Our earlier report indicated that active vitamin D3 inhibited epithelial-mesenchymal transition (EMT) in bleomycin (BLM)-induced pulmonary fibrosis. The objective of this study was to further investigate whether vitamin D deficiency exacerbates BLM-induced pulmonary fibrosis.Cyp27b1+/+, Cyp27b1+/− and Cyp27b1−/− mice were fed with standard diet. For pulmonary fibrosis, mice were intratracheally instilled with a single dose of BLM (1.5 mg/kg). Serum 25(OH) D level was measured. Pulmonary collagen deposition was assessed by Sirius red staining. EMT was measured and transforming growth factor-beta (TGF-β)/Smad3 signaling was evaluated in the lungs of BLM-treated mice.This study consists of two independent experiments. Experiment 1, male mice were fed with vitamin D deficient (VDD) fodder. Experiment 2, Cyp27b1 gene knockout, leading to active vitamin D3 deficiency, exacerbated BLM-induced pulmonary fibrosis. Moreover, Cyp27b1 gene knockout aggravated pulmonary TGF-β/Smad2/3 activation and subsequent EMT in BLM-induced lung fibrosis.The relative weight of lungs was elevated in BLM-treated mice. Col1α1 and Col1α2, two collagen protein genes, were upregulated, and collagen deposition, as determined by Sirius red staining, was observed in the lungs of BLM-treated mice. E-cadherin, an epithelial marker, was downregulated. By contrast, vimentin and α-SMA, two EMT markers, were upregulated in the lungs of BLM-treated mice. Pulmonary TGF-β/Smad3 signaling was activated in BLM-induced lung fibrosis. Further analysis showed that feeding VDD diet, leading to vitamin D deficiency, aggravated elevation of BLM-induced relative lung weight. Moreover, feeding VDD diet aggravated BLM-induced TGF-β/Smad3 activation and subsequent EMT in the lungs. In addition, feeding VDD diet exacerbated BLM-induced pulmonary fibrosis. Additional experiment showed that Vitamin D deficiency exacerbates BLM-induced pulmonary fibrosis partially through aggravating TGF-β/Smad2/3-mediated EMT in the lungs. Idiopathic pulmonary fibrosis (IPF) has characteristics of fibroblast proliferation and collagen-based extracellular matrix reconstruction , 2. The 3, vitamin D3 is then transformed by CYP27B1 to form 1,25 (OH)2D3, the active form of vitamin D3. 1,25 (OH)2D3, exerts its biological function by activating vitamin D receptor (VDR) [3 alleviated BLM-induced pulmonary interstitial fibrosis [Vitamin D is essential for uptake of calcium and metabolism of bone. The children with vitamin D deficiency have greater risks of rickets . In the or (VDR) , 17. Sevfibrosis , 19. On fibrosis , 21. Sevfibrosis , 23. Accfibrosis . Moreovefibrosis . NeverthCyp27b1 gene knockout. The objective of this study was to investigate whether vitamin D deficiency aggravates BLM-induced pulmonary fibrosis using two different animal models. Our results indicated that both feeding VDD diet and Cyp27b1 gene knockout exacerbated BLM-induced pulmonary fibrosis. Mechanistically, vitamin D deficiency exacerbated pulmonary fibrosis partially through aggravating BLM-induced TGF-β/Smad2/3-mediated EMT in the lungs.In the present study, we established two mouse models of vitamin D deficiency through either feeding vitamin D deficient (VDD) diet or Bleomycin (BLM) was from Hisun Pfizer Pharmaceuticals Co. Ltd. . 25(OH) D detection kit was from DIAsource . TRIzol reagent was from Molecular Research Center, Inc. . RNase-free DNase was purchased from Promega Corporation . ECL detection kit and Bicinchoninic acid (BCA) protein assay reagents were from Thermo Scientific . Antibodies against Lamin A/C, β-actin, E-cadherin and vimentin were from Cell Signaling Technology . Antibodies against α-SMA and Phospho-Smad3 were from Abcam .Cyp27b1+/+) and Cyp27b1 knockout (Cyp27b1−/−) mice were from Beijing Vital River. Mating wild-type mice with Cyp27b1 knockout mice produced Cyp27b1+/+, Cyp27b1+/− and Cyp27b1−/− mice. The genotype was confirmed by PCR analysis. All animals were fed with standard diet and water freely. The circadian rhythm of 12 h light and 12 h darkness was maintained. Six weeks after birth, mice each strain were randomly divided into 2 subgroups: Ctrl and BLM. In the BLM subgroup, mice were intratracheally instilled with BLM (1.5 mg/kg). In the Ctrl subgroup, mice were intratracheally instilled with equal volume of saline. All animals were sacrificed 2 weeks after BLM