and cholangiocarcinoma is crucial . In IgG4(36).Two types of hepatic parenchymal involvement have been reported in IgG4-RD. IgG4-related hepatopathy presents as heterogenous lesions in the liver parenchyma of patients with AIP or IgG4-SC. Histological findings include portal inflammation, large bile duct damage, portal sclerosis, lobular hepatitis, and cholestasis, which can be explained as the secondary cause of sclerosing cholangitis or obstruction of the extrahepatic biliary tract (37).IgG4-related autoimmune hepatitis is clinicopathologically similar to classical autoimmune hepatitis with high-grade necro-inflammatory activity, except for elevated serum IgG4 levels and increased IgG4-positive plasma cell infiltration in the liver (38).Two types of IgG4-related gastrointestinal disease have been identified. The first one involves a gastrointestinal lesion accompanied by marked thickening of the esophageal and gastric walls consisting of dense fibrosis with abundant IgG4-positive plasma cell infiltration, typically with submucosal spread. The second one is IgG4-related pseudotumor with a polypoid or mass-like form occurring in the stomach, colon, or major duodenal papilla (39).Notohara et al. reported that IgG4-related gastrointestinal disease is found in the context of IgG4-RD. Two unique histological patterns are striated inflammatory lesions in the muscularis propria giving rise to thickening and bottom-heavy plasmacytosis in the mucosa (14). Newly proposed diagnostic criteria for IgG4-related dacryoadenitis and sialadenitis (40) include symmetrical swelling of the lachrymal, parotid, or submandibular glands for at least three months along with sonographic features of the submandibular glands, such as “hypoechoic areas of the nodal pattern with high vascularity” and/or “hypoechoic areas of reticular patterns surrounding normal parenchyma.” Differential diagnosis is necessary to distinguish IgG4-related dacryoadenitis and sialadenitis from Sjögren’s syndrome, sarcoidosis, Castleman disease, Wegener’s granulomatosis, lymphoma, and cancer. Fewer sicca symptoms are seen, and anti-SSA and SS-B antibodies are rarely positive. In IgG4-related sialadenitis, the submandibular glands are more commonly affected than the parotid glands (1).Both Mikulicz’s disease, which consists of bilateral symmetrical swelling of the lacrimal and salivary glands, and Küttner’s tumor, a type of chronic sclerosing sialadenitis presenting with firm, asymmetrical swelling of the submandibular gland, are currently recognized as forms of IgG4-RD (42).In IgG4-related dacryoadenitis, other tissues besides the lacrimal glands, such as extraocular muscles, orbital fat tissue, the eyelids, the trigeminal nerve, and the nasolacrimal duct, may be involved . Clinica(43). Hanaoka et al. (44) reported that all 10 patients in their study with IgG4-related rhinosinusitis also had multiple forms of IgG4-RD. The clinical symptoms of IgG4-related chronic rhinosinusitis include nasal obstruction, nasal discharge, postnasal discharge, hyposmia, and dull headache. Radiological findings include obstruction of the way of elimination, thickening of the sinus mucous membrane, and fluid in the sinus. A deviated nasal septum, polyps, and edema of the mucous membrane are frequently seen in nasal endoscopy.IgG4-RD also involves nasal manifestations with chronic rhinosinusitis. Moteki et al. reported that IgG4-related rhinosinusitis occurred in 32% of 31 patients with IgG4-RD (45). Shimizuguchi et al. (46) detected hypothyroidism in 8 (10%) of 77 patients with AIP and subclinical and central hypothyroidism in six and two patients, respectively. Thyroid diseases associated with IgG4 include autoimmune thyroid disease and Riedel’s thyroiditis. According to the 2021 diagnostic criteria for IgG4-related thyroid disease (47), IgG4-related thyroiditis is diagnosed definitively if all of the following criteria are met: enlargement of the thyroid, hypoechoic lesions in the thyroid on ultrasound, elevated serum IgG4 levels, and histopathological findings of the thyroid lesion. IgG4-related thyroiditis is frequently confused with a malignancy because of the intense sclerosis of the thyroid, resulting in a hard texture on palpation compounded frequently by associated compressive symptoms.Patients with AIP are highly positive for antithyroglobulin antibodies and susceptible to hypothyroidism (48).The main symptoms of IgG4-related hypophysitis result from a mass lesion in the pituitary gland, hypopituitarism, or from central diabetes insipidus. Also frequently found are decreased levels of one or more hormones from the anterior pituitary gland or the affected organs, decreased response of anterior pituitary hormones on a stimulation test, and diffuse enlargement of the pituitary gland and/or stalk on imaging studies (49). Low-density hypertrophic thickening or soft tissue masses around the aorta and its major branches, sometimes accompanied by luminal dilatation or aneurysm formation, and soft tissue masses in the renal pelvic wall that are sometimes associated with hydronephrosis are characteristic abdominal radiological findings of IgG4-related periaortitis/periarteritis and retroperitoneal fibrosis ((51).IgG4-RD is reportedly the cause of as many as two-thirds of all cases of idiopathic retroperitoneal fibrosis, also known as Ormond’s disease fibrosis . It is i(52), rupturing of affected vessels after steroid therapy has also been reported (53). The risks and benefits of steroid therapy for periarterial lesions remain controversial (51).Severe complications include aneurysm formation, vessel rupture, and renal dysfunction resulting from hydronephrosis. Although the first two complications are known to occur in the absence of steroid therapy (51).As hydronephrosis caused by IgG4-related retroperitoneal fibrosis is known to lead to irreversible renal impairment, starting steroid therapy early in combination with urological surgical interventions, such as stent placement or nephrostomy, is important, depending on the presence of a urinary tract infection or renal impairment (55).IgG4-related respiratory disease is associated with a limited number of subjective symptoms and is usually detected in patients with extra-thoracic IgG4-RD lesions during work-up. In some patients, IgG4-related respiratory disease can manifest nonspecific clinical symptoms, which can delay the diagnosis. On chest CT, the most common abdominal shadow is hilar and mediastinal lymphadenopathy, followed by thickening of the bronchial wall, bronchovascular bundles, and interlobular septal wall, nodular and infiltrative shadows, and pleural thickening/effusion (58). As treatment delay is associated with partial scarring of the kidneys on imaging studies, steroid therapy should be initiated as soon as possible after IgG4-TIN is diagnosed (56).The most representative lesion in IgG4-related kidney disease is plasma cell-rich tubulointerstitial nephritis (TIN); membranous glomerulonephritis is also a representative glomerular lesion. Decreased