Various indicators were used to study the bacterial response to these drugs, including monitoring reactive oxygen species (ROS) and cell membrane permeability by flow cytometry, cell arrangement, and whole-genome RNA and protein sequencing. Enhanced conjugation correlated well with increased production of ROS and cell membrane permeability. Additionally, these non-antibiotic pharmaceuticals induced responses similar to those detected when bacteria are exposed to antibiotics, such as inducing the SOS response and enhancing efflux pumps. The findings advance understanding of the transfer of antibiotic resistance genes, emphasizing the concern that non-antibiotic, human-targeted pharmaceuticals enhance the spread of antibiotic resistance among bacterial populations.Antibiotic resistance is a global threat to public health. The use of antibiotics at sub-inhibitory concentrations has been recognized as an important factor in disseminating antibiotic resistance via horizontal gene transfer. Although non-antibiotic, human-targeted pharmaceuticals are widely used by society , the potential contribution to the spread of antibiotic resistance is not clear. Here, we report that commonly consumed, non-antibiotic pharmaceuticals, including nonsteroidal anti-inflammatories , a lipid-lowering drug (gemfibrozil), and a  Increasingly, antimicrobial resistance is a major threat to public health, causing 700,000 deaths worldwide each year . BacteriStreptococcus pneumoniae [Acinetobacter baylyi), the transfer of free plasmids could be enhanced by non-antibiotic pharmaceuticals. However, it remains unknown whether non-antibiotic pharmaceuticals promote conjugation between two intra- or intergenera populations, which is of particular clinical concern as conjugative multidrug resistance plasmids allow rapid expression of multidrug resistance phenotypes, thus facilitating the emergence and spread of antibiotic-resistant bacteria [It is commonly accepted that the emergence and spread of antibiotic resistance is largely due to intensive applications of antibiotics in clinical, veterinary, and agricultural settings . Exposureumoniae . Althougeumoniae , 10, theeumoniae  screenedeumoniae . We preveumoniae . This prbacteria . In addiβ-blocker (propranolol), and a contrast medium (iopromide). These drugs are used in a wide range of clinical settings including for pain/fever-relief, treatment of inflammation, lipid control, heart disease, and diagnostic medicine. All these pharmaceuticals are on the World Health Organization List of Essential Medicines, and are widely consumed. For example, worldwide there are 30 million users of NSAIDs daily, and over 100 million consumers of NSAIDs annually in the USA alone [In this study, we aimed to investigate the potential of different types of commonly consumed non-antibiotic, human-targeted pharmaceuticals for promoting conjugative transfer of plasmid-borne ARGs. To this end, we established two conjugative models consisting of both intragenera and intergenera conjugations, in which ARGs were harbored on environmentally relevant conjugative plasmid RP4 , or clinSA alone . Once coSA alone –19. In aSA alone –22. ThusSA alone , 24.Escherichia coli K-12 LE392 with plasmid RP4  was the donor [Pseudomonas putida KT2440 , was the donor for Model-2 [E. coli J53 with resistance to sodium azide was the recipient for Model-2 [Two conjugation models were applied in this study, i.e., environmentally relevant Model-1, and clinically relevant Model-2. In Model-1, he donor . Pseudomloputida ) with hiloputida , 28. E.  Model-2 . E. coli Model-2 . CultureBacterial MICs for antibiotics and non-antibiotic pharmaceuticals were determined according to previous methods , 29. MIC8 cfu/mL were mixed 1:1 to establish the PBS-based conjugative mating system (pH=7.2), using a total volume of 1 mL. Various concentrations of non-antibiotic pharmaceuticals were added to the mating system, including clinically and environmentally relevant concentrations, and sub-MIC levels, i.e., 0.005, 0.05, 0.5, 5, 50 mg/L for ibuprofen, naproxen, gemfibrozil, diclofenac, propranolol, and 0.01, 0.1, 1, 5, 50 mg/L for iopromide [μL of the mixture was spread onto LB agar selection plates containing antibiotics to enumerate transconjugants, with details described in Text SThis study established two mating models to investigate if non-antibiotic pharmaceuticals could promote gene transfer. In the environmentally relevant conjugation model (Model-1), donor and recipient both at a concentration of 10opromide , 21, 24.For Model-2, donor and recipient bacterial strains were both grown to an OD600nm value of 1.8, and mixed 1:2 in LB broth according to a previous study . The nonIn addition to the above matings, further sets of Model-1 were established with the addition of 100 μM ROS scavenger, thiourea. The conjugative transfer ratio was calculated from the number of transconjugant colonies divided by the number of recipients. As no nutrients were provided during the mating process, the growth of donor, recipient, and transconjugant was neglected.E. coli MG1655 with chloramphenicol resistance was the recipient [TM m Endo agar plates (to distinguish E. coli and P. putida) with the appropriate antibiotics, as described in Text STo test the reverse transfer process, transconjugants obtained from Model-1 were used as the new donor, while a mutant strain of ecipient . The conThe conjugative plasmids applied in this study are both large, i.e., 60.09 kb for RP4, and 137.57 kb for pMS6198A, and none of the recipient bacterial strains were competent cells. Thus, the bacterial transformation was ruled out .traF gene of plasmid RP4 was amplified by PCR, and the amplicons were observed using 1% agarose gel electrophoresis. To further verify the identity of the plasmid, PCR was applied for detection of the tetA and blaTEM from the RP4 plasmid. Similarly, the presence of pMS6198A in transconjugants from Model-2 was determined by genome DNA extraction followed by PCR using primers for blaNDM. PCR primers and conditions are described in Text SModel-1 transconjugants growing on selective plates were randomly picked, cultured, and stored in 25% glycerol at −80 °C. The plasmids of transconjugants were extracted using the Invitrogen PureLink Quick Plasmid Miniprep Kit . The specific TEM  operated at 80 kV was used to observe the effect of pharmaceuticals on bacterial cells. Conjugation experiments were performed as described above, and TEM samples were collected after 8-h mating with either 0.5 mg/L ibuprofen, naproxen, gemfibrozil, diclofenac, propranolol, or 1.0 mg/L iopromide. Sample preparations were performed according to standard procedures as previously described , and detThe fluorescence method was used to analyse ROS generation and cell membrane permeability, as described in Text STo further verify whether ROS is crucial to the conjugation process, conjugation Model-1 and ROS generation were additionally conducted under anaerobic conditions. The assays were the same as that under aerobic conditions, except that oxygen in LB or PBS was depleted and the experiments were conducted in an anaerobic chamber .®, Germany), with an extra bead-beating step for cell lysis [E. coli K-12 (NC_000913), P. putida KT2440 (NC_002947), and IncPα RP4 plasmid (L27758), obtained from the National Center for Biotechnology Information (NCBI). Regarding the bioinformatics pipeline, NGS QC Toolkit (v2.3.3) [2 fold-change ≥1.0 or ≤ −1.0 with both P value and false discovery rate  < 0.05 was used to distinguish the differentially expressed genes.In order to analyze the gene expression levels during the conjugative process, conjugation experiments (Model-1) were performed as described above, and RNA was extracted after 2-h mating with either 0.5 mg/L ibuprofen, naproxen, gemfibrozil, diclofenac, propranolol, or 1.0 mg/L iopromide. As bacterial mRNA expression responds quickly to external stress, a 2-h mating time was chosen, as per previously , 33. Totll lysis . Triplicll lysis . The dat(v2.3.3) , SeqAlto(v2.3.3) , and Cuf(v2.3.3)  were app(v2.3.3) , 37. Theq value less than 0.01 was used to identify the proteins with significantly different expression levels. Based on the number of proteins showing significant variations, 8 h was selected as the best exposure time for the proteomic analysis. Thus, another set of conjugation experiments was established as described above using the 8 h mating period in the presence of either ibuprofen, naproxen, gemfibrozil, diclofenac, or propranolol, each at 0.5 mg/L, or with iopromide at 1.0 mg/L. Following that, for each of the conjugation experiments, the proteins were extracted, peptides prepared, and proteomic analyses were performed as described above.Conjugation experiments (Model-1) were established as described above to compare proteins expressed in the donor and recipient bacteria during the absence and presence of the non-antibiotic pharmaceuticals. Initially, the optimal length of exposure period was examined for the conjugations when exposed to either 0.5 mg/L gemfibrozil or propranolol. Total proteins from the mixture of donor and recipient bacteria were extracted after 2, 4, 6, and 8 h mating as described previously . For pepR2 > 0.9. Further, Pearson correlation was applied to calculate correlation coefficient r, which was significant if P-value was less than 0.05.Correlation tests were conducted to identify whether the phenotypic data  were concentration-dependent. Linear regressions were performed for log-transformed concentration and the corresponding phenotypic data. A concentration-dependence was seen when t-tests were performed and Benjamini–Hochberg correction method was applied for multiple comparisons [P-values less than 0.05 were considered to be statistically significant. All the experiments were conducted in triplicate.Data were expressed as mean ± standard deviation (SD). SPSS for Mac version 25.0 was applied for data analysis. Independent-sample parisons . P-valueE. coli LE392 as the donor with the conjugative RP4 plasmid harboring multiple resistance genes against tetracycline, kanamycin, and ampicillin. Pseudomonas putida KT2440, with high tolerance towards chloramphenicol, was the recipient in Model-1 [To evaluate the effects of six non-antibiotic pharmaceuticals on environmentally relevant conjugation, we used  Model-1 . During  Model-1 , 23, 24)P = 1 × 10−8–6 × 10−4)  . It should be noted that ibuprofen, naproxen and gemfibrozil were dissolved in ethanol due to their limited solubility in water. In order to rule out the effect caused by the solvent, another set of conjugation experiments without any solvent was established with the dosage of ibuprofen , naproxen  and gemfibrozil , which were the highest concentrations that can be dissolved in MilliQ water. A significant increase in both absolute transconjugant number and transfer ratio were also observed , the fold change increase in the absolute number of transconjugants was significant (tetA and blaTEM genes (both short and long primers applied) also indicated the transconjugant plasmids harbored the donor genes   Fig. .P = 0.052–0.989)  , and 1.6 ± 0.2- to 3.4 ± 0.7-fold (P = 6 × 10−6–0.005), respectively. In contrast, iopromide did not significantly change the clinically relevant conjugation (P = 0.051–0.109). The successful transfer of pMS6198A was verified by PCR of inherent blaNDM  to validate whether the non-antibiotic pharmaceuticals could also promote conjugative transfer, in which ecipient . SimilarIt should be noted that ethanol was used to dissolve ibuprofen, naproxen, and gemfibrozil due to their limited solubility in water. However, we can rule out the effects of ethanol on the conjugative process for the following reasons: (i) The transconjugant number and the calculated conjugation ratio were compared with their corresponding solvent control; (ii) In the environmentally relevant conjugation model (Model-1), dose-response effects were observed for these three pharmaceuticals dissolved in ethanol. The increased conjugative ratio with the elevated level of non-antibiotic pharmaceuticals indicated that pharmaceuticals themselves were enhancing the conjugation, rather than quenching the effects of solvent; (iii) When these three pharmaceuticals were directly dissolved in MilliQ water at maximal concentration, concentrations of 0.005 and 0.05 mg/L still promoted conjugation; and (iv) In the clinically relevant conjugation model (Model-2), where all the non-antibiotic pharmaceuticals were dissolved in DMSO, five pharmaceuticals enhanced the conjugation in comparison with the DMSO control group. Thus, it was confirmed that the non-antibiotic pharmaceuticals promoted conjugative transfer of ARGs, regardless of the solvent.P = 