instillation. Serum 25(OH) D level was measured. The middle and lower lobes of right lung were sheared for histopathology and immunohistochemistry. The remainder tissues were collected for Western blots and real-time RT-PCR. The protocols of all animal experiments complied with the guidelines for humanitarian therapy by the Association of Laboratory Animal Sciences of Anhui Medical University and the Center for Laboratory Animal Sciences.The present study consists of two independent experiments. Experiment 1, male C57BL/6 J mice (4-week old) were from Beijing Vital River. All animals were kept with temperatures of 20–25 °C and humidity of 50%. All animals were fed standard diet and water freely. The circadian rhythm of 12 h light and 12 h darkness was maintained. After one week of adaptation, 40 mice were randomly divided into two groups: vitamin D deficient (VDD) and control (Ctrl). Mice of the VDD group were fed with vitamin D deficient fodder. Mice of the Ctrl group were fed with standard fodder. Five weeks later, mice of the Ctrl group and the VDD group were randomly divided into two subgroups, respectively (10 mice each subgroup): Ctrl, bleomycin (BLM), VDD, VDD + BLM. In the BLM and VDD + BLM groups, animals were intratracheally instilled with BLM (1.5 mg/kg). In the Ctrl and VDD groups, animals were treated with equal volume of saline in the same way. Experiment 2, wild-type (N = 6).Lung tissue was fixed with 4% paraformaldehyde, embedded in paraffin. Each wax block was sliced continuously and stained with hematoxylin and eosin (H&E) to evaluate the histological manifestations. The inflammation was evaluated using the score of 0–5 according to previous study . The numLung was cut into sections (5 μm thick) according to the standard protocol , 25. TheTo extract total protein from lung tissue, 450 μl buffer with PMSF and 50 mg lung tissue were used. The supernate was collected after 35 min grinding on ice and centrifugated for 15 min. The protein concentration of total pulmonary lysate was measured with the BCA protein assay reagents. In order to extract nuclear protein from lung tissue, 300 mg lung tissue was homogenized with 4 ml buffer A . SupernaTRIzol reagent was used to collect total RNA from mouse lungs. After precipitating and washing, total RNA was determined by OD value to quantity as 0.5 μg/μl. RNA was then reverse-transcribed with AMV after digestion with RNA-free DNase. Specific primers Table  and GoTaP < 0.05 and P < 0.01 were determined to demonstrate statistically significant. All statistical analyses were carried out with SPSS 19.0 software.The raw data were presented as means ± S.E.M. ANOVA was used to calculate differences between different groups. All data were evaluated for significance using non-parametric tests techniques when those were not normally distributed. Col1α1 and Col1α2) mRNAs were measured by real-time RT-PCR. As expected, pulmonary Col1α1 and Col1α2 mRNAs were obviously upregulated in BLM-treated mice .Serum 25(OH) D concentrations were evaluated. It showed that serum 25 (OH) D concentration was significantly lower in VDD group than that of control group Fig. a. NucleaThe influences of vitamin D deficiency on BLM-induced EMT were evaluated. It showed that, pulmonary E-cadherin, an epithelial marker, was obviously downregulated in BLM-treated group Fig. a and b. Tgf-β1 mRNA was significantly upregulated in BLM-treated mice  than that of BLM-treated Cyp27b1 wild-type mice (2.36 ± 0.27), with the most collagen deposition in BLM-treated Cyp27b1−/− mice (7.83 ± 0.45) .As shown in Fig. ice Fig. b. Moreovice Fig. c. PulmonCyp27b1 gene knockout on BLM-induced EMT were evaluated. As expected, pulmonary E-cadherin, an epithelial marker, was obviously downregulated in BLM-treated Cyp27b1 wild-type mice D3 into 1,25(OH)2D3, an active form of vitamin D3 [Cyp27b1 gene knockout results in active vitamin D3 deficiency [3 deficiency exacerbated radiation-induced bone marrow injury [3 deficiency on BLM-evoked EMT and pulmonary fibrosis using a model of Cyp27b1 gene knockout mice. Of interest, Cyp27b1 knockout aggravated collagen deposition, as accessed by Sirius red staining, in BLM-induced pulmonary fibrosis. In addition, Cyp27b1 knockout aggravated BLM-induced downregulation of E-cadherin in the lungs. By contrast, Cyp27b1 knockout aggravated BLM-induced upregulation of α-SMA in the