renal function, mild proteinuria, and mild hematuria are typical clinical features of IgG4-related kidney disease, but most kidney lesions are incidentally detected during the examination for other organ involvement using contrast-enhanced CT in patients already known to have IgG4-RD. Abnormal renal radiological findings include multiple, low-density lesions on enhanced CT ; diffuse(60). Satou et al. suggested that IgG4-related lymphadenopathy consists of five histological subtypes and proposed exclusion criteria to distinguish them as follows: continued elevation of serum CRP levels, elevated serum IgA and IgM levels, pathological findings of a sheet-like proliferation pattern of mature plasma cells, high degree of hemosiderin deposition, and neutrophilic infiltration (61).Enlarged lymph nodes are usually asymptomatic and therefore sometimes detected incidentally on imaging examinations. Lymph node lesions in IL-6 syndromes, such as plasma cell-type Castleman disease and rheumatoid arthritis, mimic IgG4-related lymphadenopathy (62).IgG4-related hypertrophic pachymeningitis is restricted to the dura and causes headaches by its traction, as well as irritation of the trigeminal nerve and higher central roots (63).IgG4-related cholecystitis is seen in about half of patients with IgG4-SC as a diffuse thickening of the gallbladder wall with a smooth inner surface (64).IgG4-related skin lesions present as erythematous, itchy plaques, or subcutaneous nodules of the head and neck, particularly in the periauricular, cheek, and mandibular areas, which are frequently accompanied by other extracutaneous forms of IgG4-RD (65).IgG4-related prostatitis is occasionally accompanied by IgG4-RD in other organs and is detected incidentally on PET. Some patients experience a resolution of urinary symptoms after steroid treatment (66).IgG4-related mastitis is an IgG4-related inflammatory pseudotumor of the breast and should be differentiated radiologically from breast cancer and histologically from mucosa-associated lymphoid tissue lymphoma (67).IgG4-related mesenteritis shows a hypoattenuating mass with or without encasement of mesenteric vessels in the mesentery, and its preoperative diagnosis is quite difficult (68).In IgG4-related mediastinitis, compression of vital, mediastinal structures can result from proliferation of invasive fibrous tissue within the mediastinum (24) recommend prednisolone 0.6 mg/kg/day  for three months to induce remission. Clinical improvement after the start of steroid therapy is typically rapid, so follow-up serological and imaging assessments should be conducted about two weeks later. As patients with IgG4-RD frequently experience a relapse during steroid tapering or cessation, maintenance therapy with low-dose prednisolone (2.5-5 mg per day) for 6-36 months is usually given in Japan to maintain remission and prevent relapses ((69) and a randomized controlled trial (70) showed that low-dose maintenance steroid treatment was effective in reducing the relapse rate in patients with AIP. A poor response to steroid therapy should increase the possibility of another diagnosis, particularly cancer. The response to steroid can vary depending on the affected organ and degree of fibrosis. If left uncontrolled, several types of IgG4-RD can lead to irreversible damage to certain organs, such as aortitis, retroperitoneal fibrosis, sclerosing cholangitis, TIN, pachymeningitis, and pericarditis (25). Thus, urgent treatment is recommended in such cases.It is essential that an accurate diagnosis of IgG4-RD is confirmed before starting steroid treatment. Steroids are the first-line treatment for IgG4-RD. The Japanese guidelines relapses . A retro(24). However, a retrospective study comparing immunosuppressants with steroid monotherapy in the treatment of AIP relapses found no significant difference in relapse-free survival between the treatments (71).Steroid readministration or dose-increase has been shown to be effective in cases of relapsed IgG4-RD. When the steroid dose cannot be tapered because of a persistently active disease, the addition of immunosuppressants, such as azathioprine, mycophenolate mofetil, or 6-mercaptopurine, is appropriate (71). In a prospective study, a disease response was observed in 97% of patients with IgG4-RD who were treated with rituximab. The effects of rituximab in IgG4-RD have been attributed, at least in part, to a failure of repletion of short-lived plasma blasts or plasma cells that produce IgG4 (72).Retrospective studies have suggested that B-cell depletion with rituximab (an anti-CD20 antibody) is effective, even in patients for whom treatment with immunosuppressants is unsuccessful (24). Steroid therapy leads to improved pancreatic endocrine and exocrine function in about half of all patients with AIP and to improved salivary and lacrimal gland function in patients with IgG4-related sialadenitis and dacryoadenitis (24).The short-term clinical, morphological, and functional outcomes of most patients with IgG4-RD who receive steroid therapy are good (73). The risk of malignancy is also reportedly high in patients with IgG4-RD (75). Although there are a few reports of AIP developing into pancreatic cancer, it is unclear whether a relationship exists between these diseases (74). However, because IgG4-RD occurs predominantly in older men and steroid therapy is immunosuppressive, imaging studies, and serum tumor markers should be assessed periodically during follow-up.However, long-term outcomes, including relapse, developed fibrosis, and associated malignancy, have yet to be clearly defined. For example, pancreatic juice stasis resulting from incompletely resolved narrowing of the main pancreatic duct can cause pancreatic stones Only 18 years have passed since IgG4-RD was introduced as a novel disease entity, and the epidemiology, pathophysiology, and long-term prognosis of this disease are still unclear. Greater awareness of this protean disease is necessary to ensure an earlier diagnosis to prevent severe organ damage and disabling tissue fibrosis. The discovery of more sensitive and specific biomarkers would be a step forward in diagnosing this disease. Further, because steroid therapy involves tapering and toxicity risks, new treatment strategies, including rituximab, need to be tested in international randomized controlled clinical trials.NoneThis work was supported by a grant from the the Japanese Ministry of Health, Labour and Welfare Research Program on Rare and Intractable Diseases (No. JPMH20FC1040).This work was supported by a grant from the Japanese Ministry of Health, Labour and Welfare Research Program on Rare and Intractable Diseases (No. JPMH20FC1040).The author declares substantial contributions to the conception or design of the work, or acquisition, analysis, or interpretation of data for the work; drafting the work or revising it critically for important intellectual content; final approval of the revision to be published; and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.