1×10−8–0.038). In addition, the transconjugant was able to transfer the plasmid containing multidrug resistance genes, becoming a new source of ARGs.Collectively, it was concluded that non-antibiotic pharmaceuticals (excepting for iopromide) at environmentally and clinically relevant concentrations, significantly increased conjugative transfer of multiresistance genes (P = 2 × 10−7–0.041) , and higher ROS levels were detected with increasing concentrations of pharmaceuticals. In contrast, the effects of ibuprofen, naproxen and gemfibrozil on ROS were not concentration dependent (P > 0.05). This indicated that these non-antibiotic pharmaceuticals at concentrations as low as 0.005 mg/L increased ROS generation, albeit in a non-linear trend. It should also be noted that the ethanol solvent did not increase ROS generation.ROS are natural byproducts of bacterial metabolism. However, under environmental stress, ROS production may increase dramatically, and this may enhance conjugative transfer , 27. In P = 1 × 10−6–0.045, Fig. P = 5 × 10−5–0.007) in the presence of the scavenger. For example, with 0.05 mg/L gemfibrozil and naproxen, the transfer ratio decreased from 7 ± 0.7-fold and 4 ± 0.6-fold to only 1.2 ± 0.1-fold and 1.3 ± 0.2-fold, respectively, when the scavenger was added. In addition, no significant increase was observed in the transfer ratio between the controls (no drug) and the scavenger-dosed drug groups (P = 0.052–0.94). It should be noted that no significant variations of ROS generation or transfer ratio were observed between the non-pharmaceutical control groups with and without thiourea dosage. These indicated that ROS scavenger thiourea itself did not affect ROS generation or conjugative transfer ratio.ROS scavenger, thiourea, significantly decreased the effect of non-antibiotic pharmaceuticals in both donor and recipient bacteria . For example, expression of SodC was enhanced 4.7 ± 0.6-fold when exposed to 0.5 mg/L propranolol. Correspondingly, genes coding for hydroperoxide reductase (ahpC and ahpF), oxidative demethylase , superoxide dismutase (sodB and sodC) and superoxide response (soxS) increased by 1.1 ± 0.3- to 4.8 ± 0.9-fold with exposure to pharmaceuticals. These genes are involved in the bacterial response to high-level oxidative stress [oxyR, and superoxide dismutase regulators, sodA and sodB, was significantly enhanced with exposure to each of the non-antibiotic pharmaceuticals.Moreover, in the conjugation experiments, expression levels of RNA and protein were compared between the non-antibiotic pharmaceutical-dosed groups and the control groups  of the donor and recipient bacteria. This was conducted to further understand the effects of these pharmaceuticals on conjugation. It was seen that pharmaceuticals significantly upregulated ROS production-related proteins and genes in both donor and recipient Fig. –S11. Fore stress , 42  , while iopromide had no effect (P = 0.041–0.107) . The results matched well with the conjugative transfer changes detected, where the ratio increased with increasing ibuprofen concentrations   Fig. . The impons Fig. . For theμm (n = 100 for each group), while the addition of non-antibiotic pharmaceuticals (except iopromide) decreased the distance to 0.23–0.32 μm (P = 2 × 10−5) . During the conjugation process direct donor and recipient cell contact is necessary for plasmid transfer [Using TEM, we examined the effect of the pharmaceuticals on cell morphology and arrangement during the conjugation periods. During exposure to the pharmaceuticals (except iopromide) cell membranes were partially damaged, and the cell arrangement became more compact with cells drawn closer to each other . For example, the abundance of OmpC and OmpF in the donor bacteria, and OmpA, OprH, OprL and OprQ in the recipient bacteria, significantly increased (up to 2.4 ± 0.6-fold) in all of the five pharmaceutical-dosed groups , and these may also contribute to the increased cell membrane permeability.Moreover, the variations in cell membrane permeability induced by non-antibiotic pharmaceuticals were supported by the analyses of both RNA and protein. Core genes and proteins related to cell membrane structure and function showed significant changes with exposure to non-antibiotic pharmaceuticals . Significant increases in pilin gene expression were not detected for iopromide exposure, although we observed decreased expression of traB, traE and traF.During the conjugation process, the plasmid is transferred through a pilin bridge, and the pilin-related genes in RP4 plasmid include and traP . With exfli, fim, pil, yad, ybg in E. coli, and fli, pil, flg in P. putida [Another contributing factor to conjugation is the direct cell-to-cell contact , where f. putida –59. In tlexA, umu, yeb in the donor, with a total of five genes being affected [Antibiotics at sub-inhibitory concentrations are known to promote horizontal dissemination of antibiotic resistance, associated with the bacterial SOS response , 8. In taffected . With exmdt, usp, kdg in the donor bacterium, and czc and ttg in the recipient bacterium [In addition to the SOS response, the non-antibiotic pharmaceuticals also enhanced expression of efflux pumps, increased levels of universal stress, and elevated levels of repressor genes which regulate antibiotic-sensitivity. Core operons regulating these effects are acterium , 62. OveThe spread of antibiotic resistance has posed a serious global threat to public health. Among the dissemination pathways of antibiotic resistance, conjugation, belonging to HGT, possesses significant ecological and evolutionary implications. This is because that conjugation enables the transfer of mobile genetic elements between bacteria, and allows bacterial instantaneous acquisition of multidrug resistance phenotypes from another population , and thuPharmaceuticals are being consumed at increasing levels each year, with a 5.8% annual growth in the global pharmaceuticals market, which was worth $935 billion in 2017, and will reach $1170 billion in 2021 . Among tIn this study, we demonstrated that these five commonly consumed non-antibiotic pharmaceuticals  can enhance the spread of antibiotic resistance through conjugation at both clinically and environmentally relevant concentrations. Based on two conjugative mating models, this pattern was repeated with two intra- or intergenera populations. This was seen in the enhancement of both absolute transconjugant number and conjugative transfer ratio. The fold changes observed (up to 8-fold) in this study were comparable with- or lower than the conjugation effects caused by sub-inhibitory antibiotics. However, considering human consumption of non-antibiotic pharmaceuticals is wide-spread, the effects caused by these drugs warrant more attention. Moreover, non-antibiotic pharmaceuticals can cause multifaceted effects on the emergence and spread of antibiotic resistance, by increasing expression of efflux pump genes , and proAdditionally, through bacterial culture-, fluorescence-, and advanced molecular methods this study explored the underlying mechanisms related to increased gene transfer , 33. TheIn addition to ROS, cell membrane permeability also contributes to enhanced bacterial conjugation. Elevated cell membrane permeability was detected in both the donor and recipient cells with exposure to ibuprofen, naproxen, gemfibrozil, diclofenac, and propranolol, correlating well with the phenotypic conjugative transfer. The outer membrane of Gram-negative bacteria is considered to be a semi-permeable barrier, where increased permeability could enable increased entry of plasmids , 75. TralexA, umuC, umuD and soxR), universal stress (Usp), the efflux pump , and antibiotic-sensitivity (KdgR). Other in vivo studies show that some pharmaceuticals can cause stress in cells. For example in humans, ibuprofen enhances oxidative stress in plasma during extreme exercise [Salmonella infection [Candida albicans in mouse kidney models [Interestingly, it was also found that the non-antibiotic pharmaceuticals caused antibiotic-like bacterial responses, increasing the expression of genes and proteins involved in the SOS response , which was originally isolated from a patient suffering from urinary tract infections [In this study, the in vitro conjugative mating systems were established at laboratory-scale, which cannot reflect the real effects caused by non-antibiotic pharmaceuticals in real environmental or clinical settings. Thus, in vivo animal studies or mixed culture based mating systems should be conducted in the future to evaluate the risk of non-antibiotic human-targeted pharmaceuticals in promoting the dissemination of antibiotic resistance in relevant niches . For example, we employed pMS6198A .Supporting Information


Service-Learning (SL) is an experience that allows students to (a) participate in activities co-designed in partnership by universities and local organizations and (b) reflect on the service activity in such a way as to gain an enhanced sense of responsibility. These experiences represent significant ways to meet and experience real-world contexts for students. The COVID-19 pandemic required Higher Education Institutions to rethink and shift in-presence courses to online platforms. This transition included SL courses as well. This study aimed to explore the responsibility and democratic dimensions elicited by an extreme online Service-Learning (XE-SL) experience and the perceptions of engaging in exclusive online service activities with local communities during the COVID-19 Italian national quarantine. A qualitative driven mixed-method longitudinal approach was chosen to triangulate qualitative  and quantitative (pre-post questionnaire) data from 20 university students. The findings shed a positive light on the capability of XE-SL to promote a sense of responsibility, civic engagement, and the acquirement of democratic and transferrable competencies, such as perspective-taking, adaptability, cultural background respect, global mindedness, teamwork, leadership, communication, creativity, and organizational competencies. Reflection, connection, and being agents of change for the community were perceived as the major assets of the XE-SL experience, while adapting face-to-face SL experiences to exclusively online activities evoked ambivalent feelings in students. The study suggests a rethinking of the design XE-SL and other forms of eSL with the inclusion of more structured interactive activities within community contexts to favor students’ sense of connection to the community organizations or NGOs. The COVID-19 pandemic caused abrupt changes to teaching and learning. In the immediate aftermath of the COVID-19 outbreak, the Italian Government required the suspension of in-presence learning activities at diverse levels of education. The transition from face-to-face to emergency remote teaching—ERT . At the Service-Learning is an experiential approach aimed at developing personal, professional (subject-specific), and civic and democratic competencies of students. It integrates meaningful community service or engagement into the curriculum, offering students academic credit for the learning that derives from active participation within the community and work on a real-world problem. Based on developing a sense of community responsibility (SOC-R) , SL shouNevertheless, defining transferable and democratic competencies is not an easy task. Transferable skills are also referred to as competence for life, key competence, and generic competence . AccordiDemocracy is a form of governance responsive to the views of the majority, and that requires citizens’ commitment to participate actively in the public good. The Council of Europe’s White Paper on Intercultural Dialogue  points oAccording to Barrett  (p. 9), Democratic competencies include skills, knowledge and understanding, and values and attitudes, regarded as essential for behaving appropriately and effectively in democratic and intercultural situations. Values and attitudes are considered psychological resources that can be activated, organized, and applied through behavior to respond appropriately and effectively in democratic and intercultural situations .Even if the learning capacity of SL is embedded in hands-on, experiential learning in the community, online SL learning has been developed to deal with those situations in which face-to-face SL is not practicable. Waldner et al.  identifiMany scholars claim that the benefits of eSL and XE-SL for students are equivalent to offline “traditional” SL , while oAmong benefits, eSL is reported to stimulate students’ teamwork competency , social However, with few exceptions, the literature on eSL benefits for students is anecdotal . FaulconThe problem of online teaching and learning assessment is not exclusively limited to eSL. In a systematic review of 262 studies conducted in 2020 during the “global online semester” to analyze students’ learning experiences during the pandemic, Bond et al.  project. This approach encouraged me to be even more determined”. (AB_C).In another situation, by a reflective process over the meaning of the disappointment generated by the experience, one participant was able to share with the community organizations the critical aspects to ameliorate and rethink the project for future XE-SL students and the final users .