Oryza sativa). To test if zaxinone also counteracts the growth regulatory effects of SLs in rice, we co-supplied zaxinone and the synthetic SL analog rac-GR24 to the rice SL-deficient DWARF17 (d17) mutant. Results showed that co-application of GR24 and zaxinone still rescued d17 phenotype, indicating that zaxinone and GR24 act independently in regulating root and shoot growth and development in rice.The cleavage of plant carotenoids leads to apocarotenoids, a group of metabolites including precursors of the hormones strigolactones (SLs) and abscisic acid, regulatory and signaling molecules. Zaxinone is a recently discovered apocarotenoid growth regulator that improves growth and suppress SL biosynthesis in rice ( D-ring,  that is d moiety . The biormation9 .Figure 11 However, we recently reported that canonical SLs, i.e. 4-deoxyorobanchol (4DO) and orobanchol, are important rhizospheric signals but not the predominant tillering regulators in rice (Oryza sativa).12 Consistently, the orobanchol-deficient cyp722C tomato (Solanum lycopersicum) mutant does not display high branching and dwarf phenotype.13 In fact, canonical SLs were originally identified as rhizospheric signals inducing seed germination of root parasitic plants and released into soil, particularly upon phosphate deficiency, to successfully recruit arbuscular mycorrhizal (AM) fungi for establishing AM symbiosis.14–16All types of SLs were generally considered as shoot branching inhibitors, as SL-deficient mutants are characterized by a high-branching and dwarf phenotype.18 Interestingly, its activity in improving rice performance requires functional SL biosynthesis and signaling machinery.17 In addition, rice zas mutants showed lower tillering phenotypes accompanied by increased 4DO exudation, which could be restored by exogenous zaxinone application.19 Rice contains five MAX1 enzymes, among which Os900 mediates the formation of the rice canonical SLs, 4DO and orobanchol. So far, max1-900 (Os900) mutant lines are the first rice SL-biosynthetic mutants that do not exhibit a pronounced SL-deficiency-related architectural phenotype.12 This observation implies that 4-DO and orobanchol are not the main regulator of rice shoot architecture and that this trait is rather determined by non-canonical SLs.20 The functionality of the SL signaling pathway in the Os900 mutants was confirmed by zaxinone application that led to improved root and shoot growth12 and reduced the transcript level of SL biosynthetic genes.21 However, the question how zaxinone causes this effect on SL biosynthesis remained elusive. In this communication, we asked whether zaxinone generally acts as antagonist of SL in regulating growth and development of rice at early stages, particularly tillering.The apocarotenoid growth-regulator zaxinone, produced by Zaxinone Synthase (ZAS) in rice, was identified as a negative and positive regulator of SL biosynthesis in rice and Arabidopsis, respectively.d17 (ccd7) mutant with 2.5 μM zaxinone, 1 μM rac-GR24, a synthetic SL analog1 or with a combination of both  according to the published protocol.25 7-day-old seedlings were transferred into black falcon tubes filled half-strength modified Hoagland nutrient solution (nutrient compositions listed in25) with adjusted pH to 5.8 with KOH.d17 seedlings, 7-day-old rice seedlings were grown hydroponically in 1/2 Hoagland nutrient solution containing 2.5 µM zaxinone , 1 µM rac-GR24 , or the corresponding volume of the solvent  for 2 weeks. The solution was changed twice per week, adding the chemical at each renewal.For investigating the effect of zaxinone and GR24 on 23 and the reads were aligned to the O. sativa genome v7.0 . Data processing and analysis were performed using the LSTrAP workflow,26 and differential gene expression (DGE), read counts from HTSeq, were analyzed by DESeq2.27RNAseq dataset was extracted from Wang et al.


Immunoglobulin G4-related disease (IgG4-RD) has recently been well recognized and Kuttner tumor is known to be a chronic sclerosing sialadenitis, representing the focal manifestation of IgG4-RD, in the submandibular gland (SMG). This study is to evaluate the immunologic features of IgG4-related Kuttner tumor in the SMG. We retrospectively chose 13 patients who were confirmed as having Kuttner tumor by surgical biopsy between May 2012 and January 2019. The fine-needle aspiration cytology, serum antibody levels , IgG serum levels , and immunohistochemical findings for IgG and IgG4-positive plasma cells were reviewed. The cytologic results found that 7 of the 9 cases were reported as chronic sialoadenitis, and the other 3 as benign lymphoproliferative lesion. The serum levels of autoantibodies, Sjögren-syndrome-related antigen A/Ro-Ab and Sjögren-syndrome-related antigen A/Ro-La, showed all normal values of serum level. The serum level of IgG was increased in only 4 among the cases. However, the IgG4 levels were significantly increased in 11 among the cases. In all the patients who received resection of SMG, immunohistochemical findings showed all positive for IgG4-RD, with elevated numbers of IgG and IgG4-positive plasma cells. The evaluation of IgG4 serum level should be very informative for the diagnosis of this tumor before surgery. Fine-needle aspiration cytology with ultrasound guidance are not conclusive in this study. The immunological study including IgG4 serum level should be required for proper diagnosis and treatment, with clinical features of the Kuttner tumor. The level of evidence was IV. This tumor should be differentiated from other benign or malignant tumors in the SMG. The salivary gland tumors show a great deal of morphological diversity and mimic the clinical appearance of malignancy in the salivary gland. The pathogenesis of Kuttner tumor in the SMG is not very well understood. Three factors have been mainly suggested: sialolith, inflammation of the salivary duct, and autoimmune reactions. Sialolithiasis by accumulation of calcium salts had been proposed as the most common cause for Kuttner tumor of the SMG.,4 However, a stone in the SMG may not be found in many cases of Kuttner tumor. The salivary duct obstruction leads to excessive accumulation or retention of ductal secretions, which result in chronic inflammations.,6 Immunoglobulin G4-related disease (IgG4-RD) is a recently recognized inflammatory disease in multiple organ systems. The salivary glands, particularly in the SMG, are frequently involved in the head and neck region. Nowadays, Kuttner tumor is known to be a chronic sclerosing sialadenitis, representing the focal manifestation of IgG4-RD.Kuttner tumor is a benign inflammatory condition of the salivary gland predominantly involving the submandibular gland (SMG). This tumor may occur in other major and minor salivary glands, unilaterally or bilaterally, including the sublingual and parotid glands.–9 The differential diagnoses include sialadenitis, Mikulicz syndrome, benign lymphoepithelial lesion, Kimura disease, lymphoma, and neoplasms of the salivary glands.,11 For the proper diagnosis of Kuttner tumor of the SMG, surgeons mainly depend on preoperative ultrasonography with fine-needle aspiration cytology (FNAC) examinations. In the ultrasonogram, Kuttner tumor shows diffuse involvement with multiple hypoechoic lesions. Focal lesions are seen as hypoechoic, heterogeneous masses with a radial-branching vascular pattern within. The FNAC findings shows cells greatly reduced in number along with scattered tubular ducts against a backdrop of lymphoplasmacytic infiltration and fibrous deposits. There may be a reduced but moderate number of cells and ducts enveloped in fibrous sheaths, as well as fibrous proliferation of the gland’s septa. However, FNAC findings may not be specific for Kuttner tumor, and ultrasonography may exhibit similar imaging findings as neoplasm of the SMG. So diagnosis should require adjunct consideration of both the ultrasonogram and the clinical presentation. We therefore evaluate the immunologic features of IgG4-related Kuttner tumor to aid in proper diagnosis.The clinical features of Kuttner tumor have diverse signs and symptoms, and diagnostic challenges significantly remained underrecognized and underscored for IgG4-RD. Kuttner tumor presents as a firm and painless enlargement of the SMG, and patients are advised to have surgical resection of the involved glandular tissue because of suspicion of possible benign or malignant tumors.We retrospectively chose 13 patients who were confirmed as having Kuttner tumor by surgical biopsy between May 2012 and January 2019 at the Department of Otolaryngology-HNS. We obtained proper consent from the patients in keeping with the mandate of the Declaration of Helsinki. Institutional Review Board was not required as it was a retrospective review. We had selected only 13 cases diagnosed as Kuttner tumor by SMG biopsies in this study period because many cases presenting with swelling of SMGs were diagnosed by clinical findings without biopsies. The diagnosis was made on the basis of clinical data, imaging, and histopathologic findings. For histological analysis, 4-μm formalin-fixed, paraffin-embedded sections were prepared and stained with hematoxylin and eosin. For immunohistochemical study, anti-IgG rabbit polyclonal antibody was used to evaluate the molecule of IgG . Further, anti-IgG4 mouse monoclonal antibody was used to evaluate the molecule of IgG4 . Marked plasmacyte infiltration, defined as >10 IgG4+ cells per high-power field (HPF) and a >40% ratio of IgG4+/IgG+ cells were considered as positive findings. They did not complain of drying eyes or mouth, but felt some discomfort on palpation of the masses and also postprandial. Their past medical histories were insignificant. Physical examination showed palpable submandibular masses unilaterally or bilaterally. To measure the IgG serum subclass, IgG and IgG4 serum levels were evaluated with the consent of our patients. Other serological data about the autoantibodies (Sjögren-syndrome-related antigen A [SSA]/Ro-antibody [Ab] and SSA/La-Ab) were also evaluated. The patient’s data was investigated using Excel software.The distributions of the patients are summarized in Table The following variables were assessed: FNAC findings, serum autoantibody levels , IgG serum levels , and immunohistochemical findings for IgG and IgG4 plasma cells.For the FNAC studies, we had 10 cases of FNAC among the 13 cases. Of the 10 cases, 7 were reported as chronic sialoadenitis, and 3 as benign lymphoproliferative lesion. Unfortunately, there was no case suspicious of chronic sclerosing sialoadenitis or SMG tumors, perhaps because fine-needle aspiration was used, not core-needle biopsy.Figure Figure Figure  Systemic IgG4-RD more commonly occurs in older males, in a 1:0.77 male-to-female ratio. But there is no gender predilection; that is, there is equal male and female prevalence for IgG4 involvement in the head and neck.Recently, Kuttner tumor is known to be chronic sclerosing sialadenitis, representing the focal manifestation of IgG4-RD especially in the salivary glands. It can occur as part of systemic or isolated diseases. MALT lymphoma has similar imaging findings and always requires exclusion, because it can also occur secondarily to IgG4-RD within the salivary glands. Wah and John reported several features of FNAC for Kuttner tumor: relatively low cellularity, probably attributable to the fibrosis; scattered ductal structures with paucity or absence of acini; ducts intimately surrounded by collagen sheaths or lymphoid cells; small, isolated fragments of fibrous stroma; and moderate to large numbers of lymphoid cells that lack definite atypia. However, Chai et al have reported similar FNAC findings in chronic sialadenitis, broadly similar to those described for chronic sialadenitis or Kuttner tumor. Furthermore, recent study showed no correlation of IgG4+ plasma cells in histopathology sections with FNAC smears. Our FNAC results are benign lymphoproliferative lesion or reactive lymphadenitis, but are not diagnostic for the IgG4-RD within the SMG.On the diagnostic procedures, FNAC analysis under ultrasound guidance is sometimes useful to confirm the diagnosis without recourse to organ excision, particularly when there is involvement of the SMGs. However, the patients are advised to have surgical resection of the SMG because of suspicion of possible benign or malignant tumors. It is only from postsurgical histopathology of the excised mass that the diagnosis of Kuttner tumor is definitively made. The key histopathological feature is the proportion of IgG4-positive