The general population has increasingly become the key contributor to irrational antibiotic use in China, which fuels the emergence of antibiotic resistance. This study aimed to estimate the prevalence of the general population’s irrational use behaviors of antibiotics and identify the potential reasons behind them. A systematic review and meta-analysis were performed concerning four main behaviors relevant to easy access and irrational use of antibiotics and common misunderstandings among the population about antibiotics. Four databases were searched, and studies published before 28 February 2021 were retrieved. Medium and high-level quality studies were included. Random effects meta-analysis was performed to calculate the prevalence of the general population’s irrational behaviors and misunderstandings relevant to antibiotic use. A total of 8468 studies were retrieved and 78 met the criteria and were included. The synthesis showed the public can easily obtain unnecessary antibiotics, with an estimated 37% (95% CI: 29–46) of the population demanding antibiotics from physicians and 47% (95% CI: 38–57) purchasing non-prescription antibiotics from pharmacies. This situation is severe in the western area of China. People also commonly inappropriately use antibiotics by not following antibiotic prescriptions  and preventatively use antibiotics for non-indicated diseases . Misunderstanding of antibiotic use was also popular among people, including incorrect antibiotic recognition, wrong antibiotic use indication, inappropriate usage, and ignorance of potential adverse outcomes. Over-and inappropriate use of antibiotics is evident in China and a multifaceted antibiotic strategy targeted at the general population is urgently required. In the past several decades, antibiotic resistance (AR) has been increasingly recognized as one of the biggest threats to public health and economic development worldwide, as it makes common infectious diseases difficult to treat and results in increased mortality and treatment cost ,2,3. It To address this issue, a call for whole society engagement from one health perspective is required, covering everyone from all sectors and disciplines who should be responsible for preserving the effectiveness of the antibiotic agents . Among tHowever, people’s irrational use behaviors relevant to antibiotics are prevalent around the world ,21,22,23As one of the largest consumers of antibiotics worldwide, China has been long criticized for its over and irrational use. Due to introducing a series of restrictive policies in the past decade, physician’s overuse of antibiotics has been significantly reduced . HoweverTo address this issue, it is important to understand to what extent people will behave irrationally when using antibiotics and the potential reasons behind them. In the past decade, increasing studies reported the prevalence of the general population’s irrational use of antibiotics as well as the potential factors contributing to it. Results from these studies showed high degrees of variability of findings, settings, participants, and other study characteristics. Though one recent study has qualitatively synthesized the factors influencing the general population’s irrational use of antibiotics , it is sTherefore, this systematic review aimed to synthesize the prevalence of irrational use behaviors as well as potential reasons relevant to antibiotics in China.To understand the general population’s irrational use behaviors and potential reasons behind them, a review framework was developed , followiBased on the World Health Organization , four kiIn terms of potential reasons for behaviors, the definition of responsible antibiotic use was adopted . To ensuFour databases were searched to identify relevant studies published before 28 February 2021, including the China National Knowledge Infrastructure (CNKI), VIP database, WANFANG database, and Chinese Biomedical Literature Service System (CBLSS). The development of search strategies followed participant, intervention, comparison, and outcome (PICOs) principles and search terms included “antibiotic”, “the general population”, “irrational use”, “behavior”, “factor” and their synonyms, combined with Boolean operators , study period, type of participant , quality level (moderate/high), and study size. Q test and Ifindings . All stan = 39,181), central , and western  areas, covering 25 of the 34 provinces in China. The participants were rural residents , urban residents , rural and urban residents (n = 2946), students , children’s parents (n = 7153), customers of pharmacies (n = 2168), and others (n = 4662)  .n = 30), followed by non-adherence to antibiotic prescriptions (n = 30), non-prescription purchasing (n = 29), and prophylactic use of antibiotics (n = 20). In terms of potential reasons of these behaviors, roughly half of the included studies surveyed the public’s incorrect recognition of antibiotics (n = 39), followed by people’s misconception of incorrect indication , incorrect usage , and potential adverse outcomes .Among the four kinds of irrational use behaviors of people, demanding antibiotic prescription was surveyed by nearly half of the included studies . The most common source of bias was from sample coverage , for which most articles did not report a justification for whether they achieved a sufficient coverage of the identified sample.p < 0.001), different types of participants (p < 0.001), and different study sizes (p < 0.001). Studies from the western area of China reported the highest pooled proportions , followed by the eastern , and central areas . The highest proportion of behavior for demanding antibiotic prescriptions from physicians was observed in the customers of pharmacies , followed by urban residents , rural residents , students , and children’s parents   of the general population have demanded antibiotic prescriptions from their physicians . Subgrou: 12–29) .2 = 7688.51, p < 0.001, I2 = 99.62%). The multivariable meta–regression yielded a significant multivariate model, explaining more than half of the heterogeneity , which indicated that study period (p = 0.016) and regions (p = 0.001–0.016) significantly influenced the pooled estimate of the general population’s behavior regarding demanding antibiotic prescriptions.Substantial heterogeneity was observed among studies (χp < 0.001), with estimates of 34% (95% CI: 25–444), 51% (95% CI: 36–65), and 70% (95% CI: 51–86), respectively. Different types of participants also showed significantly varied pooled proportions of non–prescription antibiotic purchasing behavior (p < 0.001), with the highest proportion observed in pharmacy customers , followed by urban residents , and students   of the general population have purchased antibiotics without prescriptions . A signi: 35–62) .p < 0.001), with the highest pooled proportion of this kind of behavior found in rural residents  while the lowest in children’s parents . In addition, different qualities of studies also reported different prevalence of non–adherence to antibiotic prescriptions of the public (p < 0.001), with high–quality studies reporting a pooled proportion of 63% (95% CI: 61–65) while moderate quality studies reported 50% (95% CI: 43–57)  .The overall pooled estimate demonstrated that 35% (95% CI: 29–42) of the general population have prophylactically used antibiotics to prevent infections . The subThe robustness of these pooled estimates was confirmed by sensitivity analysis. The previously obtained estimates were