“I cannot say that my contribution to society is inexistent or that my commitment is worth nothing. On the contrary, by experiencing the critical aspects of this project, I was able to share a reflection with the organization on what can be improved for improving the experience both for XE-SL students and users”. (MGS_B).Strictly related to responsibility, the positive exchange and reciprocity seemed to be critical elements to raise and internalize a sense of responsibility for the community. The experience helped students to also reflect on their level of engagement and conclude that everyone can be an agent of change.“I realized that I do too little for others, and I would like to do more. I understand now that everyone can help to make things better”. (CR_B).Even if in its virtual form, human interaction helped students raise their sense of responsibility and reinforce a call for community actions. Moreover, the community dimension became a place to present themselves and their stories, a valuable repository of shared narrations.“Receiving positive feedback from the children [target community] made me feel welcomed. During the meeting, I was able to make my modest contribution by sharing my story. This made me feel useful. Today my words are finally valuable for someone, which was important to me. I am fully aware that community starts from us as individuals; being updated on political, social, and economic issues, being active listeners of the needs that society brings, fighting for them, and promoting the recognition of fundamental rights and the empowerment of marginalized and underserved communities”. (AB_C).The responsibility dimension also emerged concerning the pandemic situation. XE-SL represented an opportunity to start a reflection, considered a process to be still “rooted to the ground”, on solidarity and social responsibility in a broader sense.“Reflecting on social responsibility, during this emergency we are living as individuals and collective, is perhaps one of those few things that allow me to stay rooted to the ground. Social responsibility means to become aware and ‘force’ awareness even when our body or our mind would rather hide under the blanket with our eyes closed until the pandemic ends. It means to open the path to new ways of learning from, and changing, what we do”. (ME_B).Finally, one participant stressed how the experience helped him understand the importance of advocating for relevant issues.“Thanks to XE-SL, I realized that battles cannot be fought only in my head or at the bar with friends. There are places where concrete actions are implemented in the community. We stand up for it even with strangers, having the courage to say, “I’m not going to take this anymore””. (GR_C).Qualitative data showed participants’ reflection and awareness of deployed and learned competencies throughout the experience. Engaging with the community organizations’ activities and projects elicited different democratic competencies, namely students’ perspective-taking, personal beliefs, cultural background respect, and adaptability.“I’ve learned that, to understand things deeply, you need to put yourself in others’ shoes. And that’s what I did. Doing so, I understood the complex reality of community organizations and that some of the things that I considered as constructive contributions [ameliorative suggestions] to the community might become work overload for community practitioners [require too many additional resources and competencies to be implemented]”. (AB_C).“I learned how to approach and respect new people and especially people of other nationalities and cultures, realizing that my contribution can be useful to other people”. (CR_B).“Coming from three different fields of study, in the beginning, our conversations [as XE-SL students] were complex, and sometimes I had the feeling that we did not understand each other fully, as if we spoke different languages. During the experience, conversations became clearer, and differences were no longer relevant. I think it was satisfying for everyone and helped us to create a positive team climate”. (AF_B).Intertwined with democratic competencies, excerpts emphasized the group dimension and the related transferable teamwork and leadership competencies trained and strengthened during XE-SL. Overall, working in a group represented an opportunity to grow and learn how to better master group dynamics.“I’ve learned the importance of reciprocity when working in a group. When we were all engaged positively, virtuous circles were established. This also motivated other students and community members to be committed and reciprocate”. (AF_B).“From this [XE-SL] experience, I’ve learned the importance and beauty of collaborating with my XE-SL colleague. I believe that collaboration is a competence that will be very useful and fundamental in the future. I hope to feel so at ease with a future team in my job as well”. (VL_C).“Sometimes, within a group, different ideas can emerge, and they might not always be perceived correctly by others. I think that the secret is to always talk about it and find compromises. By doing this, you can reach personal and collective growth”. (EL_C).One participant found an additional meaning to teamwork during the challenging times of the pandemic. To her account, “In a period like this [the COVID-19 Italian lockdown], working together is not only a way to optimize work, but a human need” (ME_B).Participants reported other transferrable competencies deriving from the experience. Students trained their communication competence, learning to present themselves in new contexts and overcoming shyness and sharing their needs.“Being a shy person, XE-SL helped me to be more confident in meeting strangers. I had the chance to present things in front of an audience, talk to people I did not know, share my ideas and thoughts in the clearest way possible. In general, the university is helping me in this; but the support of XE-SL has been essential for me”. (LD_B).“I’ve learned to share my needs with others and don’t leave space to the unsaid to build and maintain a good communication”. (AF_B).Performing different activities and conveying personal competencies and passions toward XE-SL projects helped participants be aware of their creativity. One student realized how his passion for music could be used to create a product for children.“The thing that surprised me the most was my creativity. I didn’t think I could create a story for children with visual disabilities [final product of the XE-SL project] like this”. (GR_C).XE-SL offered students a guided experience inside organizational realities, practicing collaboration, time management, and, in a broader sense, organizational competencies.“I have learned what it means to be there for others, listening and sharing opinions in a work context. I’ve also learned to work in a group, respect deadlines, and collaborate to contribute to individual and collective well-being”. (GA_B).p = 0.001), between SOCR pre and GM post , between SOCR post and GM pre , and between SOCR post and GM post . The significant positive correlation between these two constructs can be referred to as the social responsibility dimension that both scales evocate. Moreover, the data showed a general negative correlation between GM and adaptability (Ad) both in the pre  and post ; and the negative correlations of sense of community responsibility (SOCR) with Ad pre  and post . This phenomenon might be explained by the fact that the scales are differentiated by internally cognitive (Ad) versus externally social (GM and SOCR) focuses  association or at least devote my time to other associations. For example, I know very well a community organization in my city and, even if I had the opportunity, I never gave my contribution. Probably because I was afraid of not having specific skills. This XE-SL activity made me realize that commitment is precious and that everyone has some skill to offer”. (CR_B).The experience also represented a way to “learn more and more about online meetings” (AF_B), and become acquainted with new applications thanks to the help of others.“I learned how to use a new social media application. The site supervisors spent time explaining how the application worked and all its different functions”. (LD_B).According to participants’ reflexive journals, the main limitation was the online dimension of the experience. The virtual connection prevented students from being in-presence, sharing places, and took away "some of the magic" of living in the community spaces. Nevertheless, students also acknowledged the potential of being online to feel less alone, still be productive, be somehow together, and actively collaborate.“Doing it from home maybe took away ‘some of the magic’ of being in-presence. Still, it allowed us to join a close-knit group and actively collaborate”. (TC_B).“I think that the online dimension limits the experience. However, it is the only way to keep us together today, be productive, and feel less alone. Still, I think it distracts us from reality”. (VL_B).The COVID-19 pandemic led governments worldwide to swiftly implement measures  to reduce disease transmission. This transition meant taking lectures and student activities from face-to-face mode to online teaching and learning .This study shed insights into the XE-SL experiences of Italian university students during the COVID-19 lockdown and is unique for several reasons. First, a scarcity of studies investigated the impact of university students’ learning experience under COVID-19. Second, few studies focused on XE-SL courses, which entails a strong experiential component. Third, only few of the existing studies adopted validated assessment tools or a mixed-method approach akin to the present study. Lastly, while some research investigated the sense of responsibility dimension during the pandemic in Italy and the engagement with the community , there aThe study, indeed, aimed to explore the responsibility and democratic dimensions elicited by the XE-SL experience and the perceptions of engaging in exclusive online service activities with local communities that were inaccessible elsewhere. To analyze these aspects, we conducted a qualitative-driven mixed-method study. Students’ reflexive journals and pre-post surveys were used to collect data.Regarding the first research question (RQ1), the findings suggest that XE-SL had a role in enhancing students’ sense of responsibility toward the community, representing an eye-opening experience for students, especially during the COVID-19 lockdown situation. It helped participants become aware of local organizations and their mission, being part of the change and bringing new perspectives to organizations while understanding the relevance of standing up and advocating for relevant issues. These dimensions of responsibility corroborate previous findings  and are For the second research question (RQ2), qualitative data offered a clear perspective that XE-SL was a place to strengthen democratic competencies and learn and deploy new ones. The findings also disclosed the reflection of other transferable competencies  that are not strictly related to the democratic dimension. These findings confirm and add to the existing literature on the role of XE-SL, and eSL in general, to foster transferable competencies such as teamwork ,19,45. QRegarding the third research question (RQ3), qualitative data showed that students tend to reflect on the positive aspects of XE-SL and what they can do rather than focus on the limits. XE-SL assets can be divided into three main categories. The first is “reflection,” perceived as a good practice to be integrated into everyday life, given its capability to raise awareness on things and processes. Reflecting on the learning and the service-empowered students to assess individual learning goals while collaborating with others to make meaning of their XE-SL experience, and acknowledging the shared/cooperative dimension of the learning process. This finding stresses the central role of reflection in eSL and SL courses ,46. The About perceived