lungs. Finally, Cyp27b1 gene knockout aggravated BLM-induced pulmonary Smad2 and Smad3 phosphorylation. These results provide additional evidence that active vitamin D3 deficiency exacerbates Smad2/3 activation and subsequent EMT in BLM-induced pulmonary fibrosis.tamin D3 , 32. So ficiency , 34. An w injury . In the Cyp27b1 gene knockout mice. Therefore, our present study does not exclude the direct role of VDR on EMT. Further study is necessary to explore whether long-term vitamin D deficiency induces pulmonary fibrosis through directly regulating EMT in the lungs.The mechanism through which vitamin D deficiency aggravates TGF-β/Smad2/3-mediated EMT during BLM-induced pulmonary fibrosis remains obscure. An earlier report showed that active vitamin D3 inhibited renal fibrosis through promoting direct interaction VDR and Smad3 . A recenVDR as a regulator of EMT during BLM-induced pulmonary fibrosis may have preventive and therapeutic implications. According to an epidemiological report, vitamin D deficiency was positively associated with the mortality of patients with IPF . A recenCyp27b1 gene knockout, leading to active vitamin D3 deficiency, exacerbated BLM-induced pulmonary fibrosis. In addition, Cyp27b1 gene knockout aggravated pulmonary TGF-β/Smad2/3 activation and subsequent EMT during BLM-induced lung fibrosis. Data above provide evidence that vitamin D deficiency exacerbates BLM-induced pulmonary fibrosis partially through aggravating TGF-β/Smad2/3-mediated EMT in the lungs.In summary, our present experiment investigated the effects of vitamin D deficiency on BLM-induced pulmonary fibrosis using two mouse models. We showed that feeding VDD diet, leading to vitamin D deficiency, exacerbated BLM-induced pulmonary fibrosis. Moreover, vitamin D deficiency aggravated TGF-β/Smad2/3 activation and subsequent EMT during BLM-induced pulmonary fibrosis. We found that


Taking consecutive antibiotic use into account is of importance to obtain insight in treatment within disease episodes, use of 2nd- and 3rd-choice antibiotics, therapy failure and/or side effects. Nevertheless, studies dealing with consecutive antibiotic use are scarce. We aimed at evaluating switch patterns in antibiotic use in the outpatient setting in the Netherlands.Outpatient antibiotic dispensing data was processed to antibiotic treatment episodes consisting of single prescriptions or consecutive prescriptions (2006 to 2014). Consecutive prescriptions were categorised into prolongations and switches. Switches were further analysed to obtain antibiotic switch percentages and trends over time. Outcomes were compared with recommendations of Dutch guidelines.A total of 43,179,867 antibiotic prescriptions were included in the analysis, consisting of single prescriptions (95%), prolongations (2%) and switches (3%). The highest switch percentages were found for trimethoprim (7.6%) and nitrofurantoin (5.4%). For fosfomycin, ciprofloxacin, flucloxacillin and trimethoprim we found the highest yearly increase in switching. Amoxicillin/clavulanic acid was most often used as second antibiotic in a switch. A surprisingly high number of 2nd- and 3rd-choice antibiotics are prescribed as first antibiotic in a treatment.Although the actual reason for a switch is unknown, switch patterns can reveal problems concerning treatment failure and guideline adherence. In general, switch percentages of antibiotics in the Netherlands are low. The data contributes to the knowledge regarding antibiotic switch patterns in the outpatient setting. The medical impact and economic burden of antibiotic resistance is tremendous and increasing annually . AntibioA wide variety of indicators exist in literature to quantify and qualify antimicrobial use, without consensus on the most appropriate indicators to be used –10. OverTo retrieve reliable results on consecutive antibiotic use, the index date of the antibiotic and the duration of the therapy have to be well documented and a unique patient identifier is needed. Therefore only databases containing high quality prescription or dispensing data are suitable for the analysis of consecutive antibiotic use in the outpatient setting.So far, only a few studies deal with consecutive antibiotic use –14. ThesBesides calculating treatment failure for individual antibiotics, it is of great interest to visualize antibiotic switches