similar to the results from sensitivity analysis, with non–prescription antibiotic purchasing ranging from 45.86 to 48.64%, demanding antibiotic prescriptions from 36.48 to 39.30%, non–adherence to antibiotic prescriptions from 47.55 to 50.20%, and prophylactic use of antibiotics from 33.19 to 37.53%.The proportion of people who showed different kinds of misconceptions of antibiotic use was extracted and their pooled proportions were synthesized, covering antibiotic recognition, correct indication, appropriate usage, and potential adverse outcomes.The overall pooled proportion showed that nearly 41% (95% CI: 33–49) of the general population were unable to correctly recognize common antibiotics. People were also unable to correctly understand indications for antibiotic use, the pooled estimate of 53% (95% CI: 44–62) of participants believed that antibiotics can kill viruses and 47% (95% CI: 36–58) believed that most colds need antibiotics. People also commonly hold misconceptions about antibiotic usage, a pooled proportion of 44% (95% CI: 31–56) of the population thought that combined use of antibiotics was more effective than use of one antibiotic and 49% (95% CI: 35–64) believed that they should follow antibiotic prescriptions in dosing and duration. In terms of potential adverse outcomes of antibiotic use, the pooled estimates showed that 41% (95% CI: 33–50) of people did not understand that over and inappropriate use of antibiotics leads to antibiotic resistance and 45% (95% CI: 21–71) did not recognize that use of antibiotics may result in adverse reaction .The infections caused by antibiotic–resistance bacteria are increasingly difficult to treat and the consequences of AR, such as extra hospitalizations and deaths, are self–evident ,6. BasedA huge variation of the public’s irrational use behaviors in different geographical regions was also identified based on subgroup analysis. People from western regions of China were more likely to show behaviors resulting in obtaining unnecessary antibiotics. Other than the fact that people reported less demanding of antibiotics from physicians over time, no significant declining trend was identified in people’s irrational use behaviors of antibiotics in the past decades.The potential reasons for the prevalent over and irrational antibiotic use behaviors of the general population may lie on their misconception of antibiotics, which is also shown to be popular in the current study. It is estimated that roughly half of participants are unable to recognize common antibiotics, understand in what situation antibiotics are required, show sufficient knowledge of appropriate antibiotic usage, and understand adverse outcomes of antibiotics. These misunderstandings may lead to expectations of unnecessary antibiotics from the public and hinder people’s rational use of these medicines.In the current study, a review framework was developed based on the WHO’s highlights of the general population’s irrational use behaviors of antibiotics. The definition of rational use of antibiotics was also referred to help identify potential reasons for the general population’s irrational use behaviors. Based on the review framework, a comprehensive search strategy was generated, covering four kinds of common inappropriate use behaviors and four aspects of misunderstanding of antibiotics among the general population in China. In addition, we carefully followed the guidelines of the systematic reviews of prevalence and incidence from JBI. Quality assessment was applied to ensure only studies with high and moderate quality were included. We also performed subgroup analysis and sensitivity analysis to ensure the robustness of the results.There are also some limitations. First, the quality of the included studies may be underestimated due to the restrictions on the length of text in Chinese journals, causing details of methodology to possibly not be reported ,115. SecThe prevalence of irrational antibiotic use behaviors among the general population confirmed that they play a role in fueling overuse of antibiotics in China. The results of this study correspond with that from another study synthesizing data from publications in English , in whicIt is worth noting that people were less likely to demand antibiotics from physicians over time in China, which was similar when compared with results from a survey in 14 countries that patient antibiotic expectation for respiratory tract infections decreased over time . HoweverOn the other hand, non–prescription purchasing from pharmacies is increasingly become an important outlet to fulfill peoples’ unnecessary demand for antibiotics . This siIn addition, the association between regional economic development and overuse of antibiotics has been established , which iIn terms of rational use of antibiotics, the pooled proportion of people who do not follow antibiotic prescription is similar compared with the results in 2006 (44%)  and thisThe general population’s misconception of antibiotic use is evident in China. In terms of knowledge of indication of antibiotic use, the results in the current study are similar compared with the estimates worldwide that around half of people are not able to correctly answer that antibiotics cannot cure colds and they should not be indicated for viral infection . HoweverTo reduce irrational use of antibiotics, several national campaigns targeted at the public have been implemented worldwide. These interventions focused on educating people on the rational use of antibiotics via printed materials or mass media  and wereFirst, a more comprehensive surveillance and tracking system of antibiotic prescriptions may be developed. The existing national surveillance system of antibiotic use in China concentrates on public hospitals. Information regarding primary healthcare and other facilities  are limited, despite irrational antibiotic being estimated to be more serious in these places. This system also needs to be further linked with pharmacy dispensing data, tracking the end of antibiotic prescriptions, to help irregular sales of antibiotics in both online and offline pharmacies. Through its effect on the general population’s behaviors, this kind of system has been initiated in Jinan, Shandong Province . In addiIn addition, continuous training on antibiotic use and communication skills of physicians is required, especially for those in primary care, private clinics, and prescribing on the internet . Such trFinally, given the effectiveness of several national campaigns worldwide that significantly improve the general population’s awareness of rational antibiotic use ,147, sucIrrational use behaviors and misconceptions relevant to antibiotics among the general population is evident in China. People can easily obtain unnecessary antibiotics by demanding them from physicians or purchasing from pharmacies without prescriptions. They also showed popular behaviors of non–adherence to prescription or preventative use for non–indicated diseases, for example, the cold. Roughly half of people are unable to recognize common antibiotics, know the correct indication, understand rational usage, and realize the potential adverse outcomes. To address this issue, a comprehensive strategy is required, including construction of a systematic surveillance and tracking system of antibiotic prescriptions, a training program for physicians of rational use of antibiotics and communication skills, and a national campaign on the general population to improve their awareness.