plasma cells in the involved tissue, which could be diagnostic for excluding malignancy, especially including lymphoma.,19 It protects against bacterial and viral infections. The subclasses of immunoglobulin differ in their biological features and target specificity and are clinically interesting for potential immunodeficiencies and IgG4-RDs.,19 Elevation of serum IgG4 was previously thought to be a key diagnostic finding, but patients with systemic disease may have normal serum levels, up to 50%. Furthermore, although relatively sensitive, the specificity of elevated IgG4 serum levels is low and not significant in many other, disparate diseases.IgG is the most abundant type of antibody  and is found in all body fluids. The causes of a raised IgG include IgG myeloma, chronic liver disease, sarcoidosis, autoimmune disease, and parasitic diseases.,18Testing of IgG subclass levels may be indicated in patients with clinical evidence of a possible immunodeficiency or normal concentrations of total serum IgG. The IgG4 assay is best for IgG4-RD testing. Clinically, the causes of a reduced IgG serum level include acquired immunodeficiency, hereditary immunodeficiency, protein-losing syndromes, Waldenstrom macroglobulinemia, and non-IgG myeloma. Subsequent studies showed that anti-Ro/SSA antibodies may be present in patients with other autoimmune diseases, including systemic sclerosis, idiopathic inflammatory myopathies, primary biliary cholangitis, and rheumatoid arthritis. In contrast to anti-Ro antibodies, which may be present in a variety of autoimmune diseases, anti-La antibodies are specific for the diagnosis of SLE and Sjögren syndrome. In addition, as with anti-Ro antibodies, anti-La antibodies may be detected in the mothers of children who are born with neonatal lupus syndrome. The combination of anti-Ro and anti-La antibodies is relatively specific for the diagnoses of SLE and Sjögren syndrome.Although the causes of autoantibody production are not well understood, it has been suggested that some autoantibodies are caused by a genetic predisposition combined with an environmental trigger, such as a virus or a prolonged exposure to certain toxic chemicals. Autoantibodies directed against Ro/SSA and La/SSB autoantigens were originally identified in patients with Sjögren syndrome and systemic lupus erythematosus (SLE).Routine neck computed tomography and magnetic resonance imaging (MRI) show an enlarged focal mass or diffuse infiltrating lesions. However, computed tomography and MRI are also less specific, and there is subtle enlargement of the involved gland, relative to the adjacent normal parenchyma. MRI may reveal a relatively hypointense mass on T2-weighted sequences that is indistinguishable from lymphoma.,13 The number of IgG4-positive cells per HPF is counted and averaged over 3 fields; in general, more than 50 per HPF is required. In addition, the proportion of IgG4-positive to total IgG-positive plasma cells is estimated; in general, a ratio of 40% is accepted as diagnostic. In our cases, all patients were confirmed as having Kuttner tumor by showing elevated numbers of IgG4-positive plasma cells in the involved tissue. Furthermore, the IgG4 serum levels were significantly increased in 11 of 12 cases. However, elevated serum IgG4 concentrations could also be found in patients with atopic dermatitis, pemphigus, asthma, and multicentric Castleman disease. This study has a limitation in relatively small sample size with and retrospective study design. Therefore, prospective study with large sample size is required in the near future.Unfortunately, for the confirmed diagnosis of Kuttner tumor, open biopsy or excision should be performed to rule out other benign lesions or malignancies, including a lymphoma. For the histological features, it needs to present at least 2 of 3 histological features, such as dense lymphoplasmacytic infiltrate, storiform fibrosis, and obliterative phlebitis. In most cases, the diagnosis is reached with evidence of the lymphoplasmacytic infiltrate and fibrosis. Immunohistochemical staining needs to present elevated numbers of IgG4-positive plasma cells within tissue in Kuttner tumor related to IgG4-RD.IgG4-related Kuttner tumor in the SMG is a recently recognized inflammatory disease with typical immunohistopathological findings. Imaging studies may exhibit similar findings to those of other benign lesions in the SMG, and FNAC findings may not be specific for Kuttner tumor. Immunological study, including serum IgG and IgG4 levels, should be required for proper diagnosis with the clinical features of the Kuttner tumor. Especially, this study might be very informative for patients with Kuttner tumor, which has mild symptom avoiding improper surgical resection of the SMG. Further, this study expected to provide more information about Kuttner tumor to physicians and researchers.Supervision: Yong Tae Hong.Writing – original draft: Su Geun Kim.Writing – review & editing: Yong Tae Hong, Chan Mi Lee.


We aimed to investigate the predictors of relapse in immunoglobulin G4-related disease (IgG4-RD), focusing on the serum IgG4 levels during initial treatment.n = 13) and non-relapsed (n = 44) groups. Multivariate Cox regression analysis was used to assess the predictors for relapse. We performed a Kaplan–Meier analysis with a log-rank test to evaluate the cumulative relapse rate for two years.We retrospectively recruited 57 patients with IgG4-RD who were treated with immunosuppressants and elevated serum IgG4 levels in a tertiary hospital between January 2011 and December 2020. They were followed up for ≥ 6 months after initiation of immunosuppressive therapy. Clinical and laboratory findings including serum IgG4 levels  were compared between relapsed (p = 0.019). Central nervous system involvement was associated with the relapse, with a hazard ratio of 21.130 (p = 0.015). The cumulative relapse rate for two years was lower in the normal serum IgG4 group at six months than in the elevated serum IgG4 group at six months (p = 0.027).Median serum IgG4 levels at baseline were 321 mg/dL in the relapsed group and 299 mg/dL in the non-relapsed group. Serum IgG4 levels were normalized after six months in five (38.5%) relapsed and 28 (63.6%) non-relapsed patients. In multivariate Cox regression analysis, the normalization of serum IgG4 levels at six months was associated with a lower risk of relapse, with a hazard ratio of 0.232 (Our study suggests that normalization of serum IgG4 levels during immunosuppressive treatment for IgG4-RD independently predicts relapse-free outcomes. Thus, monitoring serum IgG4 levels might be used as a marker of prognosis. Immunoglobulin G4-related disease (IgG4-RD) is a systemic fibro-inflammatory condition of unknown etiology . This diPolyclonal hypergammaglobulinemia is related to various diseases including liver disease, autoimmune disease, infection, and malignancy . PatientThis study was conducted on patients with active IgG4-RD who were treated with immunosuppressants and had elevated serum IgG4 levels at a tertiary referral center in South Korea between January 2011 and December 2020. All patients met the 2011 IgG4-RD diagnostic criteria : (1) a cWe retrospectively reviewed the electronic medical records of these patients at the time of the initial treatment, including age, sex, diagnostic classification, disease duration, affected organs, and treatment regimen. The IgG4-RD responder index (RI), which is a score for each organ system and serum IgG4 level, was calculated to assess the disease activity . LaboratAt six months of initial treatment, the clinical and serological response was assessed. The clinical response included the IgG4-RD RI score, > 50% decline in IgG4-RD RI score, and remission. Remission was defined as meeting all of the following : (1) > 5U test. Categorical data were expressed as numbers (percentages) and compared using the chi-squared test and Fisher’s exact test. Wilcoxon signed-rank test was used to compare paired data of serum IgG4 levels within the two groups at baseline and six months. We evaluated the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of normalized serum IgG4 level for the prediction of relapse. Univariate and multivariate Cox proportional hazard models were conducted to assess the hazard ratio (HR) and 95% confidence interval (CI) for the predictor of relapse in IgG4-RD. Multivariate Cox proportional hazard models include variables with a p-value < 0.2 in the univariate Cox analysis, and were adjusted for age, sex, baseline IgG4-RD RI score, baseline serum IgG4 level, and medication. We performed the Kaplan–Meier analysis with the log-rank test to evaluate the cumulative relapse rates for two years in groups with normalized and elevated serum IgG4 levels at six months. A p-value of < 0.05 was considered to be statistically significant. We used SPSS software, version 24.0, for all analyses.Continuous variables were represented as median (interquartile range [IQR]) and analyzed using the Mann–Whitney This study was conducted in accordance with the Declaration of Helsinki principles and was approved by the Institutional Review Board of Asan Medical Center  (IRB number: 2020–1771). The Institutional Review Board of Asan Medical Center waived informed consent because of the retrospective nature of the study.We enrolled a total of 57 active IgG4-RD patients with elevated serum IgG4 levels at baseline. According to the 2011 diagnostic criteria of IgG4-RD, 24 (42.1%) and 33 (57.9%) patients were diagnosed with definite and possible cases, respectively. Forty (70.2%) patients fulfilled the 2019 ACR/EULAR classification criteria. Forty-five (78.9%) patients were male, and the median age at the start of treatment was 61  years. The median IgG4-RD RI score was 12 , and the organ distribution was as follows: pancreas (66.7%), lymph node (52.6%), retroperitoneum/aorta (29.8%), biliary tree (28.1%), salivary gland (26.3%), lacrimal gland (17.5%), kidney (15.8%), lung (10.5%), and central nervous system including meninges (3.5%). According to the ICDC for autoimmune pancreatitis, 32 (84.2%) of 38 patients with pancreatic involvement were definitive cases, and six (15.8%) were probable cases. The initial dose of prednisolone was median 30  mg/day. Median serum IgG4 levels at baseline were 305  mg/dL. Thirty-seven (64.9%) patients showed serum IgG4 levels ≥ 2x the upper normal of limit (UNL), of which 12 (21.1%) patients had serum IgG4 levels ≥ 5x the UNL. The median follow-up duration was 17.2  months. Thirteen (22.8%) patients experienced relapse, with a median time to relapse of 16.8  months.p = 0.309). The proportion of patients with serum IgG4 levels greater than twice the UNL was also not different between relapsed and non-relapsed groups .The baseline characteristics of the relapsed and non-relapsed groups are summarized in p = 0.324) and acquisition of remission . The treatment regimen, including prednisolone and/or azathioprine, did not differ between the two groups. In terms of serological changes, the median serum IgG4 levels declined at six months in both groups . However, the relapsed group showed higher median serum IgG4 levels at six months than the non-relapsed group (p = 0.033). Likewise, the proportion of patients with normalized serum IgG4 levels at six months tended to be low in the relapsed group compared with that of the non-relapsed group . When evaluating the performance of normalized serum IgG4 levels for the relapse-free outcome, the sensitivity and specificity were 63.6% and 61.5%, respectively. While the PPV was 84.8%, the NPV was 33.3%.p = 0.036) was associated with the relapse-free outcome. In addition, central nervous system involvement  was possible factor associated with the relapse. After adjusting for age, sex, baseline IgG4-RD RI score, baseline serum IgG4 level, prednisolone dose, and azathioprine use, the multivariate analysis revealed that central nervous system involvement  was associated with an increased risk of relapse. In contrast, normalized serum IgG4 level at six months was significantly associated with a lower risk of relapse .We performed Cox regression analyses to identify the risk factors for relapse in our cohort of IgG4-RD patients . In the p = 0.027).As shown in Identifying relapse risk in IgG4-RD is essential for maintaining a favorable outcome during the follow-up period. Although several prognostic factors have been suggested, the relationship between the relapse and the changes in serum IgG4 levels is not clear. We demonstrated that patients with normalized serum IgG4 levels