and focus on second prescriptions within an antibiotic treatment. It is of importance whether 2nd/3rd choice antibiotics are prescribed as first one, which is often inappropriate, or as second one within an infectious disease episode, which could be appropriate for severe infections, or to treat pathogens resistant to the 1st choice antibiotic. Therefore, studies dealing with consecutive antibiotic use can provide valuable insight. We aimed at evaluating switch patterns in antibiotic use in the outpatient setting in the Netherlands.Data on outpatient antibiotic use in the Netherlands was obtained from the Foundation for Pharmaceutical Statistics (SFK) for the period 2006–2014. The SFK collects dispensing data from participating Dutch community pharmacies. The participation rate increased from 90% in 2006 to 95% in 2014. The dataset contained a pharmacy number, a unique patient number, the anatomical therapeutic chemical classification code (ATC-5) of the drug , the staThe study population consists of all patients who filled a prescription for oral antibiotics (ATC J01) at a SFK participating Dutch community pharmacy from January 1, 2006 to December 31, 2014. As the SFK covers 95% of pharmacies, the study population is a proper representative of the Dutch population.SFK data, provided in Microsoft Office Excel® 2007, was processed to SPSS statistics 22 and organised per year. Only antibiotics for systemic use (ATC-code J01) were included. All antibiotic prescriptions were sorted by patient number. A stop date for each prescription was calculated using the start date, the number of dispensed units and the prescribed dose, resulting in the duration of the course. The total duration of antibiotic therapy for all patients was calculated for each successive year. The antibiotic treatment episodes were composed of single prescriptions and consecutive prescriptions. A consecutive prescription was defined as a successive antibiotic prescription for the same patient within the period from prescribing the first antibiotic up to 3 days after the calculated end date of the first antibiotic prescription. Consecutive prescriptions were further categorised as prolongation, or switch. A prolongation was defined as a repeat prescription of the same antibiotic, and a switch was defined as a consecutive prescription with another antibiotic.Prescriptions for one patient with the same start date, single antibiotic prescriptions with a duration > 14 days and patients with an annual exposure to antibiotics of more than 8 weeks were excluded . Numbers and distribution of antibiotic treatment episodes per year are shown in Table The distribution of single prescriptions, prolongations and switches for the nine most common used antibiotics is shown in Table Trimethoprim and nitrofurantoin showed relatively high switch percentages of 7.6 and 5.4% in 2014. In other words, a high percentage of initial prescriptions of these drugs were followed by a second antibiotic prescription. The switch percentages for the other antibiotics were between 2 and 4%. The time-trend from 2006 to 2014 in yearly switch percentages (from the first given antibiotic) is shown in Fig. Figure n = 1,414,747) in the year 2014. In this figure the most common switch patterns can be seen at a glance. For nitrofurantoin these concerned switches to fosfomycin (24%), ciprofloxacin (23%), trimethoprim (17%) or amoxicillin/clavulanic acid (17%). Switches after first use of amoxicillin most often go to one of the macrolides, azithromycin and clarithromycin (31%), amoxicillin/clavulanic acid (25%) or doxycycline (19%).Figure To the best of our knowledge, this is the first study giving an overview of all first and second prescriptions within antibiotic treatment episodes from a national database of outpatient antibiotic use. The vast majority of antibiotics, including antibiotics that are generally considered as second- or third-choice treatments, are prescribed as first antibiotic in a treatment. This finding indicates that the initial choice of the prescriber is not always in line with recommendations in guidelines.Previously conducted studies regarding consecutive antibiotic use report high treatment failure rates , 12. A sE. coli, K. pneumonia and P. mirabilis, the predominant species causing urinary tract infections in the Netherlands [We found the highest switch