Precast concrete system has been widely used in modern day constructions due to its high efficiency in both production time and cost. However, because of the way it is constructed (with flat and dense surface), problems with sound reflection and transmission often exist. It is known that increasing of damping property of materials can reduce the transmission of impact sound and vibration which could lead to an improvement in sound insulation performance. In this study, a type of Viscoelastic Polymer Sheet (VPS) was introduced and attached to concrete precast panels with an aim to improve damping property of precast concrete panels. Seven precast concrete specimens with various patterns and attachment position of VPS were prepared. Effect of patterns and locations of attaching VPS on damping property are investigated and discussed. Precast concrete; Viscoelastic polymer; Damping; Sound Insulation. More people find themselves living in apartments or condominiums instead of traditional houses. However, there are quite a number of problems when it comes to living in an apartment block, especially when it is built in cubical form with precast concrete material. Common problems that arise are related to temperature and sound. In the case of thermal problems, the utilization of materials with high thermal insulation properties such as lightweight concrete, cellular concrete panel, or concrete panels containing thermal capture agencies  can be used to ease out and provide comfort , 2, 3, 4In the case of sound properties, an attention has been paid to the application of viscoelastic polymer materials (VPM) for vibration control. The applications of VPM to reduce vibration and sound induced vibration can be found in several industries. For example, the VPM sheet was used to reduce vibration happened to a steel wall structures of oil rig building . A sheetHowever, in the field of civil engineering, investigations related to damping are focused mostly on structural components subjected to vibration, seismic, cyclic or fatigue loadings. Only a few number of studies has been investigated on the use of VPM to improve damping and sound properties. For example, Lee et al.  proposedSince there are very few studies related to the application of VPM sheet to improve damping and sound properties of concrete material, and most of which are focused on using coated aggregates or rubber particles as a part of concrete constituent ingredients, therefore there is obviously a lack of knowledge on the direct application of VPM attached to the surface of concrete panels. This paper aims to propose an alternative technique to increase the damping property of the precast concrete panel by using VPM sheet (VPS). Seven precast concrete panels were built as the specimens. VPS strips were attached to the specimens in different patterns and different surface constrain. Damping lost factor is measured and calculated in according to the guideline given by ASTM E756 and C215.2The aim of this study is to find a use of VPS to increase the damping loss factor by strategically attach VPS to precast concrete panels. VPS referred to in this study was a type of high adhesion butyl rubber adhesive tape coated with aluminum film. There two surface constraint conditions were introduced; 1) surface attachment - VPS was attached directly on the surface of the specimens to provide a free damping condition and 2) Mid-plate insertion - a VPS was inserted at the middle layer of the specimens to provide a constrained layer see .Figure 1Apart from the surface constraints, the pattern of attachment is also considered. Using the information obtained from the next section  of a plate is measured. The left side of The FRF However, an alternative index so-called damping loss factor 44.1There were seven precast concrete with the dimension of 300 × 300 × 50 mm were prepared as the test specimens. The mix proportion of concrete was set at 1.00/0.62/2.47/3.00 by weight (cement/water/fine/coarse). The mixing process began by dry mixing cement and sand, later the water was added to form mortar slurry. Lastly, coarse aggregates were added and the mixing continued for further 1–2 min. The specimens passed the mechanical property test in according to the international standard such as compressive strength (BS 1881 part 4), unit weight of fresh concrete (ASTM C138), slump of fresh concrete (ASTM C143), water absorption and density of concrete (ASTM C642) and the results on concrete properties are given in The fresh mixed concrete was transferred into molds and compacted by a vibrating table the were covered by plastic sheets for more than 24 h. After that, the specimens were removed from the mold and wrapped with plastic sheets and placed in under the room temperature for more than 28 days. The specimens then were attached with the VPS resulting in 6 combinations from the three attachment patterns and two surface constraint conditions as described in Section 4.2The modal testing was performed to measure the mode shapes, natural frequencies, and the damping loss factors of the specimens. In the test, the surface of each specimen was divided into 36 square patches of the size 50 × 50 mm. An accelerometer was placed at the center of the top left corner patch then the impact test was performed by hitting the hammer at the center of all patches see . The ham5st mode, 1,909 Hz +/- 2.5% for the 2nd mode, and 2,599 Hz +/- 4% for the 3rd mode. This implies that adding of VPS will not significantly change the stiffness of the specimens.The test results are shown in The damping loss factor of the controlled specimen is use as a reference to be improved. By considering the surface constraints, the results suggested that there is no significant improvement of the damping loss factor in all specimens with the surface attachment in comparison to the control specimen. A significant improvement is found in case of mid-plate insertion. Viscoelastic materials behave similarly to viscous fluid such that they resist the shearing motion. Attaching the VPS directly to the specimens with one side able to move freely does not provide strong shearing motion and results in non-significant improvement of the damping loss factor. The shearing motion can be stronger when the constrained surface is added and should result in the improvement of the damping loss factor. This was confirmed by the experimental results in case of mid-plate insertion.By considering the attachment pattern, the improvement can be ranked from high to low as ‘Corner’, ‘X’, and ‘Cross’ corresponding to the damping loss factor at 2.47, 2.20, and 1.90 times higher than that of the control specimen, respectively.η2 = 0.047, η3 = 0.044) and ‘X’ pattern  are higher than that of the ‘Cross’ pattern . This is because the ‘X’ pattern crosses less numbers of nodal lines than the others.As mentioned in section 6This paper presents an alternative technique to improve damping loss factor by attaching viscoelastic material sheet. This will lead to some applications in sound and vibration insulation of precast concrete panels. The precast concrete specimen with combination of VPS attachment technique were prepared and tested. The results show that the mid-plate insertion of VPS is more effective in improving the damping loss factor of precast panels in comparison to the surface attachment. It was also shown that the damping loss factor can be improved from 1.93 to 2.47 times in comparison to the control specimen depending on the attachment patterns. It can be concluded that the attachment pattern also plays an important role in the improvement of the damping loss factor. The ‘Corner’ pattern was found to be the most effective in improving the damping loss factor among the others. Future investigation on the effect of VPS on sound properties of precast concrete panels is recommended to determine the relationship between damping and sound property.Khemapat Tontiwattanakul: Conceived and designed the experiments; Performed the experiments; Wrote the paper.Jirawin Sanguansin: Performed the experiments; Analyzed and interpreted the data.Vatanavongs Ratanavaraha & Vanchai Sata: Contributed reagents, materials, analysis tools or data.Suchart Limkatanyu: Analyzed and interpreted the data.Piti Sukontasukkul: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.National Research Council of Thailand and by King Mongkut's University of Technology North Bangkok (KMUTNB-BasicR-64-09) and by the Thailand Research Fund (RTA6280012 & MSD61I0078).This work was supported by the Data included in article/supplementary material/referenced in article.The authors declare no conflict of interest.No additional information is available for this paper.