at six months of treatment had a low risk of relapse in IgG4-RD. In contrast, central nervous system involvement was the risk factor associated with disease relapse. This study proposes the persistent elevation of serum IgG4 levels despite initial treatment as a predictor of the relapse in IgG4-RD.We reported that approximately one-fourth of patients experienced relapse more than once over 24 months, and the overall cumulative relapse rate was 7.2% in 12 months and 35.7% in 24 months. Previous studies indicated the relapse rates of IgG4-RD ranging from 23% to 58%, depending on the disease characteristics and treatment regimen of the cohorts , 17. In et al., the increase in serum IgG4 levels by more than 20%, 50%, or 70% compared to remission was not associated with the relapse [There have been clinical prediction markers for the relapse of IgG4-RD, such as age, male, IgG4-RD RI score, blood eosinophils, and glucocorticoid regimen –20. Elev relapse . Further relapse . On the  relapse . Similar relapse , 25. Thi relapse , 11, 26. relapse . This diDespite the usefulness of serum IgG4 levels for diagnosis and disease activity in IgG4-RD, it is not fully understood what elevated serum IgG4 levels imply in the pathogenesis. However, in a study that used an adoptive transfer mouse model, the administration of IgG4 from patients with IgG4-RD resulted in the disease . In a reet al. reported that patients with head and neck involvement showed a favorable relapse-free outcome at 12 months compared to patients with other organ involvement [This study also suggested central nervous system involvement such as meningitis as a risk factor of the relapse. Clinical and laboratory features and prognosis of IgG4-RD may vary depending on the involved organ –36. Lanzolvement . Althougolvement , 38, a colvement . ConsideThere are some limitations to this study. First, this was a retrospective study conducted in a single center. There may have been informational bias in the medical records that could affect the results. For example, some laboratory parameters were not measured, including gamma-globulins via serum protein electrophoresis. Next, we included only the patients with elevated serum IgG4 levels at baseline to evaluate the significance of normalized serum IgG4 levels. Although most patients in previous studies also showed elevated serum IgG4 levels, baseline serum IgG4 level itself is associated with the disease relapse. However, given that patients with normal serum IgG4 levels were excluded, the prognostic value of change in serum IgG4 level may have been further strengthened. This point should be considered when interpreting the results of this study. Finally, we defined relapse as recurrence or worsening as determined via imaging findings or the deterioration of biochemical parameters. Clinical symptoms were not used in the definition of relapse, though additional evaluations including via imaging were performed if relapse was suspected. Nevertheless, a possible underestimation of relapsed cases cannot be excluded.In conclusion, serum IgG4 level is a useful marker to diagnose and predict the IgG4-RD. Although there is interest in the clinical implication of a condition with persistently elevated serum IgG4 levels, the association with relapse is not clear. This study showed that serum IgG4 level during initial treatment was significantly associated with relapse of IgG4-RD. Patients whose serum IgG4 level has not been normalized after initial treatment should be monitored for the disease activity during the follow-up period.S1 Data(XLSX)Click here for additional data file.


The usage of antibiotics has been, and remains, a topic of utmost importance; on the one hand, for animal breeders, and on the other hand, for food safety. Although many countries have established strict rules for using antibiotics in animal husbandry for the food industry, their misuse and irregularities in compliance with withdrawal periods are still identified. In addition to animal-origin foods that may cause antibiotic residue problems, more and more non-animal-origin foods with this type of non-compliance are identified. In this context, we aim to summarize the available information regarding the presence of antibiotic residues in food products, obtained in various parts of the world, as well as the impact of consumption of food with antibiotic residues on consumer health. We also aim to present the methods of analysis that are currently used to determine antibiotic residues in food, as well as methods that are characterized by the speed of obtaining results or by the possibility of identifying very small amounts of residues. Antibiotics are produced by living organisms or in the laboratory and have the capacity to kill or inhibit the growth of microorganisms . Even ifThe practice of using antibiotics is still widespread for prophylactic and curative purposes, and the problem of their remnants in animal products, groundwater, soil, and feed has caused (and is still causing) worldwide concern, generating vast costs to combat antibiotic resistance . In ordeMost antibiotic residues can be found in a large range of food products, both animal and vegetal . AlthougThe sources of antibiotics in humans are two-fold: first, prescriptions for people , and, second, the substances used in animal breeding (tetracycilies and sulfonamides). Antibiotics can cause serious effects on human health that have led to the introduction of maximum residue limits (MRL) in food safety legislation ,12. LongYang et al. (2021) and Huang et al. (2020) mentioned that antibiotics have become widespread in the environment due to their extensive and long-term use, influencing both human health and the system, due to the emergence of antibiotic resistance ,15. SaeeThis review focuses on three aspects: (1) antibiotic use in livestock; (2) antibiotic residues in food and methods of detection; and (3) the impact of consumption of food with antibiotic residues on consumer health.Livestock farming plays a very important role in the agriculture of the European Community. Achieving the best results largely depends on the use of safe and good quality feed. Free movement of safe and good quality food and feed is a key aspect of the internal market, and contributes significantly to the health and well-being of consumers .In terms of animals of economic interest and poultry farms, various active substances are used, including antibiotics, in order to maintain the health of the animals and to have a better yield for breeding. Antibiotics can be administered via feed or by intramuscular injection ,19. MediRegulation (EU) 6/2019 establish rules for the authorization of use of veterinary medicinal products in feeding stuffs, including the manufacture, distribution, advertising, and surveillance of such products . Feed buRecent studies have shown that a significant percentage of all manufacturers add antibiotics to healthy animals’ feed to prevent, rather than cure, diseases ,23,24,25According to different researchers, reliable data regarding the quantity and patterns of use, dose, and frequency, are not very accurate ,28,29. DThe presence of antibiotics in feed is not desirable due to the antibiotic resistance that the bodies can later acquire. Therefore, the elimination of antibiotics from feed, but improvements in the productivity of pigs and poultry, has been achieved through the use of feed additives, such as omega 3, immunoglobulin, organic and inorganic acids, zinc oxide, yeast derived β glucans, essential oils, prebiotics, probiotics, threonine, cysteine, and herbs and spices ,32. On tAdvertising for medicated feeding stuffs addressed persons who cannot properly assess the risks associated with their use, and may lead to improper or excessive use of the medicinal product, which may harm public or animal health or the environment .Animal health management is mainly based on disease prevention, compliance with hygiene conditions, and the correct application of disinfection actions. Preventive use of synthetic chemical allopathic medicines is forbitten in order to obtain organic products. In case antibiotics are compulsory, treatment should be carefully managed through minimal use with a high efficiency. In such cases, in order to ensure the integrity of organic production for consumers, an official withdrawal period after the use of such medicinal products requires a longer period of time. In organic production, the use of antibiotics as growth or production stimulants, coccidiostats, hormones, etc., is not permitted .Dozens of scientific papers have been published over the years, focusing on antibiotic residues in food and feed. Antibiotics can be naturally produced by living organisms or they can be synthetically produced in a laboratory. The main role of antibiotics is to inhibit or kill microorganism growth ,34. AntiIn order to ensure food safety for consumers, more and more studies have attempted to find effective and rapid methods for the detection antibiotic residues in feed and food . ConsumeSeveral studies have shown the presence of antibiotic residues in various types of food .Although food products of animal origin are considered to be the major source of antibiotic residues, studies in the literature have shown that even non-animal origin products represent an important problem regarding this type of contamination. The main sources of these compounds in agriculture are irrigation water with antibiotics traces due to inappropriate recycling processes or the use of manure as soil amendments, which leads to spreading antibiotics through the food chain . As a reIn recent years, research has been conducted in order to study the relation between intake of antibiotics in edible crops due to poorly management of wastewater used in irrigations and from manure ,59,60,61Daucus carota), lettuce (Lactuca sativa), and radish (Rhaphanus sativus) due to the same type of fertilization. The conclusion of the study was that three crops absorbed relatively higher amounts of gentamicin  than streptomycin (large molecule), and that the levels of antibiotics in plant tissues increased when increasing the antibiotic concentration in manure ..1].Most antibiotics cause side effects in humans  134]..134].A large number of studies refer to the management of antibiotic residues, antibiotic-resistant bacteria (ARB), and antibiotic resistance genes (ARG) can be found in dairy manure and may contribute to the spread of antibiotic resistance (AR). More than 60 ARGs can be found in milk manure (including β-lactam and tetracycline resistance genes), although correlations with antibiotic use, residues, and ARBs have been inconsistent, possibly due to sampling and analytical limitations. Antibiotic resistance genes often persist through these systems, although optimal management and a higher operating temperature may facilitate their attenuation ,139,140.Worldwide, and under the auspices of the World Health Organization (WHO), lectures and interactive broadcasts are organized to emphasize the importance of judicious administration of antibiotics in both human and veterinary use, or in agriculture. People should be aware of the need for antibiotics to be used correctly and to reduce the abuse of antibiotics. In addition, national programs aimed at the screening of antibiotic residues in various types of food are being continuously updated.The issue of the presence of antibiotic residues in food is intensely debated. Numerous research studies have highlighted the irrational use of antibiotics and the risk of problems with consumer antibiotic resistance, spread by foods with antibiotic residues.The concentration and type of antibiotic found in the form of residues varies depending on the geographical area and the type of food analyzed. Available studies present antibiotic residues in all food groups: meat and meat products, milk and dairy products, eggs, honey, and non-animal-origin products.Although alarm signals are drawn regarding irrational antibiotic use, exceeding applicable legal requirements are identified. While the European Union has clearly established limits for antibiotic residues, in the United States, legislation does not include such values, with a deadline set for 2023 to draw up these legislative requirements. Additionally, in recent years, awareness of the irrational use of antibiotics programs have been launched, but still it is necessary to develop these in order to increase the understanding of producers and consumers regarding the use of antibiotics. These measures should be implemented worldwide.