limits, participants did not explicitly and univocally address this aspect. Nevertheless, adapting face-to-face SL experiences to exclusively online activities evoked ambivalent feelings in students and provided valuable opportunities for students to serve communities in diverse ways while enhancing their online and social media competencies and literacy .On the one hand, they acknowledged the possibility of being agents of change and answering community needs while gaining new and strengthened competencies. On the other hand, the online dimension prevented them from fully experiencing the situated and territorial dimension of communities and, therefore, can be presented as the major limit to the XE-SL experience. The COVID-19 pandemic, and the national quarantine, required students to face their own personal challenges in their communities and social systems. Moreover, students had to confront the abrupt shift to online forms of learning and civic engagement all at once. We can hypothesize that these factors also played a central role for some participants in perceiving the XE dimension of SL as a limit, especially during the first stages of the XE-SL experience, as students may have lacked time to internalize what was taking place around them.The study shed a positive light on the capability of SL in its extreme online form to promote a sense of responsibility, civic engagement, and the acquirement of democratic and transferrable competencies, such as perspective-taking, adaptability, cultural background respect, global mindedness, teamwork, leadership, communication, creativity, and organizational competencies.The reflection and connection promoted and sustained by the XE-SL are fundamental aspects for participants that perceive them as added values for their academic, personal, and future professional lives.The use of a mixed-method and longitudinal pre-post data collection enabled data triangulation. It also deepened the understanding of phenomena, i.e., to formulate the response shift bias effect hypothesis on the nonsignificant decrement in some variables taken under inspection in the quantitative study.A question remains open. Would students stick to the XE-SL format if not forced to stay home by the national lockdown?The qualitative excerpts stressed that the central perceived limit was the exclusive online dimension of the experience. This result might have been influenced by the frustration of the pandemic experience of being forced to stay home. In a way, every form of online experience might have been perceived as limited in a time when connections could only take place online. The findings suggest that the extreme online form is not suited for everyone or every situation but that more hybrid forms of eSL could be more engaging and perceived as less limiting. On this, instructors and community partners (or site supervisors) can play a relevant role in designing XE-SL courses that foresee the possibility of including face-to-face service activities when required , thus underpinning a good quality of participation, and fostering student engagement ,50. To aThis study had some limitations. The main limitation was the small sample size. Having twenty students enrolled in this XE-SL course also represented a positive thing for the SL academic staff and the community partners. Students decided to be engaged with the communities no matter what. Nevertheless, having a small sample prevented us from computing more sophisticated quantitative analysis and generalizing results.Our study contributes to the existing literature by adding further responsibility and democratic benefits of XE-SL experiences. The dissonant findings underline the relevance of adopting a mixed-method design to approach SL experiences to interpret students’ experiences better and implement XE-SL (and SL) practice. Moreover, the quality of participation and ownership dimensions should be incorporated as relevant eSL elements in nurturing a positive development of students’ sense of responsibility and democratic competencies.Our study also suggests some practical implications. The use of online technology entails the possibility of overcoming physical restrictions and offering more inclusive learning to students, but the lack of the on-site component is perceived as “the missing piece” of the experience. Therefore, more structured activities are suggested to ease the interaction and connection with community organizations and NGOs. A way to favor this would be to rethink the timeframe of XE-SL courses, adjusting the number of total service hours accordingly to the SL form—that is, face-to-face SL, hybrid eSL, and XE-SL.This study also opens to future research on XE-SL experiences in nonemergency contexts, comparing XE-SL to eSL and SL experiences with a longitudinal mixed-method design, understanding eventual significant differences, or corroborating existing literature that claims no differences . This woTo conclude, our study proves the goodness and efficacy of XE-SL in sustaining and fostering students’ sense of ownership and responsibility, even in the hard times of the COVID-19 national quarantine. XE-SL allowed students to experience and live community contexts, standing  together by staying  apart.


Moreover, similar SERS peak shifts in the control experiments using CB[n]s but with smaller cavity sizes suggested the occurrence of the same guest complexations among CB[5], CB[6], and CB[7] with MV2+. Hence, an unconventional exclusive complexation model is proposed between CB[7] and MV2+ on the surface of Au NPs, distinct from the well-known 1:1 inclusion complexation model in aqueous solutions. In summary, new insights into the fundamental understanding of host–guest interactions at nanostructured interfaces were obtained by SERS, which might be useful for applications related to host–guest chemistry in engineered nanomaterials.Interfacial host–guest complexation offers a versatile way to functionalize nanomaterials. However, the complicated interfacial environment and trace amounts of components present at the interface make the study of interfacial complexation very difficult. Herein, taking the advantages of near-single-molecule level sensitivity and molecular fingerprint of surface-enhanced Raman spectroscopy (SERS), we reveal that a cooperative effect between cucurbit[7]uril (CB[7]) and methyl viologen (MV In recent studies, host–guest systems have been used on solid surfaces to functionalize nanomaterials12. The results showed that the host–guest complexation behavior on surfaces may differ from those observed in solutions. For instance, host–guest complexation on surfaces may greatly enhance compared with solutions. This can be explained by the preorganization of ligands on surfaces, as well as the synergistic effects induced by multiple noncovalent interactions5. Besides, competitive adsorptions on surfaces may lead to unpredictable surface environments, thereby affecting the interfacial host–guest complexation. As a result, it is important to better understand the interfacial behavior of host–guest systems for their wider and more robust applications in nanomaterials. However, the complex surface effects and trace amounts of components present on the surface make the study of interfacial complexation more difficult13.Host–guest chemistry offers a reversible and versatile way to achieve high-fidelity recognition between host and guest molecules; therefore, it is widely employed in homogeneous solutions16, and cyclic voltammetry18 have been used to monitor the host–guest complexation at interfaces. However, these methods often require the use of chromophores or redox-active motifs and suffer from background interferences generated by nanoparticles (NPs). Nuclear magnetic resonance (NMR) spectroscopy provides information about ligand-shell morphology or ligand exchange on metal NPs21, but suffers from relatively high detection limit and background interference caused by residual agents in solutions. Alternatively, surface-enhanced Raman spectroscopy (SERS) provides rich molecular vibrational information along with many advantages in terms of ultrahigh sensitivity, label-free and in situ detection, distinction among ligand types, and exclusive detection of adsorbates present on the surface23. Furthermore, SERS peaks of host and/or guest could shift upon host–guest complexation27, beneficial for investigating host–guest complexation at the interface. Therefore, SERS is a practical and powerful in situ technique for revealing interfacial host–guest complexation phenomena.Several techniques such as extinction spectroscopy, fluorescence spectroscopy2+2I−) at Au NP-water interface  and methyl viologen  induced an instant aggregation of Au NPs  and MVNPs Fig. . The timNPs Fig. , accompamin Fig. . Though ent Fig. . The muc2+2I−. The second ζ-potential was recorded as a function of MV2+2I− concentration with and without CB[7]. As shown in Fig. 2+2I− led to a variation in ζ-potential more significantly than separately. To confirm the cooperative effect, two ζ-potential values were calculated and compared. The first is the change in ζ-potential (Δζmix) on the addition of the mixture of CB[7] and MV2+2I−, and the second is the sum in the variations of ζ-potential (Δζsum) caused solely by CB[7] and MV2+2I−. The data indicate that the value of Δζmix  Raman intensities  already caused the intense aggregation of Au NPs (Scheme 2+ complexes in the form that one CB[7] molecule binds two adsorbed MV2+ molecules from adjacent Au NPs. As the content of CB[7] increased (1:8 < CB[7]:MV2+2I−≤1:2), more CB[7] would bind to the adsorbed MV2+. This results in electrostatic repulsion among Au NPs and partially cleavage of the linkers, thereby reducing the aggregation of Au NPs (Scheme 2+2I−. Excess CB[7] (CB[7]:MV2+2I− > 1:2) would completely inhibit the adsorption of MV2+2I− on Au NPs surface, thereby eliminating the cooperative effect between MV2+2I− and CB[7] and further reducing the aggregation of Au NPs , guest cations (MV2+ and Na+) and hosts  on the surface. Moreover, an exclusion complex model between CB[7] and MV2+ distinct from that in aqueous solutions was proposed based on the SERS results. These findings provide new insights into the fundamental understanding of host–guest interactions at the solid–liquid interface, promising for applications in host–guest chemistry for engineered nanomaterials.In summary, the interfacial interactions of CB[n]-based host–guest system on Au NPs surfaces were systematically studied by SERS. The ultrahigh sensitivity and rich molecular vibrational information provided by SERS allowed the determination of unreported cooperative adsorptions among counter anions (I2+2I−) and methyl viologen dichloride (MV2+2Cl−) were purchased from J&K Chemical. CB[7], CB[6], and sodium 3-(trimethylsilyl)-1-propanesulfonate [1H NMR standard used for D2O solvent] were obtained from Sigma-Aldrich. Methyl viologen dibromide (MV2+2Br−) was synthesized following literature45. High-purity water  was used throughout the studies.Methyl viologen diiodide  once and resuspended in 1.4 mL H2O after the removal of all the supernatant.In all, 50 nm Au NPs were prepared using the citrate reduction method reported by Lee and Meisel2O followed by dilution to 50 μM and then to 10 μM.All stock solutions (2 mM) were prepared in 4 mL HThe ultraviolet-visible (UV−vis) spectra were recorded using a Shimada UV-2550 spectrophotometer. In all, 100 µL Au NPs and 300 µL of reagents were mixed and immediately placed in a cuvette with a one-millimeter optical path. The time-dependent spectra were acquired at 1 min intervals for 5 min.The ζ-potential was collected using a Malvern Zetasizer Nano ZS Instrument. First, 200 µL Au NPs and 600 µL reagents were mixed and then immediately placed into a cuvette. The temperature was set as 25°C, and every sample was tested three times.The SERS data were collected using a Renishaw Invia instrument . First, calculated volumes of stock solutions were mixed in a 96-well plate, and then 50 µL of concentrated Au NPs was added and mixed through pipetting a dozen times. The total volume was adjusted to 200 µL, and SERS measurements were immediately carried out after mixing by focusing the laser beam directly on the sample suspension.TEM images were viewed using a JEM-1400  and scanning TEM images were collected using a Talos F200 instrument .NMR spectra were collected using a Bruker AVANCE III-500 MHz NMR unless otherwise stated.Supplementary information