We report a rare case of dismemberment and encasement from our catchment area. Various body parts were encased in concrete and thrown into a small stream behind the perpetrators’ farm and visualized with postmortem computed tomography (pmCT) and mobile X-ray device. For reconstruction purposes the head and paw of a pig were encased in concrete in a comparable manner to an actual victim and regularly examined with imaging techniques, to figure out the value of such efforts in cases where concrete is used and to understand changes in the animal’s body over time.The mutilation and encasement of corpses are rare in daily forensic work, but when they occur, close cooperation between different disciplines, such as legal medicine and forensic anthropology, is necessary to obtain the most valuable results. One forensic examination method is the radiological evaluation of victims or body parts by postmortem CT (pmCT) and X-ray images. In relation to a case described in this paper, an empirical study was conducted to figure out the value of radiological imaging and the ability to visualize and temporally classify changes in a corpse encased in concrete. For this purpose, the head and paw of a pig were encased in concrete and scanned regularly over a period of one year. Body parts such as the head and paw are clearly visible on X-ray images. Although decay-related changes are shown, a specific minimum time interval cannot yet be found, as these changes occur continuously in lesser amounts. Cases of encasement and dismemberment are rarely seen in our catchment area in North Germany and, therefore, pose significant local challenges. When they do occur, their circumstances and dynamics are of great interest.In the forensic context, body disposal, or “dumping” describes the process of disposing of a corpse. According to the literature, the most common method of disposal is dumping the body in water , while bDismemberment can be divided into defensive and offensive types. Defensive dismemberment is characterized by a systematic approach, which serves the purpose of hiding the body. Offensive dismemberment, on the other hand, is often characterized by an indiscriminate approach and the mutilation of the genitals ,5. HowevDismemberment cases tend to be underreported ,7. The fn = 18) in Los Angeles. Investigators used both metal detectors and radiological equipment to find the body or body parts inside the concrete. Specific to these cases, the authors reported difficulties in estimating the postmortem interval (PMI), but also the positive effects of concrete in terms of preserving the body for longer and the possibility of making negative casts of, for example, the hands and face, thus aiding the identification process.A study by Toms et al.  examinedAn article by Dressler and Madea  describeIn another study in Germany, the authors described six cases where corpses were encased in concrete and/or bricks. The results of these cases also showed slowed decomposition compared to non-sheathed cases, and therefore difficulties in an uncritical estimate of PMI. These victims were all immured or concreted into floors or walls; therefore, only discovered after confessions of the perpetrators or police investigations. However, no radiological evidence was used in any of the cases .Dedouit et al.  used CT Aside from concrete, quicklime is another material often used to disguise the victim. In the case described, it was also used for the torso. The literature search revealed two studies on the effects of quicklime on buried corpses ,21.In crime scene investigations of dismembered bodies, there are technical modalities that can help specialists in the identification and analysis of body parts. Particularly in the case of entrapped victims, there are obvious difficulties in finding the victims in the first place, and then excavating them safely to preserve clues as much as possible.A study by Ruffell et al. suggests a specific search strategy in trapped or walled-in victims, including, for example, geophysics, search dogs and the use of imaging such as X-rays, ultrasound, or gamma rays, followed by drilling and the use of an endoscope .One possibility for cement blocks with unknown contents, as in our case, is a mobile X-ray device, e.g., the so-called ScanVan . This device is normally used to scan luggage at airports and national borders but can also identify and localize the contents of concrete blocks already at the site of discovery . AnotherA few years ago, a man made a statement about the possible murder of a person who had been missing for two years. In this statement, an indoor riding arena was mentioned as a possible location of the body, which led to a police search at the victim’s property.A human torso was found at the described location. During a later search for the remaining body parts, several concrete blocks were found in a stream behind the farm. As the specialists suspected that they might hold the missing parts, the blocks were scanned with a mobile X-ray machine of the Hamburg customs department.With the help of this mobile X-ray device, the body parts could already be localized in the concrete on site . To illustrate gunshot wounds but not to hit the pig’s head, a projectile, provided by the colleagues from the Hamburg customs department was placed in an orbit. The pig was slaughtered for food on 8 September 2020 and dismembered afterwards using butcher blades. The head and paw provided, which were considered waste, were encased the next day.This box was then repeatedly examined with imaging techniques—including the ScanVan of the Hamburg customs department and the pmCT of the Institute of Legal Medicine in Hamburg and stored on the roof of the Institute of Legal Medicine in between. Over the course of a year, the concrete block was scanned about every other week and a total of twenty-six times. The position of the pig’s head and paw inside the concrete is shown in In addition to the radiological examinations, weather data were also collected, as the experiment was set up on the roof of the forensic institute from 23 September 2020, and was thus exposed to the ambient weather. These data were taken from a weather station in Hamburg Fuhlsbüttel, as this is the closest weather station to our institute (air distance: approx. 6 km) ,25, and The X-ray device from the Hamburg customs department has a lower radiation density than conventional human X-ray devices. The colors depend on the density of the material being X-rayed. These colors can be used to draw conclusions regarding the scanned material. In general, organic materials are shown as orange, and inorganic material, for example metal and concrete, are blue. Furthermore, bones, plastic, aluminum, salts, etc., are shown in green, and anything black could not be penetrated by the radiation due to its high density. The pig’s head and paw appear blue because they are covered by the concrete and therefore appear inorganic due to the increased density of the layer above and below.For a better comparison with similar studies, Galloway and Megyesi’s models were used for the decomposition stages once the remains were removed from the concrete ,27. GallThe effectiveness of the so-called backscatter device was also assessed as part of this study. This is a portable X-ray device, which is used by Hamburg customs to search for drugs behind or inside obstacles such as doors or containers.The backscatter device could not penetrate deep enough into the concrete and therefore proved ineffective in this forensic setting.In general, the outlines of the head and paw were clearly visible on both the X-ray and pmCT. On the pmCT, one could additionally see differences between air, osseous structures, and soft tissues, while on the ScanVan image, different compositions such as organic or non-organic material were made visible with different colors.The projectile, located in the eye socket of the pig was easily distinguishable from the surrounding structures. Its position was stable over the years and clearly visible in every image. Even after the pig’s head was gouged out after 12 months, it was still in place.As time progressed, the accumulation and continuous redistribution of air bubbles around the carcass