percentages for trimethoprim and nitrofurantoin. These antibiotics are predominantly used to treat urinary tract infections and the switch percentages indicate that initial treatment with these antibiotics is quite often followed by another antibiotic. According to the Dutch national guidelines, nitrofurantoin is the first choice treatment for uncomplicated urinary tract infections in the outpatient setting . Fosfomyherlands . It was herlands . Howeverherlands . In the herlands .For ciprofloxacin we found a yearly increase in total use and a corresponding annual increase of 0.08% in its switch percentage. This is worrisome as it could reflect increased bacterial resistance to ciprofloxacin. Ciprofloxacin often is a 3rd choice treatment in Dutch primary care and reserved for severe infections and hospitalised patients, and resistance in primary care jeopardises this aim.Not surprisingly, amoxicillin/clavulanic acid is the drug that is most often used as second antibiotic in a switch. It is used after a wide variety of first prescriptions, including nitrofurantoin, amoxicillin, doxycycline, flucloxacillin, a macrolide and ciprofloxacin. A second prescription of either azithromycin or clarithromycin, amoxicillin/clavulanic acid or doxycycline after initial amoxicillin are all plausible changes in treatment strategy.Our data indicate that fosfomycin and ciprofloxacin are increasingly used as second drug in an antibiotic treatment course. This could be the result of high switching from trimethoprim and nitrofurantoin and the increase in cystitis episodes . TreatmeThe main strength of the study is the use of a nationwide database containing over 90% of total dispensed antibiotics. The use of a database containing pharmacy dispensing data instead of prescription data results in more valid estimations of consumed antibiotics. Because patients in the Netherlands are often linked to one pharmacy, it is possible to monitor subsequent antibiotic use based on pharmacy data. A recent study showed that only 2% of patients regularly switch between pharmacies . In contHowever, there are also limitations to our study that need to be addressed. First, the indication for prescription was not available. With the indication it would have been possible to specifically identify those switches that deviate from national treatment guidelines. Second, the data only contains outpatient pharmacy data. Data on treatment failures leading to hospital admission, visits to the emergency department, or antibiotics previously used in the hospital setting were not available and could not be incorporated. Finally, we were not able to determine the actual reason for the antibiotic switches. Although we speculate that an antibiotic switch reflects ineffectiveness of the treatment, possibly as a result of bacterial resistance, there can be several other explanations. Switches can also be a result of doctors’ behaviour , patients’ behaviour , or progression of the infection itself (including complications), or availability of the results of in vitro antimicrobial susceptibility testing. The evidence linking antibiotic treatment failure and antibiotic resistance is considered to be weak , 21. TheThe dataset could be enriched by linking pharmacy data with clinical patient data, the antibiotic indication, outcomes of antimicrobial susceptibility tests, clinical outcomes and complications. However, there are security, privacy and technical issues that need to be addressed before such linking can be performed. Another challenge is to study seasonal and demographic differences in switch percentages for antibiotics. For European comparison studies, we encourage others to also evaluate trends in consecutive antibiotic use and switching patterns. These issues can provide even more detailed information that can help policymaking.The vast majority of antibiotics are prescribed as single courses, also antibiotics that are generally considered as 2nd- or 3rd-choice treatment. This is not in line with current guidelines. Switch percentages of antibiotics are low with an average percentage of 3%. The highest switch percentages were found for trimethoprim and nitrofurantoin. The most common antibiotic switches are in line with guideline recommendations. The data contributes to the knowledge regarding antibiotic switch patterns in the outpatient setting.