NRAMP) genes encode proteins with low substrate specificity, important for maintaining metal cross homeostasis in the cell. The role of these proteins in tobacco, an important crop plant with wide application in the tobacco industry as well as in phytoremediation of metal-contaminated soils, remains unknown. Here, we identified NtNRAMP3, the closest homologue to NRAMP3 proteins from other plant species, and functionally characterized it. A NtNRAMP3-GFP fusion protein was localized to the plasma membrane in tobacco epidermal cells. Expression of NtNRAMP3 in yeast was able to rescue the growth of Fe and Mn uptake defective Δfet3fet4 and Δsmf1 mutant yeast strains, respectively. Furthermore, NtNRAMP3 expression in wild-type Saccharomyces cerevisiae DY1457 yeast strain increased sensitivity to elevated concentrations of iron (Fe), manganese (Mn), copper (Cu), cobalt (Co), nickel (Ni), and cadmium (Cd). Taken together, these results point to a possible role in the uptake of metals. NtNRAMP3 was expressed in the leaves and to a lesser extent in the roots of tobacco plants. Its expression occurred mainly under control conditions and decreased very sharply in deficiency and excess of the tested metals. GUS-based analysis of the site-specific activity of the NtNRAMP3 promoter showed that it was primarily expressed in the xylem of leaf blades. Overall, our data indicate that the main function of NtNRAMP3 is to maintain cross homeostasis of Fe, Mn, Co, Cu, and Ni  in leaves under control conditions by controlling xylem unloading.Natural resistance-associated macrophage protein ( Excess metals taken up from the soil are accumulated by most plants in their leaves, however, the efficiency of accumulation in these organs differs among them. Those capable of storing very large amounts are frequently used in the phytoremediation of metal-contaminated soil. Tobacco is one such species. It is a high biomass plant effective in translocating metal from the root to the shoot, especially zinc (Zn) and cadmium (Cd), which is an important determinant of accumulation in leaves , showed that they likely participate in the cell-specific accumulation of Zn in the palisade parenchyma of tobacco leaves , similarly to NtZIP1-like and NtZIP11 , Zn2+, copper (Cu2+), iron (Fe2+), Cd2+, nickel (Ni2+), and cobalt (Co2+), into the cytoplasm , 100 μM Cd , 1,000 μM Fe/EDTA (GmNRAMP6a), or 200 μM Mn (GmNRAMP5a)  and then propagated in the greenhouse of the University of Warsaw.Wild-type (WT) tobacco −2 s−1, fluorescent Flora tubes ] 250 μMol mSeeds were surface sterilized in 8% sodium hypochlorite (w/v) for 2 min, washed with distilled water, then germinated and grown on vertically positioned Petri dishes containing quarter-strength Knop's medium, 2% sucrose (w/v), and 1% agar (w/v). After 3 weeks seedlings were transferred to hydroponic conditions (five plants per 2.5 L pot) and further grown according to experiment-specific schemes presented below. In all experiments, the quarter-strength Knop's medium was applied as a reference control medium  4-week-old seedlings (3 weeks on plates and 1 week on hydroponics): whole roots and all leaves were collected separately; (2) 6-week-old plants (3 weeks on plates and 3 weeks on hydroponics): whole roots and all leaves were collected separately; (3) 9-week-old plants (3 weeks on plates and 6 weeks on hydroponics); the following plant parts were collected: (a) apical part of the root (3–4 cm from the tip), (b) basal part of the root (3–4 cm from the base), (c) stem (3 cm of the middle part), (d) young leaves (two leaves of minimum 0.5 cm length counting from the top), and (e) old leaves (two leaves counting from the base). Lateral roots were not included in the analysis. The material was pooled from a total of 30 (stage 1), 15 (stage 2), or 10 (stage 3) plants, frozen in the liquid nitrogen and stored at −80°C.To determine developmental regulation of NtNRAMP3, 3-week-old WT plants were further hydroponically grown on a control medium for 2 weeks. Then, they were exposed to different regimes: (i) metal excess (200 μM Fe or 100 μM Mn or 20 μM Co or 20 μM Cu or 30 μM Ni or 50 μM Zn or 4 μM Cd); (ii) metal deficiency ; (iii) control conditions. Metal was added to the control medium as ZnSO4; Fe-EDTA; MnSO4; CoCl2; CuSO4; NiCl2 or CdCl2. After 3 days, the following organs were collected: (i) 3–4 cm fragment of the apical part of the root; (ii) 3–4 cm fragment of the basal part of the root; (iii) leaves (2nd and 3rd leaf counting from the base of the stem) without petioles and major midribs. The material was pooled from a total of 10 plants, frozen in the liquid nitrogen, and stored at −80°C.For assessment of metal status-dependent expression of NtNRAMP3 expression between leaves of tobacco exposed to a long-term treatment of elevated Zn, 3-week-old tobacco WT seedlings were grown on a liquid control medium for 18 days, then for 3 weeks at 10, 50, and 200 μM Zn. Next, all leaves (without petioles and midvein) were removed from the stem and collected in groups of two , frozen in the liquid nitrogen and stored in −80°C. The material was pooled from a total of 8 plants.To compare the NtNRAMP3 expression patterns, transgenic plants expressing pMDC163::promNtNRAMP3::GUS were subjected to two different treatments. In the first experiment, three-week-old plants were transferred from agar plates to hydroponics and grown under control conditions for 7 days. Whole seedlings were used to determine GUS activity. In the second experiment, 5-week-old plants grown at control conditions (3 weeks on agar plates and 2 weeks on hydroponics) were exposed to 200 μM Zn for 3 days. In the end, the second leaf (counting from the base of the stem) was collected for GUS assay. Wild-type tobacco plants were used as a negative control.To examine the tissue-specific NtNRAMP3 (previously annotated as NtNRAMP3-like) was identified, and NtNRAMP3 was reported as a candidate gene encoding metal transporter potentially involved in zinc transport in tobacco leaves . After screening with the FGENESH+ tool , the full-length NtNRAMP3 putative sequence was identified within the scaffold AWOK01S026429.A partial genomic sequence of NtNRAMP3 (containing STOP codon or not) was amplified from cDNA transcribed from total RNA using appropriate primers . Finally, two constructs were obtained—pENTR/D-TOPO::NtNRAMP3 and pENTR/D-TOPO::NtNRAMP3-STOP (STOP codon was included).The full-length open reading frame (ORF) of  primers  by PCR wpENTR/D-TOPO::NtNRAMP3 was recombined with the pMDC43 vector  using LR reaction . The resulting plasmid pMDC43::GFP::NtNRAMP3 contained GFP linked at the N-terminus of NtNRAMP3. The fusion protein was expressed under 35S promoter control.To prepare the construct for subcellular localization, pAG426GAL::NtNRAMP3 were generated by LR recombination between pENTR/D-TOPO::NtNRAMP3 and pAG426GAL-ccdB-EGFP plasmid.For yeast growth assay the vectors NtNRAMP3 was amplified from genomic DNA using appropriate primers . Then, to view predicted transmembrane domains of NtNRAMP3, the web-based software Protter (http://wlab.ethz.ch/protter/start/) that gathers protein features from various annotation sources, such as Uniprot, was used.The nucleotide sequence of Arabidopsis thaliana, Solanum lycopersicum, Zea mays, Theobroma caccoa, and Nicotiana) identified in ARAMEMNON, Solgenomics, MaizeSequence, Phytozome, and NCBI, using Clustal Omega . The phylogenetic analyses were conducted with the MEGA X program (https://www.megasoftware.net)   based on the sequences of NtNRAMP3  and scaffold AWOK01S026429 (primers for NtNRAMP3 promoter amplification). Primers sequences are listed in All primers used in this study were designed with the OligoAnalyzer tool (NtNRAMP3 promoter region were identified with the program PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).Regulatory elements within the pMDC163::promNtNRAMP3::GUS was stably transformed into tobacco plants using GV3101 A. tumefaciens-mediated transformation protocol for tobacco leaf disks . The T1 heterozygous lines with a segregation ratio of 3:1 (hygromycintoler: hygromycinsensit) were self-pollinated to obtain the homozygous generation (T2). Developed independent homozygous lines were tested in a preliminary histochemical GUS assay in young 4-week-old seedlings, subsequently three lines were selected for more detailed analysis.The construct NtPP2A (Nicotiana tabacum Protein Phosphatase 2 A) gene was used as an internal control, and its stability was measured in all plant tissue samples in the range of applied metal concentrations. At least three independent biological replicates were analyzed for each experiment. A minimum 2-fold change in relative gene expression level was considered significant.Total RNA extraction and quantitative real-time RT-PCR (qPCR) analysis were performed according to procedures described in Papierniak et al. . In briesmf1, Δzrt1, Δfet3fet4) Saccharomyces cerevisiae strains  to 0.2, were serially diluted , and then spotted on Petri dishes containing SC-URA+GAL solidified with 2% (w/v) agar and supplemented with required components (details below). Yeast growth was monitored for the next up to 10 days.In the study, two wild-type (DY1457 and BY4742) and three mutants  10 or 30 μM FeCl3, (ii) 30 or 100 μM ferric citrate, and/or (iii) 30 or 80 μM BPDS (bathophenanthrolinedisulfonic acid). For Δsmf1 to the medium 2.5 mM EGTA (Ethylene glycol-bis(β-aminoethyl ether)-N, N, N′, N′-tetraacetic acid) alone or with 0.1 mM MnCl2 (pH was adjusted to 6.0 with 50 mM MES or SC-URA+GAL medium of pH 5.3 was applied) were added. The Δzrt1 mutant was assayed for growth on medium containing (i) 1.0 mM EDTA (Ethylenediaminetetraacetic acid), and/or (ii) 100, 500, 600, or 700 μM ZnCl2. Wild-type strains transformed with the empty pAG426GAL vector were included as a reference control in all assays (DY1457 in experiments including Δfet3fet4 and BY4742 in experiments including Δsmf1 and Δzrt1). Mutant strains transformed with the empty pAG426GAL vector were included as a negative control in all experiments.In complementation tests, for the ΔpAG426GAL::NtNRAMP3 construct was monitored on plates containing SC-URA+GAL supplemented with (i) 5, 10, 20, 50, or 75 μM CdCl2, (ii) 50, 100, 250, 500, or 750 μM CoCl2, (iii) 1.0, 1.4, 2.0, 2.2, or 2.4 mM CuSO4, (iv) 0.2, 3.0, 4.0, 4.5, or 5.0 mM FeCl3, (v) 0.2, 2.5, 5, 7.5, or 10.0 mM MnCl2, (vi) 0.2, 0.3, 0.4, 0.5, or 0.6 mM NiCl2, (vii) 0.2, 1.0, 2.5, 5.0, or 7.5 mM ZnSO4, and (viii) 4.0, 4.5, 5.0, 5.5, or 6.0 mM ZnSO4 . DY1457 transformed with the empty pAG426GAL vector was included as a control in all assays.In sensitivity tests, the growth of DY1457 yeast transformed with A. tumefaciens  transformed with pMDC43::GFP::NtNRAMP3 were used for infiltration of 7-week-old WT tobacco leaves. To visualize the cell wall, after 24–72 h from the infiltration leaf fragments were stained with 50 μM water solution of propidium iodide (PI) for 20 min. Then, GFP  and PI  signals were detected in Nikon A1 confocal laser scanning microscope .The subcellular localization of NtNRAMP3 was determined by monitoring the transient expression of GFP-NtNRAMP3 translational fusion product in tobacco leaf epidermal cells accordingly to the previously described protocol  was performed according to Weremczuk et al. . In briek et al. . In brieNtNRAMP3-like first identified in tobacco within the scaffold AWOK01S026429 was upregulated in the leaves by 200 μM Zn . Analyses of the nucleotide and amino acid sequences provided evidence that the identified sequence is NtNRAMP3.Earlier studies showed that the partial sequence of A. thaliana, S. lycopersicum, T. cacao, Z. mays, and Nicotiana species  showed that the newly identified protein was located on the same branch as other NRAMP3 