became visible in different slices on the sagittal plane of the pmCT images. The air is exemplified as diffuse black areas marked with arrows in Over time, an odor was released from the concrete block, and, in addition, a hole was discovered on the enclosed head on 27 July 2021, which was probably caused by a bird.The pmCT scans in the axial plane see a–d, do nOn the exemplified radiographs in On 14 September 2021, the concrete was gently removed see . First, This score cannot be directly compared to a specific PMI under “normal” circumstances, as there are several factors that additionally influence the rate of decomposition, e.g., the sum of ADD, weather effects, etc.The skin and soft tissues on the skull were completely decomposed and had a slimy consistency. They were easily separated from the bone. Therefore, the head would be at the level of ten points with a bone exposure of over 50 percent of the assessed area, including moist decomposition. This part of the body obviously was at a more advanced stage of decomposition than the paw. When the head was removed from the concrete, much soft tissue remained attached to the concrete in addition to the skin, which is why the condition of the skin could not be assessed further or in more detail. The condition of the paw corresponded to the condition previously seen on the X-ray and pmCT images. The head, on the other hand, supplied a completely different picture than would have been expected after seeing the images compared to its physical integrity. The discrepancy between the anatomical impression and its ‘virtual image’ can be explained by the properties of concrete, which formed an impression of the carcass and hardened around it, and therefore, did not lose its shape when the innards dissolved.On the final pmCT/ X-ray image in This empirical study was inspired by the aforementioned case, where the mobile X-ray device of the Hamburg customs department proved to be a particularly useful, ready-to use and fast investigation tool. As a result, it can be said that X-ray and pmCT are useful for finding and identifying human/mammalian remains even in difficult media such as concrete, and can thus help to reduce periprocedural damage. Furthermore, both X-ray and pmCT were able to detect decomposition changes with increasing time, but results could only show a significantly slowed rate of decomposition in concrete compared to putrefactive changes in an open environment and could not be used to assign specific changes to a specific point in time.The total body score in combination with ADD, which are often used to assess the degree of decomposition, was not fully applicable in the present case, because normally the three scores of each table would be added, which would then result in the total body score. In this study, only two body parts were encased, insects were excluded from the decomposition process, and there was relative insulation in the concrete against temperature, humidity, and sunlight. However, the tables were still applied to the remaining body parts after the concrete had been removed, to supply some measures of comparison to similar other studies including encased carcasses.In the studies by Martin et al.  and GibeIn our study, the paw was still in the early stage of decomposition (TBS of limbs: 3), while the decomposition of the head had progressed more and was already in the advanced stage (TBS of head and neck: 10). This could be due to the higher humidity and more intense autolytic processes inside the head where there is more soft tissue and brain matter as well as air in the sinuses and pneumatized bones.This TBS result differs from the other studies, as the TBS we added only for the head and limb without the trunk is thirteen points and therefore shows more advanced decomposition in our environment.A similar finding to ours was the black discoloration of the outer skin layer and the separation of epidermis and dermis due to chemical ‘burning’ by the concrete . This caThe different TBS results in the two aforementioned studies and our study, conducted in Hamburg, could be explained by the stronger influence of external factors due to different environmental conditions. Another explanation could be the different composition of the concrete or cracks and holes that developed over time . In our Our study was the first to investigate this specific set up, with encased pig parts in combination with continuous radiological imaging. It proved the value of such methods in specific forensic cases during the investigation process on site and in the Institute of Legal Medicine. Since, in many larger cities, it is possible to request mobile X-ray devices from Customs, it is advisable to make use of these devices to ensure the safe removal from concrete and thus preservation of the body and its possible clues.X-ray and pmCT methods each showed added value and limitations. The advantages of the portable X-ray are that it can be used quickly in the field and can distinguish between varied materials such as organic and inorganic. However, it did not show as much detail as the pmCT and showed little evidence of a slow decomposition. PmCT images were better at distinguishing between air, bone, and soft tissue structures and better at suggesting decomposition changes due to the accumulation and redistribution of air. A limitation for pmCT, however, is a fixed location and thus, there is a lack of possibility or access to use the device on site. Both imaging techniques can only scan a limited range of objects in terms of weight and size and are therefore of limited value in cases where victims were encased as a complete body inside floors or walls. As a first step towards solving this problem, smaller, portable X-ray devices could be evaluated as next steps. The technical possibilities of such small devices are still limited and need to be thoroughly investigated in the future.In addition, further studies should be encouraged in the future to compare encased and non-encased specimens also from other body parts and investigate a larger sample size. Adding radiological imaging to the list of examinations, a more frequent sampling or longer duration of the experiment could result in the development of a timeline of changes and a calculation of the extent of delay in decomposition in concrete.A limitation of this study is the specific environmental setting, which makes the comparison of results only possible in similar weather conditions, regarding temperature, total precipitation, sun hours and humidity. Data on these details are available on reasonable request but were not included in full in this article to ensure its reasonable length/content ratio.In the studies cited in the introduction, as well as in the mentioned case report, combined and sufficient autopsy and radiological investigations, among others, proved to be valuable for the identification, localization, and minimization of artefacts during excavation.Radiological techniques such as X-ray and pmCT can be used to identify and localize body parts in concrete, helping to safely remove them from the substrate and thus reduce periprocedural damage. In contrast, assessing the state of decomposition and obtaining indications of the postmortem interval is hardly possible even with the inclusion of postmortem imaging. A minimal decay could be detected on both X-ray and pmCT, but it was not possible to assign a specific time point because these changes occurred continuously and in lesser amounts. X-ray and pmCT both have their value and limitations in such cases, including the possibility of detailed imaging of different structures in different planes on CT and the portability and use of the X-ray device in the field. The limitations of this include the stationary location of the pmCT and the less detailed image of the X-ray. In addition, the images did not reflect the actual situation of the head, which could be explained by the properties of the concrete, forming an impression around the carcass.In summary, although radiological imaging modalities can be helpful in locating, identifying, and assessing encased and dismembered victims, such crime scenes still pose various difficulties and remain a challenge even for experts.