Antibiotic stewardship interventions are being implemented across different healthcare settings. We report the findings of a global survey of healthcare professionals on the implementation of antibiotic stewardship programmes.p-values were considered as two-tailed. Significance was set at p-value of < 0.05.Learners of a Massive Online Open Course (MOOC) on antibiotic stewardship were invited to complete an online survey on the core available organisational resources for stewardship. The categorical variables were analysed using chi-squared test, and Likert questions were analysed using an ordinal regression model. The The response rate was 55% (505/920), from 53 countries. The responders were 36% (182) doctors, 26% (130) pharmacists, 18% (89) nurses and 20% (104) other . Post-graduate training in infection management and stewardship was reported by 56% of doctors compared with 43%  nurses and 35%  of pharmacists. Hospitals were significantly  more likely to have antibiotic policies, when compared to primary care. A surveillance mechanism for antibiotic consumption was reported in 58% (104/178) of teaching hospitals and 62% (98/159) of regional hospitals. Antimicrobial resistance, patient needs, policy, peer influence and specialty level culture and practices were deemed important determinants for decision-making.Postgraduate training and support in antibiotic prescribing remains low amongst nurses and pharmacists. Whilst antibiotic policies and committees are established in most institutions, surveillance of antibiotic use is not. The impact of specialty level culture, and peer influence appears to be important factors of antibiotic prescribing. The threat of antimicrobial resistance is driving initiatives across the one health care economy , 2. Whilhttps://www.futurelearn.com/courses/antimicrobial-stewardship). Since then it has been translated to Spanish, Russian and Mandarin (2017) with several runs each year. The MOOC has reached a global audience and the feedback so far suggests it was effective in engaging with participants on the topic of stewardship [To address the global challenges of implementing antibiotic stewardship, in 2015, an international group of researchers and clinicians developed a six-week Massive Open Online Course targeting a global audience . The MOOwardship . There awardship . RecogniTo investigate the existing stewardship activities, including education and training, in the participants’ organisations and countries and to explore what influenced the learners’ own antibiotic prescribing decision-making. a survey questionnaire  was developed using data from previous studies , and witThe questionnaire was piloted with healthcare staff in England. All learners participating in week five of the MOOC course were invited to complete the questionnaire online. The survey was placed as an optional activity halfway through the week’s course material. The invitation to participate included a study information sheet and learners were informed that completion of the survey was not mandatory with learners having the option to choose to carry on with the week material without completing the survey . All the data from the completed forms were automatically collated and extracted into an excel sheet. The data entry and collection was anonymous and no learner identifier data was collected.p-values were considered as two-tailed and a p-value of < 0.05 was set as significant. Wherever a p value of 0.000 was reported in the software, it was presented as < 0.01 in the results. All analyses were conducted in SPSS 24 , and Microsoft Excel 2013 (Microsoft).The descriptive data were analysed by institution type and profession. For the statistical analysis, responses of ‘not applicable’ were combined with ‘no’ when analysing the response to whether individuals had received postgraduate training. The categorical variables were analysed using chi-squared test. Cronbach’s alpha test was performed to test the co-efficient alpha for overall reliability of the Likert questions. The Likert questions investigating the perceived determinants of antibiotic prescribing were analysed using an ordinal regression model. All the determinants  were fitted into the model to predict their perceived influence on prescribing behaviours. The The first run of the MOOC had 15,570 unique registrations. Of these 6839 individuals from 164 countries completed at least one of the six modules. In total, 920 learners continued to, and completed week five. Of the week five learners, there were 505/920 (55%) survey respondents from 53 countries. The top five countries with the highest frequency of survey responses comprise 65% (327) of the results, summarised in Table p < 0.01) compared with pharmacists , and nurses   respondents from hospitals, with 178/505 (35%) from a teaching hospital and 159/505 (31%) from a regional hospital Table . Ten perp < 0.01)  when compared to primary care (33% 16/49). A surveillance mechanism for antibiotic consumption and prescribing was present in 58% (104/178) of teaching hospitals and 62% (98/159) of regional hospitals.Hospitals  were significantly more likely to have an institutional antibiotic related policy or guidelines when compared to primary care (p = < 0.01), Table p < 0.01). The opinion of colleagues, pharmacist recommendation, and senior doctor recommendation were ranked very closely in influence on specialty level and practices  response rate from the participants of week five of the MOOC. Most of the survey respondents, however were from the UK and Australia, and five countries comprised 65% of the respondents. This may have been due to the MOOC being in English, and therefore not accessible to non-English speakers, although, responses from the USA and Canada with English speaking populations, and with established stewardship programmes in their