The amount of steel chips generated by lathes and CNC machines is 1200 million tons per year, and they are difficult to recycle. The effect of adding steel chips without pre-cleaning (covered with production lubricants and cooling oils) on the properties of concrete was investigated. Steel waste was added as a replacement for fine aggregate in the amounts of 5%, 10% and 15% of the cement weight, which correspond with 1.1%, 2.2% and 3.3% mass of all ingredients and 0.33%, 0.66% and 0.99% volume of concrete mix, respectively. The slump cone, air content, pH value, density, compressive strength, tensile strength, tensile splitting strength, elastic modulus, Poisson’s ratio and thermal parameters were tested. It was observed that with the addition of lathe waste, the density decreased, but mechanical properties increased. With the addition of 5%, 10% and 15% metal chips, compressive strength increased by 13.9%, 20.8% and 36.3% respectively compared to plain concrete; flexural strength by 7.1%, 12.7% and 18.2%; and tensile splitting strength by 4.2%, 33.2% and 38.4%. Moreover, it was determined that with addition of steel chips, thermal diffusivity was reduced and specific heat capacity increased. With the addition of 15% metal chips, thermal diffusivity was 25.2% lower than in the reference sample, while specific heat was 23.0% higher. No effect was observed on thermal conductivity. Someemission ,13,14. Memission ,16. Thesemission ,18, and emission . Due to emission . Becauseemission . In addiIt is possible to use a variety of waste as concrete components ,25. In lWith the addition of steel chips as a replacement for fine and coarse aggregate at 22%, 33% and 44% of cement mass, Maanvit et al.  obtainedThe addition of steel waste as fibers has been tested by other researchers ,32,33,34Based on the above, it can be concluded that the results for concretes with the addition of metal chips are very different, and in order to commonly use them, further research is required. Moreover, different scientists use varied classifications of steel chips . Therefore, this study aimed to assess the effect of steel chips as a replacement for fine aggregate on the mechanical and thermal properties of concrete. Compared to other research, lathe chips were used without pre-cleaning (covered with production lubricants and cooling oils), which is new and aims to increase the use of waste materials from the production process in construction. Tests of mixed  and hardened concrete with 5, 10 and 15 wt.% of steel chips  were carried out. Steel chips in the amounts of 5, 10 and 15 wt.% of the cement weight, which correspond with 1.1%, 2.2% and 3.3% of mass of all ingredients and 0.33%, 0.66% and 0.99% of volume of concrete mix, respectively, were used. The obtained results show that it is possible to efficiently and ecologically manage lathe waste in concrete while improving its mechanical parameters.The mix was prepared using the following materials: Portland cement, aggregate, tap water, admixture and steel waste.CEM I 42.5R Portland cement according to the EN 197:1:2011  standardCrushed basalt sand with fractions of 0–4 mm was used as aggregate in the mixture, see Superplasticizer  was used to reduce the amount of water required to liquefy the mixture. The product complies with the EN 934-2:2009+A1:2012  standardCurved and slightly twisted steel chips  of post-production origin generated by CNC machine tools were used . The chiIn this study, steel chips without pre-cleaning (covered with production lubricants and cooling oils) were used. Tests of organic substance content on the chip surface carried out at 450 °C showed the presence of organic substances of post-production origin in an amount of about 6%. By exposing the material to the temperature of 900 °C, the amount of organic substances increased to about 10% . The incFour types of mixtures were tested: a reference (without an additive of steel waste) and three mixes with different amounts of steel waste. Mixture composition is shown in In the other mixtures M1–M3, , the addFirst, dry ingredients (the aggregate and steel chips) were mixed for 3 min. Then water mixed with a superplasticizer in the amount of 1% of cement weight was added. All components were mixed for 6 min, and then the mixture was poured into molds.Molds in the shapes of cubes with dimensions of 150 mm × 150 mm × 150 mm; cylinders with dimensions of 150 mm × 300 mm and beams with dimensions of 100 mm × 100 mm × 500 mm and 40 mm × 40 mm × 160 mm were used. The mixture in the mold was compacted using a vibrating table. The top of the mold was prevented from water evaporation for 24 h after sample preparation. All samples were made in laboratory conditions (at 21 °C and 50% humidity). Samples were removed from molds after 36 h. The samples were stored in water until testing on day 28 in accordance with EN 12390-2:2019 [The consistency, air content and pH value of each concrete mixture were tested.The concrete mix consistency was determined using an Abrams cone  according to EN 12350-2:2019  Figure a. The slAir content was tested using the pressure method according to EN 12350-7:2019 . The samAcidity and alkalinity (pH) values were determined for the liquid phase extracted from fresh mixtures in accordance with PN-EN 1015-3:2001+A2:2007 . The tesThe tests were carried out after 28 days of concrete hardening. Density and mechanical and thermal properties were determined for samples made from the analyzed mixtures .Density of hardened samples was determined for 150 mm × 150 mm × 150 mm cubes according to EN 12390-7:2019 . The tesTests were performed using a Zwick machine  with a force range of 0–5000 kN. Compressive strength was tested on 150 mm × 150 mm × 150 mm samples (cubes) according to EN 12390-3:2019 , see FigEach test was carried out for ten samples for each mixture.Thermal conductivity, thermal diffusivity and specific heat tests were carried out using the ISOMET2114 analyzer . The test consisted in assessing the temperature change of the tested material per heat flow impulses. The heat was generated by electric resistor heaters on the probe with a diameter of 60 mm, which was in direct contact with the concrete sample with a minimum thickness of 25 mm. It was assumed that heat propagation takes place in an unrestricted body. The temperature was recorded as a function of time. Five measurements were taken at different locations for each sample. The average of the resulting readings was taken as the final result. The test was carried out on ten samples for each mixture.The obtained results of the concrete mixture tests are shown in The mixture workability increased for mixtures M1 and M2 compared to the reference mixture . This maThe tested mixtures were S1 class (M0 and M3) and S2 class (M1 and M2). Shewalul  obtainedThe air content of tested mixtures with the addition of steel chips  increaseThe obtained results are presented in Lathe scraps are added to concrete as replacement for aggregate or as fiber. Depending on this, its addition can be calculated in relation either to the aggregate or to the cement see . In ordeWith the addition of steel chips in the amount from 5% to 15% of cement mass, the workability of the mixture decreased . This trThe less liquid consistency obtained in this study is due to the composition of the mixture. Moreover, it can be observed that for longer chips presented in this study and in ,26, simiWith the increase in addition of steel waste , concrete density decreased by 2.9%, 7.0% and 8.6% compared to the reference sample, and this correlation is linear . This waMoreover, similar observations were determined by Mohammed et al. , who obtThe same trend was observed for lathe chips used as steel fibers ,32,51. KA higher compressive strength increase (40%) was obtained by Seetharam et al.  for addiA similar increase in compressive strength in relation to the reference sample (15% and 13%) as in this study was observed by Mansi et al.  for addiThe samples with metal lathe waste addition were destroyed in the same way as the reference sample. The relation between compressive strength and the addition of chips is linear. The obtained results are in line with the observations of other scientists . For addConcrete with analogous added content to that in this study  was tested by Hemanth Tunga et al. , and theAn almost incredible increase in compressive strength with the increase in steel waste addition can be observed here, while the experimental studies of other scientists indicate a rather slight or even negligible increase. This may be related to the adopted mixture composition see .The results of the flexural strength of the samples are shown in The increase in flexural strength with increasing steel chip content is in line with the observations of other scientists . SimilarResults of splitting tensile strength tests of samples with different steel chip contents are shown in Analogously to other mechanical properties, it can be observed that the slope of the obtained curve for correlation between splitting tensile strength and lathe waste content is very similar to relationships determined by other scientists ,53, espeWith the addition of steel waste, the elasticity modulus increased from 0.6% to 6.2% compared to plain concrete (32.0 ± 0.4 GPa) . This phPoisson’s ratio for the reference sample was 0.120 ± 0.03. It can be observed that the results for samples M1–M3 were within the measurement error limit .Thermal parameters for concrete samples are presented in For a 5%, 10% and 15% addition of lathe waste, a 3.7%, 17.5% and 25.2% decrease in thermal diffusivity was obtained in relation to the reference sample. A linear correlation between steel chip content and thermal diffusivity was demonstrated . In addiFor a waste addition of 5% and 10% of cement weight, the workability of the concrete mix increased and was qualified to the S2 class. The reference samples and the samples with additions of 15% waste lathe were S1 class.The air content of tested mixtures with the addition of steel chips  increaseThe compressive strength of concrete after 28 days increased linearly from 50.4 MPa to 68.7 MPa in relation to the reference sample. For an addition of steel chips at 15% of cement weight, a 36.3% increase in compressive strength was obtained.The flexural strength increased linearly from the reference value of 10.83 MPa to 12.8 MPa. This corresponds to an 18.2% increase in flexural strength for the 15 wt.% addition of steel chips.The splitting tensile strength for the 15 wt.% additive increased by 38.4% compared to the reference sample.A slight increase in the elastic modulus from about 1% to 6% was observed for additions of metal lathe waste from 5% to 15% of cement weight.It was demonstrated that the addition of steel chips as a substitute for fine aggregate does not affect the thermal conductivity of concrete.A 3.7%, 17.5% and 25.2% decrease in thermal diffusivity was obtained for the addition of steel chips at 5%, 10% and 15% of cement weight.The specific heat for concrete with addition of 15% steel chips as a replacement for fine aggregate was higher by 23.0% compared to the reference sample.The aim of the study was to assess the possibility of using post-production waste materials in the form of lathe chips without pre-cleaning (covered with production lubricants and cooling oils) in concrete. In this study, three different contents of addition  were used as substitutes for fine aggregate. The following conclusions can be drawn on the basis of this experimental study’s results:Moreover, in this study it was determined that thanks the adoption of lathe chip content in relation to the cement content, correlations between properties and lathe chip content for different concrete mixes are similar. The slopes of the curves are approximate.Based on the obtained results and correlations between properties and lathe chip content, differences can be observed between these results and results obtained by other scientists due to the use of different cement classes, different aggregate type and fraction, type of plasticizers and the type, quality and level of contamination of the added steel chips. Moreover, it was determined that use of lathe chips without pre-cleaning (covered with production lubricants and cooling oils) as replacement for fine aggregate in the amount of 5% to 15% can improve the mechanical properties of concrete. It was particularly surprising that better results were obtained than with chips processed using the classical method entailing their melting.This paper is part of a wider research project aimed at developing environmentally friendly concrete in accordance with the principles of sustainable development and closed-loop economy by using recycled construction materials in concrete. Taking into account the improvements in the mechanical properties of concrete through the addition of chips, further testing of this material, especially fatigue testing, will be scheduled.