Existing experimental research on bond properties of the interface between Carbon Fiber Reinforced Polymer (CFRP) and damaged concrete is limited, although CFRP strengthening technology has been widely used for corroded reinforced concrete structures. This work investigated the bond behavior of CFRP to the corrosion-cracked concrete interface, in which three factors were considered for experimentation, including corrosion degree, concrete strength and concrete cover thickness. The tests were conducted by developing a self-balancing double shear lap test device. In addition, a corrosion scene was provided simultaneously to simulate the external corrosion environment. The results showed that three peeling modes of CFRP sheets were observed with respect to corrosion degrees of the steel bars. The effects of the three factors on the stripping bearing capacity and effective bond length of CFRP sheets were discussed by systematic parametric analysis. Finally, a nonlinear degenerate law of CFRP-to-concrete interface considering the corrosion degree was improved and verified in this study. In coastal areas or inland lakes with high salinity, the steel bars in reinforced concrete (RC) structures are easy to rust by chloride and carbon dioxide due to the porosity of concrete, which will lead to a reduction in load-carrying capacity and durability of RC components, as well as huge economic loss. For sustainable development, externally bonded Carbon Fiber Reinforced Polymer (CFRP) sheets have received increasing attention in retrofitting corroded RC structures . In thisMany studies about the mechanical properties of the interface between Fiber Reinforced Polymers (FRP) and concrete have been carried out in recent years ,13,14,15In this study, electro-chemical accelerated corrosion test and double shear lap test were carried out to study the combined effect of corrosion degree, concrete strength and concrete cover thickness on bond behavior of CFRP to corrosion-cracked concrete interface. Based on existing theoretical models, the factor of corrosion degree was introduced to obtain the degradation model of effective bond length and bond stress-slip relationship. The understanding gained will help in interpreting durability-oriented CFRP applications in the marine environment.The specimen configuration is shown in The selected raw materials for C30 concrete were ordinary Portland cement whose level is 42.5 grade, crushed stone (particle size in the range of 5–25 mm), medium sand and tap water. The mix proportions of C30 were cement:sand:aggregate = 1:1.49:3.17 with a water-to-cement ratio of 0.5, and the mix proportions of C50 were cement:sand:aggregate = 1:1.5:2.92 with a water-to-cement ratio of 0.41. These two mixtures of concrete had a mean compressive strength of 40.88 MPa and 53.21 MPa, respectively, determined on standard concrete cubes (150 mm × 150 mm × 150 mm), after a 28-day standard curing condition.HRB335 crescent rib steel bar with a diameter of 18 mm was used in this test. This bar has the yield strength of 335 MPa, the tensile strength of 455 MPa and Young’s modulus of 200 GPa.CFRP sheets used in this study were produced by Shanghai tongzheng building materials Company. The adhesive was a mixture of glue A and B produced by Yueqing Zhonggu Construction Technology Development Company with a ratio of 1:0.4. The mechanical properties of these two items provided by the manufacturers are shown in Fifteen corrosion specimens and two control specimens were bonded with CFRP sheets according to the manufacturer’s instructions. To ensure the bonding quality of CFRP sheets, the loose concrete layer in the bonded area was removed with a sander. After washing and wiping, a layer of adhesive was evenly coated on this prepared area with a plastic scraper, and then a CFRP sheet was put on it. The second layer adhesive was evenly brushed on the surface of CFRP sheet to completely soak it. The indoor temperature was 20–25 °C, and the curing time was set as 7 days to render full development of the adhesive strength. In the process of glue curing, the surface of CFRP sheets was pressed by hand. If there is a large hollowing area, re-apply glue and re-stick the CFRP sheets.t) [2; Based on Faraday’s Law, the target corrosion degree (time (t) :(1)t=134The actual corrosion degree of reinforcement  were put in the same temperature environment but not soaked in salt water.A total of 10 strain gauges were axially arranged along the centerline of CFRP sheets: one was placed at the unbounded zone, and nine were placed at the bonded zone b.Mukhtar et al.  presenteAs shown in Pre-loading method was adopted to ensure that two surfaces of CFRP sheet were parallel and the force acted on the center of specimens. Static loading method controlled by force was adopted with 1 kN loading grade and 3 min holding time. The test was terminated when CFRP sheets were peeled off or the self-balancing loading device could not sustain balance due to large-area peeling of the CFRP sheets.Cracks propagated along the radial direction of reinforcement a. From tAfter the double shear lap test, the corroded steel bars were taken out from the failed specimens to calculate the actual corrosion degree, as reported in The crack initiation load  in the C30-20 series were also completely peeled off. Three specimens with intermediate corrosion degree  were failed in mode B, in which bond performance may not be fully developed due to the early termination of the loading process. This indicates that concrete cracking caused by steel corrosion has a negative effect on the bond property of CFRP to concrete interface. For the severely corroded specimens, due to the excessive crack width of concrete and the adverse effect of corrosion medium penetrating into the bond surface, the peeling mode of CFRP sheets was C. Besides, CFRP fibers were partially torn in specimens C30-20-10 and C50-40-10, as shown in The combined effect of three factors on the peeling strength LData of specimens with failure mode C were removed for proposing EBL model due to the meaningless strengthening effect of these partially peeled CFRP sheets. Moreover, for the sake of safety, data of the two specimens (C30-20-5 and C50-40-10) with insufficient development of bonding properties were also excluded from the modeling process. EBL model considering corrosion was obtained by linear fitting in this study, as shown in Equation (5).2) between Equation (5) and experimental data was 0.94. This model can be used to calculate the EBL of CFRP sheets strengthening RC elements with corrosion degree of less than 12%, thus providing a reference value for deciding the actual length of the CFRP sheet.Correlation coefficient , (10) and (11), the bond stress-slip relationship of CFRP sheets and corrosion-induced cracking concrete interface can be obtained (see Equation (12)).Local slip at section The accuracy and applicability of the proposed model were further verified by other test data which were not used for the modeling process . These eCombined effects of corrosion degree and concrete strength, as well as concrete cover thickness on bond behavior of CFRP to the corrosion-cracked concrete interface were investigated by an accelerated corrosion test and a double shear lap test with a developed self-balancing device in this study.There are three stripping modes of CFRP sheets with respect to different corrosion degrees. Non-corrosive specimens and those with corrosion degrees lower than 5% showed failure mode A in which CFRP sheets were completely stripped. Three specimens with intermediate corrosion degree  displayed failure mode B, i.e., large-area peeling of CFRP sheets, which leads to the unbalance of self-balancing device. Severely corroded specimens showed mode C in which CFRP sheets were torn along the concrete crack and peeled off in pieces.When corrosion degree is less than 12%, EBL has a positive linear correlation with corrosion degree, but barely depends on concrete grade and concrete cover thickness. When corrosion degree is higher than 12%, the characteristic of failure mode C and the adverse effects of immersion condition, as well as the accumulation of corrosion medium make the EBL lower than that of the benchmark specimens.An in-depth data analysis shows that corrosion degree and concrete strength obviously affect two bond parameters of the CFRP-concrete interface, while the impact of concrete cover thickness can be ignored. A nonlinear degenerate interface law was proposed and verified with the test data in this study and also the collected data. Specifically, the maximum bond stress increases with the increase in corrosion degree. Besides, concrete strength has little effect on the maximum bond stress, whereas it has an effect on the slip corresponding to this stress.