proteins, with the closest relationship between NtomNRAMP3 and AtNRAMP3  and has both N- and C-termini located intracellularly . Others are distributed in various locations within the sequence, however, the same for certain groups of plants. Thus, in the structure of NtNRAMP3 together with NtomNRAMP3, NaNRAMP3, NsNRAMP3, and LeNRAMP3, an additional histidine was present within CTM. Furthermore, additional histidine residues, H4 and H40, were present in the N-terminus of NtNRAMP3, and also within NtomNRAMP3, NaNRAMP3, and NsNRAMP3 .pMDC43::GFP::NtNRAMP3 construct was transiently expressed in tobacco leaf epidermal cells  and complementation tests . Accordingly, NtNRAMP3 expression was tested in the selected mutants  and WT (DY1457 or BY4742) yeast strains.Natural resistance-associated macrophage proteins are typically involved in the transport of several metals, including Fe, Mn, Ni, Co, Cu, Zn, and Cd  expressing ns of Fe , Mn  was severely restricted.Next, yeast mutants defective in Fe and Mn uptake were used. The expression of fet3fet4  and Δsmft4  Zn, suggesting NtNRAMP3 involvement in plants' response to toxic zinc levels  (A previous study showed that the expression of th pair) . The higNtNRAMP3 in the regulation of the balance of several metals in plants grown under control conditions, with a possible specific role at exposure to extreme Zn-imposed stresses.Taken together, our results indicate a general role of NtNRAMP3 promoter region  is shown in cis-regulatory elements potentially involved in a plant's metal stress response, MRE1 C(T/G/C/A)C , light  and abiotic stresses (ARE) .To investigate the tissue-specific expression, tobacco was stably transformed with the NtNRAMP3 was the highest under control conditions  detected mainly in the xylem of leaves indicates that NtNRAMP3 may play an important role in the unloading of minerals carried by vessels from the roots to the leaves, thus supplying the leaf cells with essential micronutrients.The promoter activity of the Nicotiana tabacum), also frequently used for phytoremediation of metal-contaminated soil. So far, only NtNRAMP1 has been cloned from tobacco and preliminary characterization showed that the protein was targeted to the plasma membrane and contributed to Fe uptake   between VIII and IX TMD , which aFurthermore, the NtNRAMP3 sequence contains essential amino acid residues important for the transport function. These include histidines, which are also believed to contribute to the functional divergence of the NRAMP family , in the presence of elevated concentrations of all tested metals  were localized in the tonoplast   were also targeted in the plasma membrane  Baxter, . This isNtNRAMP3 tissue-specific expression found primarily in the vascular bundles of leaves  in the presence of both excess and deficiency of Fe was demonstrated for AhNRAMP3 in Arabidopsis halleri  and upon the excess of metals  . Both ofn leaves  may contn leaves  support n leaves . Furthernditions . It cannNtNRAMP3 promoter sequence  in leaves from plants exposed to high Zn. It should be noted, however, that the yeast growth assay does not exclude the possibility of transporting Zn via NtNRAMP3. As shown in NtNRAMP3 did not restore the ability of the high-affinity Zn uptake mutant zrt1 to grow on a low Zn medium. However, the growth of transformants was impaired on a medium supplemented with high Zn  under control conditions, by controlling xylem unloading and transfer of metals to neighboring leaf cells. It seems, however, that it may perform the same function when tobacco is exposed to 200 μM Zn, primarily in the 2nd and 3rd pairs of leaves. Further research is necessary to show the underlying mechanisms of metal status-dependent regulation of The original contributions presented in the study are included in the article/KK carried out all experiments, performed data analysis, and contributed to writing the manuscript. AP-W contributed to cloning, hydroponic experiments, and expression analysis. MP and AB were involved in the hydroponic experiments and GUS assays. DMA designed the study concept, coordinated the research and supervised experiments, performed data analysis, and wrote the manuscript. All authors read and approved the final version of the manuscript.This work was financially supported by National Science Center, Poland (grant HARMONIA no. NZ3/00527 to DMA).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: An estimated 30% of antibiotic prescriptions in outpatient settings may be inappropriate. Antibiotic exposure increases an individual’s risk of Clostridioides difficile infection (CDI). To assess the prevalence of community-acquired CDI (CA-CDI) among patients without recent hospitalization and to examine the influence of outpatient antibiotic exposure on the risk of acquiring CA-CDI in this population, we examined a 2-year cohort of patients seen in primary care clinics at VA community-based outpatient clinics (CBOCs) associated with a large VA medical center. Methods: All primary care visits and nonvisit antibiotic prescriptions were identified in calendar years 2018–2019 as encounters of interest. Encounters occurring Results: We identified 84,787 patients with visits meeting our criteria. In this cohort, 3,533 patients were prescribed antibiotics at their encounter of whom 5 (0.14%) developed CA-CDI. Among the 81,254 patients who were not prescribed antibiotics, 15 (0.02%) developed CA-CDI, yielding an unadjusted CA-CDI odds ratio of 7.68 . p Conclusions: Although CA-CDI episodes were infrequent among VA outpatients with a CBOC visit in 2018–2019, the odds of CA-CDI were 7-fold greater in outpatients with antibiotic exposure than outpatients without antibiotic exposure. Antibiotic stewardship interventions that emphasize adverse events as a result of care provided in the outpatient setting, rather than as events limited to acute-care settings, may mitigate CDI risk.Funding: This work was supported by the Merck Investigator Studies Program (MISP 59266 to F.P. and R.J.), and funds and facilities were provided by the Cleveland Geriatric Research Education and Clinical Center (GRECC) at the VA Northeast Ohio Healthcare System. The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.Disclosures:. All authors report no conflicts of interest relevant to this article. R.J. has received research funding from Pfizer; she has also participated in advisory boards for Pfizer and Merck.


Background: Neurofibromatosis type 2 (NF2) is a rare genetic syndrome that predisposes individuals to develop bilateral vestibular schwannomas (VSs) causing a high risk of life-threatening neurological complications. Traditional treatment options for NF2-associated VS usually cause neurological damage, and to date, there are no FDA-approved pharmacotherapies for NF2. The aim of this study was to evaluate the antitumor efficacy of Qu-Du-San-Jie (QDSJ) decoction, a traditional Chinese medicine formula, on NF2-associated VS and to investigate the potential underlying mechanisms.Methods: Ultra high-performance liquid chromatography-mass spectroscopy (UHPLC-MS) analysis was performed to identify the components of QDSJ and their targets. To determine the relationships between the putative targets of QDSJ and the differential genes of NF2-associated VS, the drug-disease crossover genes were screened using the UHPLC-MS data combined with our previous gene expression profiling data. The differentially expressed genes were imported into the STRING database to generate a PPI network. Differentially expressed gene targets and pathways were identified using GO and KEGG pathway enrichment analyses. The in vitro and in vivo drug efficacy of QDSJ decoction was tested using a patient-derived schwannoma cell line and a patient-derived xenograft mouse model, respectively. H&E staining, immunochemistry, and immunofluorescence staining were used to evaluate the cell proliferation and tumor vessels.Results: A total of 133 compounds were identified in QDSJ decoction using UHPLC-MS analysis. Network pharmacology showed that the regulation of necroptosis, apoptosis, cell cycle, angiogenesis, adherens junction, and neuroactive ligand-receptor interaction could be associated with the efficacy of QDSJ in treating NF2-associated VS. Treatment with QDSJ induced necrotic cell death and apoptosis of schwannoma cells in vitro and suppressed the tumor growth in vivo. Histopathological analysis revealed areas of cell necrosis and enlarged tumor blood vessels in the QDSJ-treated tumors. The numbers of cells positive for Cyclin D1 and Ki-67 were significantly reduced in QDSJ-treated tumors compared to control tumors. Immunofluorescence staining of CD31 and αSMA showed a decreased number and density of tumor vessels and normalized vessel structure in QDSJ-treated tumors.Conclusion: Our study demonstrates that QDSJ decoction shows significant antitumor activity against NF2-associated schwannoma and is a possible candidate for future clinical trials. Neurofibromatosis type 2 (NF2) is a rare tumor predisposition syndrome characterized by the development of multiple benign tumors, including schwannoma, meningioma, and ependymoma in the central and peripheral nervous system . BilaterUntil recently, there were no trial-proven medical treatments for NF2-associated VS. The majority of clinical trials for NF2-associated VSs have applied receptor tyrosine kinase inhibitors including nilotinib, sorafenib, everolimus, lapatinib, and erlotinib, and showed mixed response rates . Recent Traditional Chinese medicine (TCM) has shown advantages in suppressing tumor progression, relieving surgical complications, increasing the sensitivity of tumors to chemo- and radiotherapeutics, and improving the survival time and quality of life of patients with tumors. . In thisGinseng Radix Et Rhizoma, 20 g of Gastrodiae Rhizoma, 26 g of Scorpio, 10 g of Scolopendra, 16 g of Chuanxiong Rhizoma, 30 g of Chrysanthemi Indici Flos and 30 g of Hedyotis Diffusae Herba. Briefly, all the raw materials were co-decocted in water twice for 30 min each time. The amount of solvent used was ten times the total weight of the materials. The decoction was concentrated and a total of approximately 500 ml of decoction was collected. For the animal experiments, the concentrated decoction was used for oral administration. For LC-MS analysis and cell experiments, the concentrated QDSJ decoction was freeze-dried using a Pilot5-8M freeze dryer  with an extraction yield of 23%. For 125 ml of QDSJ decoction, which was equivalent to the clinical dosage per day, an average amount of 6.69 g of freeze-dried powder was collected.QDSJ decoction was prepared from 8 g of Ten milligrams of freeze-dried QDSJ powder was dissolved in water, sonicated for 30 min, and centrifuged at 1,000 rpm for 5 min. The supernatant was filtered through a 0.22 μm PTFE membrane.Chromatographic analysis was performed with a Vanquish UHPLC system . The samples were separated on a ACQUITY UPLC BEH C18  at a flow rate of 0.4 ml/min and a column temperature of 30°C. The injection volume was 5 μl. The mobile phases consisted of 0.1% aqueous formic acid (A) and acetonitrile solution (B). The gradient elution program was as follows: 90% ∼ 75% B (0 ∼ 10 min), 75% ∼ 60% B (10 ∼ 20 min), 60 ∼ 30% B (20 ∼ 23 min), 30% ∼ 30% B (23 ∼ 26 min), 30% ∼ 10% B (26 ∼ 27 min) and 10% ∼ 10% B (27 ∼ 28 min).Mass spectrometry was performed using a Q Exactive Plus mass spectrometer  equipped with an electrospray ionization source and operated with the Xcalibur (version 2.1) data acquisition software. The experimental conditions for positive ion detection were as follows: mode, HESI ion source; ionization source voltage, 3.5 kV; capillary temperature of 320°C; sheath gas and auxiliary gas, high purity nitrogen (purity >99.99%); sheath