Idiopathic retroperitoneal fibrosis is a rare fibro-inflammatory disease that can be associated with other IgG4-related diseases (IgG4-RDs). It is exceedingly uncommon to encounter this condition in a patient with Riedel’s thyroiditis (RT), another disease in the IgG4-RD family. We present the case of a 53-year-old man with a history of RT who presented for severe localized lower abdominal and suprapubic pain due to obstructive uropathy from extensive retroperitoneal fibrosis. The biopsy of the mass demonstrated fibro-inflammatory tissue, and its immunohistochemistry was notable for IgG4-positive plasma cells. This case highlights the challenge associated with the diagnosis and management of this rare manifestation of IgG4-RD. In a patient with a history of any form of IgG4-RDs, providers should be vigilant for any signs or symptoms that suggest the development of fibrosis in other organs. Retroperitoneal fibrosis is a rare fibro-inflammatory condition that can lead to the entrapment and obstruction of retroperitoneal structures including the ureters and infrarenal vessels such as the aorta and inferior vena cava . It is cRiedel’s thyroiditis (RT) is another rare, destructive fibro-inflammatory condition that is a part of the IgG4-RD spectrum and is the most common thyroid manifestation, with five of the six RT samples demonstrating IgG4-positive immunostaining in a Mayo clinic cohort from 1958 to 2008 . In anotA 53-year-old male with a history of RT previously treated with isthmectomy for compressive symptom relief presented with a one-week history of severe localized lower abdominal and suprapubic pain. He denied any fever, gastrointestinal symptoms, genitourinary symptoms, or weight loss. He was initially diagnosed with acute prostatitis and was treated with ciprofloxacin without improvement of symptoms, which prompted a second visit to the emergency room. Physical examination demonstrated a flat, soft abdomen with normal bowel sounds and no palpable masses, but with diffuse tenderness across the lower abdomen, especially in the right lower quadrant and suprapubic region. The patient underwent a non-contrast CT scan of the abdomen and pelvis with findings significant for an extensive, predominantly right-sided retroperitoneal mass encircling the aorta, inferior vena cava Figure , and proThe patient underwent right ureteral stent placement, which partially resolved hydronephrosis and restored kidney function. The CT-guided biopsy of the retroperitoneal mass revealed fibro-inflammatory tissue without specific features Figure . The immThe patient was finally diagnosed with IgG4-related systemic fibrosclerosis. Additional lab testing showed normal lactate dehydrogenase, uric acid, and IgG4 levels. He was started on high-dose prednisone at 60 mg daily. Throughout this time, he developed acute renal failure requiring additional stent placement by urology, as well as refractory pain necessitating the use of narcotics.Over the next few months, along with decrease in the size of the retroperitoneal mass on follow-up CT Figure , his anaGiven the presence of an IgG4-positive plasma cell infiltrate, retroperitoneal fibrosis, and history of RT, the diagnosis of IgG4-related retroperitoneal fibrosis in a patient with RT was made.IgG4-RD is an immune-mediated fibro-inflammatory condition capable of affecting multiple organs. Its prevalence is unknown, although the recognition of the disease continues to grow . CD4 T cIgG4-RD requires a broad differential specific to the organ system(s) involved. One of the patient groups that is at high risk of having this disease and requiring further evaluation are those with retroperitoneal fibrosis. Patients with retroperitoneal fibrosis are at a high risk of having IgG4-RD and thus necessitate further evaluation. The diagnostic evaluation of IgG4-RD includes histopathologic, clinical, serologic, and radiologic findings. IgG4 levels are elevated in the majority of patients, although a minority of patients have normal serum levels, as this patient did . Tissue Primary retroperitoneal fibrosis, whether it is truly idiopathic or associated with IgG4-RD, is treated with immunosuppressive therapy in addition to immediate relief via open surgical, percutaneous, or endoureteral urological interventions if significant obstruction is present. Corticosteroids are considered the first-line therapy . The proRetroperitoneal fibrosis is one of the most frequently encountered IgG4-RDs, and RT is the most common thyroid manifestation of these diseases, though Hashimoto’s can exhibit some of the same pathologic features. The two are rarely diagnosed in the same patient. In a patient with the diagnosis of any form of IgG4-RD, clinicians should remain vigilant for any signs or symptoms that suggest the development of fibrosis in other organs. A comprehensive diagnostic criterion is employed to diagnose IgG4-RD, and the patient detailed in this case met the criteria to diagnose the disease. Given his history, the above case likely represents a rare diagnosis of IgG4-related retroperitoneal fibrosis causing obstructive uropathy in a patient with a history of RT whose fibrosis improved with the initiation of corticosteroids.


MET amplification or METex14 skipping mutations are uncommon oncogenic events in NSCLC patients. Clinicopathological characteristics, concurrent gene alterations, and prognosis of MET TKIs in these patients are yet to be elucidated.We retrospectively analyzed the genomic profiles of 43 MET amplifications or 31 METex14 skipping mutations in NSCLC patients with no previous treatment with EGFR TKIs. Survival outcomes were analyzed in evaluable patients receiving MET TKI treatment: MET amplification cohort (n = 29) and METex14 skipping mutation cohort (n = 29).vs. 11.0 months, P = 0.043). Concurrent mutations in both cohorts resulted in a statistically significant shorter PFS . However, a statistically significant OS  was only observed in the MET amplification cohort. TP53, the most common concurrent mutation in both cohorts, was associated with worse survival outcomes as compared to the wild type. The MET amplification cohort with a concurrent PIK3CA mutation exhibited primary resistance to MET TKIs and showed disease progression (80%).Among evaluable patients, a shorter PFS was observed in the MET amplification cohort than in the METex14 skipping mutation cohort (7.0 months MET TKIs could be a better treatment option for patients with METex14 skipping mutations. Concurrent mutations may deteriorate the PFS of MET TKIs in NSCLC patients with MET amplification or METex14 skipping mutations. PIK3CA mutations may confer primary resistance to MET TKIs in patients with MET amplification. Non-small cell lung cancer (NSCLC) is the leading cause of tumor-related deaths worldwide. Molecular heterogeneity, proliferation, and metastasis in NSCLC have been associated with various driver mutations in the epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), proto-oncogene tyrosine-kinase protein ROS1), and mesenchymal epithelial transition factor receptor (MET) , and mes. MET, a MET exon 14 (METex14) skipping mutations have recently been found to be oncogenic, seen in approximately 3 to 4% of NSCLC cases, and are associated with MET amplification and overexpression –13. Respin situ hybridization (FISH) was the most commonly used method in detecting chromosomal abnormalities. In recent years, next-generation sequencing (NGS) has become a more popular method and is being integrated into routine clinical oncology practice. Targeted NGS allows the simultaneous integration of multiple genes and provides a comprehensive mutation profile of not only actionable mutations, but also other gene mutations associated with cancer development. Concurrent driver genes with sensitizing mutations have been shown to affect the survival outcomes of targeted therapies.Fluorescence de novo MET amplifications and METex14 skipping mutations.Our study aimed to elucidate the comprehensive mutation profile of patients with MET amplification or METex14 skipping mutations in NSCLC receiving MET TKI therapy by analyzing molecular factors that could contribute to their prognosis. In fact, no retrospective study has previously evaluated the efficacy of MET TKIs in the treatment of patients with Herein, we describe the survival characteristics by analyzing mutated genes and underlying primary resistance factors with MET amplification and METex14 skipping mutations.via needle biopsy of lung lesions or lymph nodes. Samples were sent to Buring Rock Biotech for molecular genotyping using NGS (56 gene or 168 gene panel). The following patients were excluded: (1) patients previously treated with other EGFR TKIs before MET detection and (2) patients with evidence of small cell lung cancer (SCLC) metastasis. This study was approved by the Institutional Review Board of Hunan Cancer Hospital. All patients provided written informed consent for the use of their data in this study.Patients diagnosed with NSCLC with MET amplification (n = 43) or METex14 skipping mutations (n = 31) from Hunan Cancer Hospital between March 2015 and October 2020 were retrospectively analyzed. Baseline MET amplification or METex14 skipping mutation status was assessed using blood or tissue samples obtained MET TKIs, including 250 mg crizotinib twice daily, 200 mg bozitinib twice daily, or 600 mg volitinib once daily, were prescribed to the MET amplification or METex14 skipping mutation cohorts. Treatment was discontinued if unacceptable toxicity, disease progression or death, patient refusal, or treatment withdrawal for any other reason, including pregnancy, were noted. Response was measured using enhanced computed tomography (CT) scans in accordance with the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 . The objCirculating cfDNA was extracted from plasma samples according to the manufacturer’s instructions using a QIAamp Circulating Nucleic Acid Kit . Similarly, tissue DNA from formalin-fixed, paraffin-embedded (FFPE) cell blocks of tumor biopsy or other cytologic samples were extracted using the QIAamp DNA FFPE tissue kit . A Qubit 2.0 Fluorometer with dsDNA HS Assay Kit  was used to quantify cfDNA and tissue DNA concentration.NGS library construction was performed using optimized protocols developed by Burning Rock Biotech . Tissue via capture-based targeted sequencing using commercial gene panels. These panels, including either the 56 gene or 168 gene panel , interrogated all exons and critical introns of classic lung cancer oncogenes ; METex14 skipping mutation cohort, 83.9% (26/31). Squamous cell carcinoma was detected in the remaining patients: MET amplification cohort, 16.3% (7/43); METex14 skipping mutation cohort, 16.1% (5/31).The disease stage upon initial diagnosis was I-IIIa in 14.0% (6/43), IIIb-IIIc in 2.3% (1/43), and IV in 83.7% (36/43) in the MET amplification cohort. On the other hand, all patients in the METex14 cohort were in the advanced stage, with 90.3% (28/31) of patients at stage IV and the remaining 9.7% (3/31) at stage IIIb-IIIc. In the MET amplification cohort, 67.4% (29/43) of the patients received MET TKI treatment . The remaining 32.6% (14/43) did not receive MET TKI therapy because of early-stage post-surgery (6/14) or refusal of treatment (8/14). Of the 29 patients receiving therapy, 82.8% (24/29) received it as a first-line regimen, while the remaining 17.2% (5/29) received it as a second-line treatment. In the METex14 skipping mutation cohort, 93.5% (29/31) of patients received MET TKIs . The remaining 6.5% (2/31) of the patients did not receive any therapy due to refusal of treatment. Of the 29 patients receiving therapy, 86.2% (25/29) received it as a first-line regimen, while the remaining 13.8% (4/29) received it as a second-line treatment. In addition, 60.5% (26/43) of the MET amplification cohort and 64.5% (20/31) of the METex14 cohort had concurrent gene mutations. Both cohorts were independent of sex, age, smoking status, and clinical stage. The clinicopathologic characteristics of the two cohorts are summarized in vs. 11.0 months, P = 0.043, vs. 20.0 months, P = 0.635; vs. 8.0 months, P = 0.556) and OS  as compared to those with high MET amplification  and 93.5% (29/31) of patients from the MET amplification or METex14 skipping mutation cohort, respectively, who received MET TKI treatment were included in the analysis. Log-rank analysis showed a statistically significant shorter PFS in the MET amplification cohort than in the METex14 skipping mutation cohort  achieved PR, 48.3% (14/29) achieved SD, and 17.2% (5/29) achieved PD, resulting in an ORR of 34.5% and a DCR of 82.8%. Of the 29 patients who received MET TKIs in the METex14 skipping mutation group, 51.7% (15/29) achieved PR, 34.5% (10/29) achieved SD, and 13.8% (4/29) achieved PD, resulting in an ORR of 51.7% and DCR of 86.2% of the MET amplification cohort and 64.5% (20/31) of the METex14 skipping mutation cohort had other concurrent mutations. In the MET amplification cohort, concurrent TP53 mutations were the most common, affecting 34.9% (15/43) of the patients. In addition, other concurrent mutations, including BRCA CN amplification (n = 8), PIK3CA mutation (n = 6), STK-11 mutation (n = 4), BRAF CN amplification (n = 3), ALK missense mutation (n = 2), RB1 CN deletions (n = 2), RECQL4 mutation (n = 1), KRAS mutation (n = 1), EGFR CN amplification (n = 1), TERT CN amplification (n = 1), SMARCA4 mutation (n = 1), NF1 mutation (n = 1), and ERBB4 CN amplification (n = 1) were also detected. Regardless, 39.5% (17/43) of the patients in the MET amplification cohort did not harbor any concurrent gene mutations. Moreover, TP53 mutation was also observed in the METex14 skipping mutation cohort, accounting for 22.6% (7/31) of the patients. Other gene mutations detected in the METex14 cohort included MET CN amplification (n = 4), BRAF CN amplification (n = 4), PIK3CA mutation (n = 3), EGFR CN amplification (n = 2), ERBB2 CN amplification (n = 2), RET CN amplification (n = 1), CDKN2A mutation (n = 1), NRAS mutation (n = 1), CCND3 mutation (n = 1), LRP1B mutation (n = 1), and LZTR1 mutation (n = 1). Regardless, 35.5% (11/31) of the patients did not harbor any concurrent mutations Figure 2In this study, we analyzed the presence or absence of concurrent mutations associated with survival outcomes to further explore other molecular factors that may influence patient prognosis. As discussed above, 26/43 patients in the MET amplification cohort were found to have concurrent gene mutations. Of these, 16 received treatment with MET TKIs . Of the remaining 17 patients without concurrent gene mutations in the MET amplification cohort, 13 received MET TKI therapy .vs. 8.0 months, P = 0.038, vs. 20.0 months, P = 0.044, vs. NR months, P = 0.022, versus NR months, P = 0.249; We compared the PFS and OS of the patients. In the MET amplification cohort who received MET TKIs, we compared patients with and without concurrent mutations . Statistically significant shorter PFS , those with a TP53 mutation (n = 10) had a statistically significant shorter PFS , those with a TP53 mutation (n = 6) had an insignificantly shorter PFS  of the MET amplification cohort, of which 5/6 have undergone MET TKI therapy (four patients achieved PD and one patient achieved PR) while 1/6 received chemotherapy. In this cohort, those with a PIK3CA mutation who received MET TKI therapy (n = 5) were found to have a statistically significant shorter PFS  of the patients were found to have PI3KCA mutations, of which 2/3 received therapy with MET TKIs (one patient achieved PD and one patient achieved PR), while one patient refused any therapy. Due to the small cohort size, the relationship between survival outcomes and PIK3CA mutation in the METex14 skipping mutation cohort could not be analyzed.Four patients from the MET amplification cohort who had concurrent PIK3CA mutations and had received MET TKIs showed progressive disease at the first radiologic assessment . Although several previous studies have reported the survival outcome in NSCLC patients with MET amplification and METex14 skipping mutation patients, no clinical study has directly compared the survival outcomes of these two genetic alterations. Our study is the first retrospective analysis of various outcomes in patients with NSCLC, with either MET amplification or METex14 skipping mutation, receiving treatment with MET TKIs. Our results suggest that MET TKI therapy in NSCLC patients with METex14 skipping mutations is more efficacious than MET amplification.Generally, patients with MET amplification or METex14 skipping mutation have a variable response to the same treatment, suggesting the presence of another factor contributing to molecular heterogeneity. Therefore, we further analyzed the comprehensive mutation profile of the patients in our cohort to identify whether a concurrent mutation would influence the clinical outcomes of these patients. Several studies have shown that concurrent mutations in lung cancer are associated with worse survival outcomes and resistance to TKIs , 33. Morversus 11.0 months, P = 0.357) and OS , as compared to TP53 wild-type patients. Despite the lack of statistical significance in the METex14 skipping mutation cohort, it is worth mentioning that those with TP53 mutations had comparatively worse prognosis than those without, indicating a clinically significant trend.In our study, 60.5% (26/43) of patients with MET amplification and 64.5% (20/31) of patients with METex14 skipping mutations harbored a concurrent gene mutation. Of these patients, those who received treatment with MET TKIs were found to have a significantly shorter PFS, as compared to those without a concurrent mutation. Studies have also shown that a concurrent TP53 mutation is associated with reduced responsiveness to TKIs and a relatively worse prognosis –38. In oIn addition to TP53, a study by Schrock et al. showed that other gene amplifications, such as EGFR, frequently occur concomitantly with METex14 alterations , which wMET is activated upon binding of HGF, leading to downstream activation of the PI3K and MAPK pathways, subsequently causing tumor proliferation, progression, and metastasis . The PI3Our present findings suggest that PIK3CA mutation may be associated with resistance to MET TKIs and is based on the following observations. In our study, 50% of the METex14 skipping mutation cohort and 80% of the MET amplification cohort harboring a PIK3CA mutation showed disease progression with MET TKI therapy, suggesting primary resistance to MET TKIs. In relation to this, five patients, specifically in the MET amplification cohort, with concurrent PIK3CA mutation receiving MET TKI therapy had a PFS of only 1.0 months. This further supported the hypothesis that PIK3CA mutations may be a molecular factor in primary resistance to MET TKIs.This study has some limitations. Due to its retrospective nature, some clinical information is incomplete, such as the exclusion of several patients due to lack of detailed information and proper follow-up. In addition, not all patient samples were tested using the 168 gene panel; some patients were tested using the 56 gene panel. Therefore, analysis of concurrent molecular factors in patients tested using the 56 gene panel was limited.After analyzing our data, we speculate the possible association of PIK3CA mutation with primary resistance to MET TKIs in NSCLC patients with MET amplification. Additional studies and experiments must be conducted to elucidate this hypothesis. Moreover, a large prospective cohort study is needed to investigate predictive and prognostic biomarkers for stratifying patients with MET-amplified and METex14-mutated lung cancers.In summary, patients with METex14 skipping mutation had a significantly longer PFS than in patients with MET amplification, indicating that MET TKIs could be a better treatment option for patients with the METex14 skipping mutation. Moreover, concurrent mutations may deteriorate the PFS in NSCLC patients with MET amplification and METex14 skipping mutations. TP53 mutations in patients with MET amplification and METex14 skipping mutations were associated with worse survival outcomes than those with the wild type. PIK3CA mutations may confer primary resistance to MET TKIs in patients with MET amplification. These findings contribute to a better understanding of the molecular factors associated with clinical outcomes of NSCLC patients with MET amplification and METex14 alterations.The datasets generated for this study are available on request to the corresponding authors.The studies involving human participants were reviewed and approved by Hunan Cancer Hospital. All patients signed the informed consent form. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.LL and JQ performed the experiment. HY, CZ, YX, and LL provided patient information. SZ collected the data. FK revised the manuscript. JQ and NY were responsible for study conception and design and acquiring financial support. All authors contributed to the article and approved the submitted version.This work was supported by grants from the National Natural Science Foundation of China (No. 81802278) and the Natural Science Foundation of Hunan Province (No.2019JJ50361).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.