gas flow rate, 35 arb; auxiliary gas flow rate, 10 arb. For negative ion detection, the following conditions were applied: mode, HESI ion source; ionization source voltage, 3 kV; capillary temperature of 320°C; sheath gas and the auxiliary gas, high purity nitrogen (purity >99.99%); sheath gas flow rate, 35 arb; and auxiliary gas flow rate, 10 arb. Data of the positive and negative ions were collected by the method of Fourier transform high-resolution full scan . Data-dependent acquisition (MS2) was performed by the method of collision-induced dissociation.The mass spectrometry data were searched with the corresponding information in the OTCML high-resolution mass spectrometry database  to identify traditional Chinese medicine components.p < 0.05 was used.Gene expression profiling analysis was performed on an independent cohort of 37 NF2-associated VSs and seven normal peripheral nerves using the Affymetrix Human Transcriptome Array 2.0 Kit , as we described previously . Sample https://pubchem.ncbi.nlm.nih.gov/). The SMILES strings were then submitted to SwissTargetPrediction (http://www.swisstargetprediction.ch) to identify the corresponding targets of QDSJ. Targets with a probability of no less than 0.05 were selected.The identified compounds in the QDSJ decoction and the Simplified Molecular-Input Line-Entry System (SMILES) strings were retrieved from PubChem (https://string-db.org/) to generate the protein-protein interaction (PPI) network. The PPI network was visualized and analyzed using Cytoscape . The cytoHubba plug-in was then used to screen the top 15 key genes based on degree scores in the PPI network.To illustrate the relationships between the putative targets of QDSJ and the DEGs of NF2-associated VS, the drug-disease crossover genes were screened. The overlapping genes were submitted to the STRING database (https://www.genome.jp/kegg/pathway.htmL) were analyzed using clusterProfiler package (version 4.2) in R software (version 3.6.2).The underlying mechanisms of these overlapping targets and corresponding Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways .BNI-VS-50 cells were seeded in 96-well plates at a density of 3,000 cells/well. QDSJ freeze-dried powder was dissolved in PBS and filtered through a 0.22 μm filter. After being treated with QDSJ for 72 h, the cells were observed using a Cytation 5 Cell Imaging multimode reader . Cell viability and IC5 cells/well. After being treated with 250 ng/ml or 350 ng/ml QDSJ or PBS for 48 h, the cells were rinsed in PBS three times and incubated for 20 min in the dark at room temperature in 2 μmol/L Calcein-AM and 5 μmol/L PI. The cells were then gently washed three times in PBS. Excited at 488 nm, the living cells appeared green  and the nuclei of the dead cells displayed red fluorescence (per PI instructions). The stained cells were then imaged under a laser scanning confocal microscope .Calcein-AM/PI staining  was used to assess the cell death of BNI-VS-50 cells after QDSJ treatment. BNI-VS-50 cells were seeded in 24-well plates at a density of 2 × 10A pSIVA apoptosis detection kit  was used to evaluate cell apoptosis. Treated with QDSJ for 48 h, the BNI-VS-50 cells were incubated with 10 µl/ml of the pSIVA-IANBD probe and 5 µl/ml of PI. Cells were observed under microscope using the green fluorescence filter for pSIVA-IANBD and the red fluorescence filter for PI visualization.BNI-VS-50 cells treated with QDSJ or PBS for 48 h were collected and then sonicated in ice-cold RIPA lysis buffer containing a protease and phosphatase inhibitor cocktail . The cell lysates were centrifuged at 12,000 × g for 15 min at 4°C, and the supernatant was collected. The protein concentrations were determined using the BCA protein quantification assay . Twenty micrograms of denatured protein was loaded onto an SDS-PAGE gel, and 5 μl of prestained protein marker was loaded as a molecular weight standard . The proteins were transferred onto PVDF membranes and incubated with the following primary antibodies: anti-Cyclin D1 , and anti-GAPDH . The membranes were then washed with 0.01% TBS-Triton X and incubated in HRP-conjugated goat anti-rabbit IgG  or goat anti-mouse IgG . The bands were visualized using a chemiluminescence reagent . Imaging was performed using Amersham Imager 680 . The bands were analyzed with Image Lab Software .ad libitum access to food and autoclaved water.All the experiments were performed in accordance with the research guidelines set forth by the ethics committee of Beijing Neurosurgical Institute for animal research . Male BALB/c nude mice (6 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology . The animals were group-housed and kept on a 12-h light and dark cycle with 6) were inoculated into the lower backs of BALB/c nude mice using a Hamilton syringe, as we described previously (n = 5/group). QDSJ decoction was administered by oral gavage every 12 h for 14 consecutive days. The dose was calculated based on the equivalent dose for humans by multiplying a factor of 0.026 for mice with reference to body surface area. Mice in the vehicle group received autoclaved water instead. Tumor growth was evaluated by monitoring tumor volume every 2 days. The tumor volume was calculated using the following formula: (length × width) × √ (length × width) × (π/6). The animals were sacrificed 24 h after the final administration. The tumor xenografts were harvested for further evaluation.BNI-VS-50 cells  for 18 min. To block endogenous peroxidase activity, the sections were treated with 3% hydrogen peroxide for 15 min. After blocking, the sections were incubated with anti-Cyclin D1 antibody  at 4°C overnight. After washing, the sections were incubated with a biotinylated secondary antibody  for 10 min followed by treatment with HRP, DAB substrate, and hematoxylin counterstaining .For immunofluorescence staining, after antigen retrieval using sodium citrate buffer, the sections were treated with 0.3% Triton-X-100 in TBS for 30 min and then blocked with TGB superblock containing 10% normal goat serum and 10% BSA. The sections were incubated with primary antibodies at 4°C overnight, including anti-Ki-67 , anti-CD31 , and anti-alpha smooth muscle Actin  antibodies. The secondary antibodies against the appropriate species, including Alexa Fluor 488-conjugated goat anti-rabbit  and Rhodamine Red-X-conjugated goat anti-mouse , were incubated for 2 h at room temperature. The sections were then treated with a True VIEW autofluorescence Quenching Kit  and mounted.https://imagej.net/Fiji). All the sections were analyzed by an investigator who was blinded to the experimental groups.Two sections per xenograft and three random fields per section were used to analyze the results. The sections were 100 μm apart from each other. H&E and IHC staining images were obtained using digital whole slide scanning  and reviewed using computer-based image analysis . Immunofluorescence images were captured using an Olympus BX53 microscope and a DP74 CCD camera . To reduce the variability in signal intensity, images were collected using the same exposure time setting, brightness, and contrast for each fluorophore. The percentages of cells that were positive for Cyclin D1 and Ki-67 expression were measured. The percentage of αSMA-positive vessels, collapsed vessels, and vessel densities were quantified for CD31 staining. The integrated optical densities (IODs) of αSMA were assessed. Images were analyzed using Fiji software . IC50 values were determined using nonlinear regression analysis of effect-log concentration curves. Graphs were produced in Prism 9, and error bars denote the standard deviation (SDs). Comparisons were interpreted as significant when associated with p < 0.05.Raw data from Fiji and Image Lab analyses were imported into Prism 9  for statistical analyses using ANOVA followed by Tukey’s post-hoc intergroup comparison or using Student’s in vitro and in vivo experiments to reveal the potential mechanism by which the QDSJ decoction affects NF2-associated VS. The chemical constituents of the QDSJ decoction were determined by UHPLC and mass spectrometry . A total of 1914 DEGs were identified using the criteria of fold change ≥ 2 and p < 0.05. Venn diagram analysis showed that QDSJ decoction shared 147 putative targets with NF2-associated VS ; and the numbers of early apoptotic cells, late apoptotic cells, and necrotic cells were 8.84-, 27.01-, and 30.06-fold higher, respectively, compared to control group 48 h after 350 μg/ml QDSJ treatment , and the tumor weight was reduced by 49.74% (0.79 ± 0.35 g in vehicle-treated mice and 0.40 ± 0.12 g in QDSJ-treated mice) after 14 days of QDSJ treatment compared to vehicle treatment (p < 0.05, p < 0.001, p < 0.05, As QDSJ improved the necrosis and apoptosis and inhibited the proliferation of BNI-VS-50 cells, we evaluated the antitumor effect of QDSJ decoction on NF2-associated VS ace area . We obseace area . The tump < 0.05, +/CD31+ cells, which indicated the pericyte-covered vessels, was significantly increased after QDSJ treatment compared to the vehicle-treated group (28.77% ± 2.58% in vehicle-treated mice and 57.90% ± 14.36% in QDSJ-treated mice, p < 0.01, Impressed with the suppressed tumor growth and cell proliferation after QDSJ treatment, and the tumor angiogenesis-related proteins, including EGFR, FGF1, FGF2, VEGRR2, and PDGFRA revealed in the top 15 key targets in the PPI network of QDSJ decoction treating NF2-associated VS, we hypothesized that QDSJ decoction would attenuate tumor angiogenesis of the NF2-associated VS. As expected, decreased vessel density after QDSJ treatment was confirmed by evaluating the areas covered by vessels (p < 0.01, p < 0.05, Vessel collapse is another characteristic of tumor vasculature, which might be exacerbated by tumor fibrosis, leading to interruption of blood flow and hypoperfused hypoxic areas . CD31 stin vitro and in vivo antitumor effects of QDSJ decoction on NF2-associated VS. Our findings indicate that QDSJ decoction may have the potential capacity to slow the growth of VSs in NF2 patients.The clinical treatment of NF2-associated schwannoma is still a challenge due to the high risk of morbidity . The curIn recent years there has been intense interest in developing chemotherapeutic strategies for patients with NF2 tumors . TCM hasTCM has been used in treating tumors relying on its multi-target characteristics targeting several crucial proteins in cancer-related signaling networks . Tumor sTumor blood vessels are a key target for inhibiting tumor progression. Poor vessel functionality caused by the improper development of tumor blood vessels leads to poor hypoxia, decreased immune cell infiltration and activity, and increased risks of metastatic dissemination . AntiangThis study revealed that QDSJ decoction exerts a multitarget antitumor effect on NF2-associated VS by increasing cell apoptosis, reducing cell proliferation, and normalizing tumor blood vasculature. The bioinformatics analyses suggested the involvement of several signaling pathways that are closely related to the nervous system and tumorigenesis. As our study is the first to validate the effect and mechanism of a Chinese medicine formula in treating NF2-associated schwannoma, further pharmacological and pharmacokinetic studies will be needed to understand the mechanisms and biological efficacy of QDSJ to optimize this therapeutic strategy.In summary, we show that QDSJ decoction suppresses tumor growth and improves vascular normalization of NF2-associated VS. Furthermore, the drug-target-pathway network elucidated by bioinformatics analyses illustrated the potential mechanisms by which QDSJ decoction affects NF2-associated VS. These results suggest that QDSJ could be a new strategy for the treatment of schwannoma.


In this paper, the issue of self-compacting concrete (SCC) with the addition of polyethylene terephthalate (PET) recycled aggregate is addressed. The PET utilized was a waste fraction in the PET-bottle-recycling process. The implementation of waste in concrete mixes has a positive impact on their environmental and social profile; however, technical requirements are not necessarily met. In this investigation, PET was used as a substitute for fine aggregate in quantities ranging from 0 to 20% in increments of 5%. Both the flow properties of SCC mixes and the hardened SCC properties  were investigated. Additionally, non-destructive tests (ultrasound and sclerometric) were performed to determine the correlation curves. The research revealed that both the flow properties and the parameters of the hardened concrete deteriorated with the PET content. Concrete with 20% PET replacement did not meet the self-compacting requirements and its compressive strength decreased by almost 50%. However, it was noted that replacing fine aggregate with PET aggregate in the amount of 5% did not significantly alter the concrete parameters and could be an attractive alternative to traditional concretes. Based on non-destructive testing, correlation curves were constructed that could be applicable to the future quality assessment of self-compacting concretes with recycled PET aggregate. The rapid consumption of multiple natural resources and the production of substantial amounts of waste by the construction industry are widely recognized to contribute to a serious environmental problem. With the development of both science and technology, including building materials, this crisis should be continuously alleviated. One of the prevailing approaches is the implementation of intelligent and energy-efficient solutions in the construction sector, such as the idea of a circular economy or new generation materials with improved durability parameters.2) and particulate matter (PM10) to the environment, as well as the depletion of natural resources. Following the sustainable development strategy, new technologies have been introduced to the construction industry for several decades, for example, eco-efficient concrete. These measures aim, among other things, to minimize the environmental impact during production, increase the durability and resistance to environmental hazards, extend structural reliability, and reduce the life cycle costs of structures.Today, concrete is the most widely used construction material due to its versatility, good general availability, favorable mechanical properties, and relatively low price. Its production increases annually (approximately 32 billion tons worldwide ) and is 2 emissions [2 and PM10, limit the consumption of natural mineral resources, and reduce post-industrial waste, which can be given a “second chance of life,” in line with the concept of a circular economy. In their final life cycle stage, these materials, such as traditional concrete, can be recycled by preparing crushed concrete to replace natural aggregate, as a highway substrate, or as a clean fill around buildings. Compared to other materials, concrete is relatively environmentally friendly; therefore, the eco-improvement of the performance characteristics of concrete must be multiphased and customized to the location of its implementation. The analysis of applied solutions taking into account the whole life cycle of the building object is considered crucial for concrete construction [In the world literature, suggested strategies for the development of eco-efficient concrete involve, among others, the replacement of Portland cement’s  mineralmissions ,4. Thus,truction ,6,7. TheAnother global concern is the depletion of natural raw materials, including sand, which is used extensively in the construction, road, and glass industries. Although Earth’s sand resources are relatively abundant, not all sand is suitable for concrete mixes. Thus, in concrete technology, the concept of substituting sand with recycled aggregates, including glass or plastic products, emerged .Due to their superior properties , plastics remain in use in many industries around the world. In the last 50 years, plastic production has increased more than 22-fold. However, given the amount of plastic consumed and the fact that not all of it can be recycled, plastic pollution is one of the fastest-growing threats in the world . PolyethIn the literature, there are various examples of the use of PET materials in concrete technology, but most often they concern PET fibers, i.e., intentionally prepared fibers (by the author or by the industry) rather than waste materials. The volume of fiber content with respect to fiber concrete is between 0.3% and 1.5%. Therefore, the procedure reuses a small amount of PET waste, and in addition, the waste must be deliberately prepared . EvaluatThis study investigated concrete mixes that involved the addition of waste from the PET bottle recycling process, which cannot be recycled, or the spectrum of their application is limited. The design of modern building materials with a reduced environmental impact and consume fewer natural resources while disposing of non-recyclable waste is a necessary step in light of climate change and the European Union requirements being introduced. The implementation of waste into a new generation concrete mix, such as self-compacting concrete (SCC) mix, would reduce the environmental impact while maintaining fine mechanical properties .In this study, the PET fines washed material acquired from a leading PET bottle recycler in Poland was used. The physical and mechanical characteristics declared by the recycler are summarized in Given the relatively wide range of dimensions of the PET flakes declared by the manufacturer (0.5 to 4.0 mm), sieve analysis was performed according to standard  without The grading curves of PET aggregate indicated that the particle size distribution for individual samples was nearly identical, and slight differences could be observed for the finest fractions. Fractions above 4.0 mm constituted approximately 5% of the material composition; fractions between 2.0 and 4.0 mm constitute 20% of the material; the largest fraction was between 1.0 and 2.0 mm, which represented almost 70% share; and the remaining 5% were fractions below 1.0 mm. The entire material was divided into three main fractions , as shown in In this research program, 5 concrete mixes with different contents of PET flakes (fraction below 2.0 mm) were prepared, which was used as the fine aggregate. The SCC control mix was prepared with a replacement of 5%, 10%, 15%, and 20% of the mass of fine aggregate (sand). The mixtures were based on Portland composite cement CEM II/B-M of a strength category of 42.5 and normal strength gain (symbol N). The cement consisted of Portland clinker and the addition of fly ash and ground-granulated blast furnace slag in the amount of 21–35% according to EN 197 . In the 3, which corresponded to a water-to-binder ratio of 0.36. The proportion of fine aggregate was fixed at 684 kg/m3 for all mixtures. The superplasticizer was dosed each time until sufficient flow parameters of the SCC were achieved. Detailed compositions of the mixtures are compiled in The binder content was fixed at 440 kg/mA total of 21 test elements (20 cubes with 150 mm sides and 1 cube with 100 mm sides) were prepared from each concrete mix, resulting in a total of 105 test elements.500) and the diameter of the slump flow after completion of the flow were measured according to [All concrete mixtures were subjected to flow properties tests immediately after the mixing process. Flowability and plastic viscosity were evaluated using the slump flow test, during which, the time to 500 mm slump flow diameter .Another important strength parameter of concrete is the splitting tensile strength. Investigations of this aspect were performed according to standard  on cube E) and Poisson’s ratio (ν) were determined over the full range of linearity of the stress–strain characteristics  of a given specimen. The modulus of elasticity was determined as the ratio of the difference in compressive stress difference  to longitudinal strain  with 54 kHz transducers with an automatic transmitter voltage. Prior to each use, the device was calibrated on a dedicated calibration rod with an ultrasound wave transit time of 25.4 μs. Measurements were performed with a coupling agent in the form of a chemically passive polyacrylate gel; both transducers’ heads were covered with a layer of gel before each measurement. The specimens were divided into two types: specimens for determining the mechanical properties of 150 mm × 150 mm × 150 mm cubes and specimens for determining the mechanical properties of 50 mm × 100 mm cylinders. For samples from both groups, the testing fields were prepared via gentle grinding. For samples in group 1, tests were made only for side surfaces in the central part of the samples, while for samples in group 2, four sections were defined: two along the direction of concrete casting and one in each of the upper and lower zones of the sample.For each test section of a given sample, 3 measurements were performed and the average values of the ultrasound wave transit velocity (v) between the transducer heads were presented as the results. The tests were performed in relation to the standards EN 12504-4  and EN 1The sclerometric tests were conducted with a Proceq N-type Schmidt sclerometer (Switzerland). The Schmidt sclerometer allows for the surface hardness of the concrete to be measured from the indentation of an incident mass after its collision with the surface. In order to ensure proper stability of the tested samples and thus accuracy of the results, the samples were mounted in a vice. The tests were performed on the same surfaces as the ultrasound examinations. The testing fields were prepared via gentle grinding to achieve smoothness and flatness. For each test surface, nine measurements were performed and their results, properly arithmetically mediated, provided the sclerometric rebound index. The tests were performed in relation to the standards EN 12504-2  and EN 1X-ray computed tomography (X-ray CT) illustrating the distribution of PET flakes in each SCC was performed for selected samples. X-ray CT was performed using a GE  Phoenix v-tome-x m device.The GE Phoenix v-tome-x m device together with VG Studio Max  software, enables the reconstruction and analysis of the internal structure of the element tested on the basis of a series of X-ray images obtained during a 360° rotation of the sample . Table 4Beam hardening correction, automatic geometry calibration, and geometry optimization algorithms were applied during the reconstruction process. After the image reconstruction, separation of the PET flakes in a given sample from the 3D model was performed.The properties of the fresh SCC mixtures were determined using filling ability (slump flow) and passing ability (L-box) tests. Furthermore, the fresh visual segregation index was assessed. On the basis of the results, all mixtures were classified according to the European Guidelines for SCC . As SCC The main finding of the flow tests was that regardless of the investigated parameter, deterioration increased with the addition of PET flakes. The amount of superplasticizer was controlled to avoid segregation of the SCC mixture. Thus, despite the higher proportion of superplasticizer, the slump flow decreased with an increased proportion of PET flakes; simultaneously, the viscosity of the mixture (the time of slump flow up to 50 cm diameter) increased. The lower the natural aggregate content in the mix, the more difficult it was to maintain sufficient stability of the mix; therefore, bleeding was observed in all mixes with the addition of PET flakes. Consequently, due to the high probability of significant segregation, the mixture with 20% PET in relation to the fine aggregate mass did not reach the self-compacting guidelines. A reduction in the stability of the mixture was also observed during the L-box test. The L-box ratio deteriorated with the addition of PET flake aggregate as a result of the higher viscosity of the mixture. The authors of other studies on self-compacting mixes with PET aggregates drew analogous conclusions ,38,39.The present research program involved the preparation of self-compacting concretes with recycled aggregate in the form of PET waste flakes. A series of tests were conducted on samples made from such concretes, including the determination of compressive strength, splitting tensile strength, modulus of elasticity, and Poisson ratio through the destructive test method. In addition, a