Cases of concurrent immunoglobulin A nephropathy (IgAN) and IgG4-related tubulointerstitial nephritis (IgG4-TIN) are rare and previous case reports have lacked important data. KDIGO suggests a treatment with systemic glucocorticoids in IgAN patients. Glucocorticoids are recommended as the first-line therapy for IgG4-TIN. The use of tacrolimus as a long-term maintenance treatment has not been described. We report the case of a man who developed IgAN and IgG4-TIN without abnormalities in extra-renal tissue, without renal function abnormalities or impairment as well, and was treated by tacrolimus as a long-term maintenance during 45 months follow-up.A 56-year-old Chinese man first presented to our hospital with the chief complaint of foamy urine for 1 year and hematuria for 3 months, with a medical history of hypertension. Testing revealed a notable increase in serum IgG4 level without abnormalities in renal function or imaging, or in dysfunction other organs. Renal biopsy showed mesangial extracellular matrix proliferation, increased mesangial cell numbers and infiltration of plasma cells. Immunofluorescence showed mesangial positivity for IgA and C3. Immunohistochemistry staining showed widespread IgG4 and increased CD38 and CD138 expression. Electron microscopy showed immune complexes located on the tubular basement membrane. He was diagnosed with IgAN and IgG4-TIN. He received glucocorticoids, leflunomide and tacrolimus to induce remission. He was given tacrolimus as long-term maintenance treatment. When tacrolimus was temporarily withdrawn, proteinuria recurred. After resuming tacrolimus therapy, he again entered complete remission. After 45 months of therapy, he remains in complete remission and the serum IgG4 level is normal.The finding of concurrent IgAN and IgG4-TIN without abnormalities in renal function, imaging or extra-renal tissue is rare and their coexistence may be coincidental. Long-term treatment with tacrolimus proved effective and he has remained in remission during 45 months follow-up. IgA nephropathy (IgAN) is the most common cause of primary glomerulonephritis worldwide , and is IgG4-related disease (IgG4-RD) is an fibroinflammatory condition involved multiple organs characterized by IgG4 positive plasma cells infiltration in the involved tissues and elevated serum IgG4 level , with a Glomerular disease in patients with IgG4-RD has been reported in the setting of IgG4-TIN, but most such patients had extrarenal involvement and multiorgan involvement –10. OnlyA 56-year-old Chinese man was admitted with the chief complaint of foamy urine for 1 year and hematuria for 3 months. Medical history was notable for hypertension for 5 years; the highest blood pressure was 180/110 mmHg. His blood pressure was poorly controlled, 140–150/90–100 mmHg, while he took candesartan irregularly.He denied diabetes, hepatitis, tuberculosis, and coronary heart diseases. His medical family history was unremarkable. He denied use of illicit drugs and exposure to pesticides and other toxins. Medications included an angiotensin-converting enzyme inhibitor. His weight was 79 kg, blood pressure was 170/90 mmHg, and physical examination was otherwise unremarkable.2 by the CKD-EPI equation)  3.4 g/d, serum total protein 75.3 g/l, serum albumin (Alb)33.5 g/l, serum creatinine (Cr) 86 umol/l, , anti-neutrophil cytoplasmic antibodies (−), IgG4 3.68 g/l, IgG 25.70 g/l, IgA 5.96 g/l, IgM 1.41 g/l, IgE 1586 IU/ml  scan and enhanced abdominal CT scan were normal. Renal ultrasound showed left kidney dimensions was 12.61 cm in height 5.58 cm in width and 5.43 cm in depth, while the right kidney dimensions were 11.22 by 5.62 by 7.86 cm. Both kidneys were slightly enlarged and hyperechoic  staining under light microscopy, one out of fourteen glomeruli showed global sclerosis. The mild to moderate mesangial proliferation was present. The two glomeruli showed ischemic shrinkage Fig. a. There Renal tubules exhibited multifocal atrophy and proteinaceous casts. Renal tubular epithelial cells showed granular and vacuolar degeneration. Focal interstitial fibrosis and interstitial edema with significant inflammatory cell infiltration, including lymphocytes and plasma cells were present, Fig. a, b. OccOn immunofluorescence staining, a pattern of lumpy-like deposition was seen in the mesangium  criteria for the diagnosis of IgG4-renal disease .Tripterygium wilfordii, angiotensin converting enzyme-inhibitor was replaced by an angiotensin receptor blocker, together with amlodipine to maintain his blood pressure below 130/80 mmHg. After 12 weeks, the prednisolone reduced gradually and proteinuria level reached complete remission (< 0.3 g/d), and Tripterygium wilfordii was administered for 18 months maintenance  therapy was initiated, based on the KDIGO guideline for IgAN  therapiePneumocystis carinii infection which requires sulfamethoxazole, which can also cause interstitial nephrititis. Rituximab therapy was not available for this patient because of his financial reasons.From the 15th month on, serum IgG4 and IgA re-elevated as well as serum albumin dropped. Tacrolimus was put on the patient as immunosuppresent based on a Cochane systematic review . Before After 3 months of tacrolimus treatment, kidney disease activity and immune indices were remitted again for 8 months Fig. . When thAt the most recent visit in June 2021, URTP remained < 0.3 g/d, IgG4 plasma was negative Figs. . AlthougThis study reported a man patient with concurrence of IgAN and IgG4-TIN without renal function abnormalities or impairment at the initial hospitalization. At admission, the patient presented marked proteinuria, the decreased serum albumin level, and normal renal function. The renal biopsy showed typical mild-moderate mesangial proliferation, predominant IgA, and C3 deposition. However, abundant monocytes infiltrated in the tubule-interstitium of the kidney biopsy and serum IgG4 level increased near two-fold. On immunohistochemistry of renal biopsy, absolute number of positive IgG4 cells was more than 10/high power field and plasma surface biomarkers were positive. Concurrence of IgAN and IgG4-TIN was diagnosed. Oral prednisone and tacrolimus showed effective for both IgAN and IgG4-TIN with over than 45 months follow up Figs. .Takako Saeki et al. firstly reported a patient biopsy proven as IgG4-TIN without prominent proteinuria and microscopic hematuria. However dominant mesangial IgA deposition is also seen in one case . This caIgAN is the most common glomerulonephritis in the world  and it i2 despite supportive care. The benefit of immunosuppressive agents remains controversy [Tripterygium wilfordii, a traditional Chinese medicine, was beneficial for numerous Chinese IgAN patients [Tripterygium wilfordii as the treatment for IgAN in first 18 months. When we followed up this patient on 15th month, tacrolimus was administered due to the elevated serum IgG4, IgA and total IgG levels, and the decreased albumin level indicating the activity of the disease. Recent meta-analysis and RCTs showed that tacrolimus was beneficial for the remission of proteinuria in patients with IgAN, showing that tacrolimus may be a promising agent for IgAN [In terms of the treatment, KDIGO suggested a treatment course of systemic glucocorticoids in IgAN patients with proteinuria above 1 g/day and eGFR higher than 50 ml/min/1.73 mtroversy . No randtroversy . Glucocotroversy , 22. Howtroversy , are reapatients . Considefor IgAN , 25–29. for IgAN . T follifor IgAN –33. Tacrfor IgAN , 25–29 ofor IgAN –33. Thesfor IgAN –36. Durifor IgAN  and to bfor IgAN , HoweverIn summary, we presented a case of a 56-year-old male without renal function abnormalities or impairment who was diagnosed as concurrence of IgAN and IgG4-TIN without extrarenal involvement and follow up 45 months. Tacrolimus was effective for the both diseases. In the early stage of IgG4-TIN without extrarenal involvement, tacrolimus was beneficial for development of the extrarenal tissue impairment by controlling serum IgG4 level and was also effective for IgAN.


FAPI-04 is a promising PET tracer for tumor imaging as well as IgG4-related disease (IgG4-RD).68Ga-FAPI PET/MR helped to diagnose IgG4-RD that involved pancreas and bile duct. A 62-year-old patient suffered from diffusive discomfort at middle upper abdomen and presented brown urine. Blood test revealed abnormal liver function and elevated IgG4 (4.830g/L↑). 18F-FDG PET showed enlarged uncinate process and dilated bile duct tree. Mild increase of FDG uptake in uncinate process and head of pancreas indicated possible pancreatic malignancy, but the clinical evidence was not sufficient and histology examination was negative. 68Ga-FAPI PET revealed prominent increased radioactivity distribution in the entire pancreas and bile duct, suggesting IgG4-RD.We herein present a case where 18F-FDG imaging is ambiguous. The presented case illustrates that 68Ga-FAPI-04 PET is helpful in improving the differential diagnosis of pancreatitis and pancreatic cancer.FAPI-04 is not only a good PET imaging tracer for tumors, but also for prominent fibroblast-mediated inflammation. FAPI imaging should be considered when the diagnosis using  Serum tumor marker test showed elevated CA199 43.9 U/ml↑(≤30 U/ml). Hepatitis B and hepatitis C tests were negative. Serum IgG4 testing was performed to screen for autoimmune hepatitis, and the result was 4.830g/L↑ (< 2.01g/L).Routine abdominal ultrasound  showed diffusive slight enlargement of the pancreas, dilated intra-/extra-hepatic bile duct, and enlarged gallbladder; space-occupying lesion at pancreas was detected.2) with clear boundary. No obvious expansion of the main pancreatic duct in the body and tail pancreas was found. Possible malignancy was considered  and MRI were performed on November 9, 2020. EUS showed uneven echo in pancreatic neck adjacent to the portal vein and superior mesenteric vein , and the possibility of malignant tumor was considered .68Ga-FAPI PET/MR imaging approved by the institutional review board in our hospital, and written informed consent was obtained from the patient. From 68Ga-FAPI PET/MR, homogenously elevated radioactivity uptake was found in the entire pancreas  and increased radioactivity was also found around dilated intrahepatic and extrahepatic bile duct   was excluded. Based on elevated IgG4 and negative biopsy histology, IgG4-related disease was considered. Meanwhile, various imaging modalities were highly suggestive of underlying malignant tumor lesions. For further differentiation, the patient was enrolled in the clinical trial of Hormone therapy was initiated thereafter. Specifically, the patient was treated with methylprednisolone and prednisolone, as well as medication for liver protection, bile evacuation, and nutritional support.After 2 weeks’ treatment, the patient’s blood test was as follows: total bilirubin 17.20 umol/L (≤26.0 umol/L), direct bilirubin 14.3 umol/L↑ (≤8.0 umol/L), total bile acids 11.7 umol/L↑(0–10.0 umol/L), glycocholic acid 6.6 mg/L (0–10.0 mg/L), alanine aminotransferase 41 U/L (9–50 U/L), aspartate aminotransferase 18 U/L (15–40 U/L), AST mitochondrial isoenzyme 1.7 U/L (≤15 U/L), alkaline phosphatase 338 U/L↑ ( 45–125 U/L), γ-glutamyl transpeptidase 478 U/L↑ (10–60 U/L), lactate dehydrogenase 116 U/l (120–250 U/l), and superoxide dismutase 192 U/mL (129–216 U/mL). Tumor marker CA199 was 10.4 U/ml (≤30 U/ml) and IgG4 was 4.220 g/L↑ (<2.01 g/L). The patient’s clinical symptoms improved significantly and liver function improved, so he was discharged on December 9, 2020.18F-FDG PET has demonstrated advantages over other anatomical imaging modalities , but there are still challenges in differentiating autoimmune pancreatitis from pancreatic cancer , expresses prominent FAP in the cancerous tissue and thus may have similar image presentation with IgG4-RD when using 18F-FDG imaging, but 68Ga-FAPI-04 imaging showed uniform increased uptake throughout the pancreas, which ruled out potential malignancy and confirmed the IgG4-RD diagnosis. This case demonstrated that 68Ga-FAPI-04 PET is more sensitive to IgG4-RD compared with 18F-FDG, and thus may be helpful in improving the differential diagnosis of pancreatitis and pancreatic cancer.In this case, inflammation in the pancreas mimicked focal malignant lesions in