series of non-destructive tests were performed, which are described in In whisker-box plots, the boxes are determined by quartiles (25% and 75%), the midpoint represents the mean of the results, and the minimum and maximum values of the test series are presented as whiskers. The results of the mechanical and deformation parameters are presented in relation to the type of mixture.Compressive strength appeared to be significantly affected by the addition of PET waste flakes, starting with a 10% replacement of the fine aggregate a. The beThe splitting tensile strength decreased with the addition of PET aggregates to the SCC b. The deYoung’s modulus determined in compression decreased with the addition of PET aggregate c. Due toThe higher the PET percentage in the mixture, the higher the Poisson ratio in compression d. Its chIn the literature, there is some research on destructive tests of self-compacting concretes with PET aggregate in terms of compressive strength, splitting tensile strength, and modulus of elasticity ,41,42,43Non-destructive test series were performed on the samples, including the determination of the rebound number and ultrasound wave transition velocity. As in the case of destructive tests, the results are presented graphically using box plots . The tes3 was recorded for SCC-P-0 and, on average, reductions of 2.4%, 3.8%, 5.7%, and 9.5% occurred for concretes with 5%, 10%, 15%, and 20% PET replacements, respectively.For both sclerometric and ultrasound tests, a decrease in the test result value was observed with increasing PET aggregate content. For ultrasound tests, the relationship of the decrease in wave transition velocity with the increase in PET flake content was more linear than for the sclerometric tests. One can also observe that the decrease in the ultrasound wave velocity was accompanied by an increase in the coefficient of variation, which was primarily caused by registering single results significantly lower than the average . The reduction in the velocity of the ultrasound wave transition for successive concretes was well correlated with the decrease in density as the proportion of PET replacement in the concretes increased. Compared to the average ultrasound wave transition velocity of SCC-P-0, reductions of 2.0%, 5.2%, 10.0%, and 15.8% on average occurred for concretes with 5%, 10%, 15%, and 20% PET replacements, respectively. In the case of the bulk density of hardened concrete, a density of 2360.3 kg/mIn the sclerometric tests, the variations were not linear; for SCC specimens without and with 5% PET replacement, the rebound numbers differed minimally and the scatter was slightly less for the control specimens. For SCC-P-10 and SCC-P-15, a significant decrease was reported relative to the control samples, and the values were also close to each other. Significantly lower results were observed, which may have been due to the location of the PET flakes close to the sample surface, which may have directly affected the decrease in surface hardness, and the highest coefficient of variation was observed here. The lowest rebound number results were recorded for SCC-P-20 specimens. Compared to the average rebound number of SCC-P-0, decreases of 0.4%, 9.6%, 9.4%, and 14.9% on average were observed for concretes with 5%, 10%, 15%, and 20% PET replacements, respectively. Similar correlations were obtained for compressive strength; hence, the conclusion is that the cause may have also been due to a significant deterioration in the flow properties of the fresh mixture.In the literature, there are individual works on non-destructive testing of self-compacting concretes ,43, althX-ray CT scans provided a view of the distribution of PET flakes in the concrete specimens. To visualize them more clearly in the figures, an analysis was performed to isolate the voxels in a given grey value range corresponding to the PET material. In concrete materials technology, compressive strength is taken as the basic mechanical characteristic. In the present study, it was found that the modification of the fine aggregate to up to 5% of the PET aggregate had no significant effect on the compressive strength of the SCC. A considerable reduction in compressive strength was observed for mixtures containing at least 10% PET flakes relative to the mass of sand, while for subsequent percentages, the effect was not as pronounced. These results were compared with data from the available literature and presented in a summary graph in The mixture containing PET granules did not show a significant decrease in compressive strength up to 10% PET granule content. This may have been related to the favorable shape of this PET aggregate, as well as the generally higher compressive strength of concrete .In general, SCC mixes with PET flakes exhibited a reduction in compressive strength, even in the case of a small replacement percentage. It should be noted that the control concretes varied in compressive strength between experiments; therefore, the comparison was made on the variations rather than the values themselves. Self-compacting concrete appeared to be more prone to a decrease in compressive strength as the proportion of PET aggregate increased. This was related to the deterioration of flow properties and the increased sensitivity of self-compacting mixtures to segregation when adequate technological rigor is not maintained. In the case of the highest replacement percentage of fine aggregate in Hama et al. , which wThe compressive strength of traditional concretes also tended to decrease as the proportion of PET in the mix increased. For lower-strength normal concretes, these reductions were more significant, although not as pronounced for self-compacting lower-strength concretes. In the case of the study by Rahmani et al. , the totA noteworthy hypothesis is that the decrease in strength of concrete with PET recycled aggregate is related to the increased softness of the material compared to natural aggregate, which was proposed by Faraj et al. . The revFurthermore, on the basis of SEM studies, it was concluded by other authors that as the proportion of PET aggregate increases, the porosity of concrete and the width of the poor quality interfacial transition zone (ITZ) increases. Kangavar et al.  reportedThe second characteristic that is of great importance for concrete composites is tensile strength. In the present study, analogous to compressive strength, it was found that substitution of fine aggregate to PET aggregate up to 5% did not significantly affect this property. The subsequent decrease in the splitting tensile strength with increasing PET content can be considered to be relatively linear. These results were compared with available literature data and presented in a summary graph in All traditional concrete mixes with PET aggregate exhibited a similar linear decrease in splitting tensile strength with an increased proportion of PET aggregate. In the case of the replacement of 5% fine aggregate with PET aggregate, both Albano et al.  and SaikThe ultrasound results obtained were similar to those reported in the literature. Albano et al.  tested nThe empty spaces created by the air pores and PET particles attenuate the ultrasound wave due to the acoustic impedance. When the ultrasound wave passes through different media , it is partially reflected and transmitted; therefore, its velocity decreases. First of all, the decrease in the ultrasound wave velocity is caused by the reduction in concrete density . Moreove2 = 0.845). The basic curve, on the other hand, seemed to be strongly underestimated relative to the results obtained. However, it should be considered that the basic curve presented in EN 13791 [f (Equation (4)), which consists of the average difference obtained between the destructive tests results of the samples and the results obtained from the basic curve k1 that depends on the number of pairs of results :The tests performed on the mixtures with different PET replacements indicated significantly varying results of ultrasonic wave transit velocity tests. Therefore, it was possible to correlate the compressive strength with the velocity of the ultrasound wave and relate these results to the standard basic curve presented in EN 13791  is the lThe shifted basic curve provides a better representation of the relationships described. There is only a single point located under the curve (thus the estimation using this function would be highly secure). The analyses carried out indicated that it was necessary to adopt a limitation of the curve application (both shifted and correlation) similarly to the basic curve. For the shifted basic curve, it was 3900 m/s to 4800 m/s, and for the acquired correlation curve, it was 3700 m/s to 4600 m/s. Crucially, the correlation function exhibited a pattern very similar to that of the basic and shifted functions in terms of direction.2 = 0.57). As in the case of ultrasound examinations, these results were summarized in a combined graph with a basic curve and a shifted basic curve , is far more beneficial from an environmental point of view than the use of granulates or specially prepared fibers. One can assume that the acquisition of PET flakes in fractions above 4.0 mm (used in the production of PET granulate) is inevitably connected with the production of waste in the form of smaller-fraction flakes and PET powder. Therefore, the costs associated with PET bottle treatment need to be borne anyway, which improves the economic profile of SCCs with PET aggregates compared to SCCs with PET fibers. However, as demonstrated in numerous studies, the use of PET fibers in small quantities (less than 2% by volume) has the potential to improve the mechanical properties of concrete, especially in terms of compressive strength and flexural strength, including SCCs ,48. NeveGiven that the world’s annual production of concrete is steadily increasing and currently reaches approximately 32 billion tons per year , and itsNowadays, SCCs are considered to be a special type of concrete used in structures with dense reinforcement and complex geometries. However, given the further aspects of environmental and social assessment, it seems inevitable that the utilization of mixtures that do not require additional technological processes (such as vibration and troweling) will increase in demand. It is essential to investigate the incorporation of waste materials into new generation concretes in order to effectively implement these solutions in the future.PET aggregates in self-compacting concrete were uniformly distributed regardless of their content in the mix.The higher the PET content in the mix, the lower the density of the concrete, and therefore, the velocity of the ultrasonic wave and the rebound number of the sclerometer. The non-destructive test results acquired relatively good correlations with the compressive strength test results.The replacement of fine aggregate with PET recycled aggregate negatively affected the parameters of both fresh mixes and hardened concretes. In the case of the concrete with the highest aggregate replacement, the decreases compared to the control concrete were 9.5%, 47.8%, 32.5%, 32.7%, and 61.7% in terms of bulk density, compressive strength, splitting tensile strength, modulus of elasticity, and Poisson ratio, respectively. The ASCC-P-20 also did not meet the requirements of self-compaction.However, the replacement of 5% of natural aggregate with PET aggregate of the appropriately selected fraction was possible for SCC and did not cause significant variations in the physical and mechanical properties: 2.4%, 1.3%, 2.4%, 5.0%, and 5.4% in terms of bulk density, compressive strength, splitting tensile strength, modulus of elasticity, and Poisson ratio, respectively.This paper discusses the possibility of using PET waste flakes of the fraction below 2.0 mm in self-compacting concrete mixtures. The presented results and their juxtaposition with data from the literature provide the following conclusions:Research on the properties of SCC mixtures with PET aggregates in the form of flakes should be further developed to determine properties such as corrosion resistance, frost resistance, water penetration resistance, the durability of concrete or bond to reinforcing steel, and the bond to subsequent layers of concrete. In addition to the differences in the obtained basic research results relative to the literature data, as important steps forward in this research, one can indicate the determination of correlation curves for non-destructive testing and the determination of the Poisson’s ratio, which is not commonly referred to, but can be useful in the future structural design using SCC with PET .