healthcare system were low. It was encouraging to see participation from nurses and pharmacists, in addition to doctors, in this survey. Though this course was targeted healthcare professionals in hospitals, it attracted learners from primary care as well as scientists, students and from other wider members of the population. This interest from the non-healthcare professional workforce in an activity about antibiotic stewardship is a positive step in raising awareness about the need to engage with the wider population on the necessity to conserve antibiotics.The participation of nurses and pharmacists in stewardship activities such as developing policy and guidelines, and reviewing antibiotic prescriptions is a positive step to a broader inclusion of healthcare professionals, globally in stewardship efforts. Although pharmacist participation in stewardship activities is the norm in some healthcare systems such as in USA, UK, Canada and Australia, in many other parts of the world, stewardship remains the responsibility of doctors . The repThe most common components of stewardship are reported to be the use of policy and guidelines, the review of antibiotic prescriptions, and the existence of dedicated committees to stewardship. The availability and reporting of antibiotic consumption and surveillance reports however, remain low at 58% in teaching hospitals, and 62% in regional hospitals, and 43% in primary care. In general, stewardship activities were significantly more likely to be present in hospitals than in primary care. Since this is a survey with responses from 53 countries, the results will reflect the heterogeneity of the participants’ national healthcare systems. Education and training in infection management and antibiotic stewardship, at the postgraduate level, remains a resource primarily available to doctors. This may be one of the reasons why it is more difficult for other healthcare professionals to become involved and participate in stewardship activities. Education and training in this field is not universal and requires further resources, particularly in healthcare settings where traditionally doctors are still considered to be the main decision makers and leaders in infection management and antibiotic stewardship.https://www.futurelearn.com/courses/antimicrobial-stewardship).When asked to rate the factors that influence antibiotic decision making, all three professions were consistent in their rankings of the key factors, namely: antimicrobial resistance, patient need, local policies. These determinants were closely followed by the influence of senior doctors, local culture and practices and personal experience as key determinants of antibiotic decision making. This finding of the influence of senior doctors and specialty level and local culture is similar to findings from in-depth qualitative studies investigating the determinants of antibiotic decision-making , 11. To This survey was conducted across a group of individuals who had registered to participate in a course on antibiotic stewardship. There will have been inherent selection bias in this group of respondents. The data is from 2015, and since then there has been further progress in supporting antibiotic stewardship programmes both internationally and nationally.This study with participants representing 53 countries provides insights into the main components of antibiotic stewardship programmes in different countries and the key factors which influence the antibiotic decision-making of key healthcare professionals. Whilst antibiotic policies and committees are established in most institutions, surveillance of antibiotic use is not universal. Postgraduate training and support in antibiotic prescribing remains low amongst nurses and pharmacists. The impact of specialty level culture, and peer influence appears to be significant factors of antibiotic decision-making. These data identify existing gaps in adequate training and education for staff involved in stewardship, and the need for greater participation of nurses and pharmacists to ensure effective and sustainable stewardship programmes.


This mini-review summarizes the applications of olefin metathesis in synthesis and functionalization of polyhedral oligomeric silsesquioxanes (POSS) and POSS-containing polymeric materials. Three types of processes, i.e., cross metathesis (CM) of vinyl-substituted POSS with terminal olefins, acyclic diene metathesis (ADMET) copolymerization of divinyl-substituted POSS with α,ω-dienes and ring-opening metathesis polymerization (ROMP) of POSS-substituted norbornene (or other ROMP susceptible cycloolefins) are discussed. Emphasis was put on the synthetic and catalytic aspects rather than on the properties and applications of synthesized materials. A number of silsesquioxane structures have been reported including random, ladder, cage and partial cage structures. Silsesquioxanes with specific cage structures are commonly referred as polyhedral oligomeric silsesquioxanes (POSS). From among POSS structures the most thoroughly studied is a cubic silsesquioxane unit, denoted also as T8. It contains an inorganic cubic core composed of eight Si atoms at the vertices, connected through O atoms along the edges, chemically bonded with eight different or similar organic substituents so that it represents a truly hybrid architecture. The cubic silsesquioxane unit is characterized by a three-dimensional nanoscopic size structure with approximate Si–Si distance equal to 0.5 nm and an approximate R–R distance of 1.5 nm , POSS are often functionalized through the chemical processes of C=C bond transformation, e.g., hydrosilylation, Heck coupling, silylative coupling and olefin metathesis.Olefin metathesis, i.e., catalytic exchange of double bonds between carbon atoms, is a powerful tool in organic synthesis. The use of metathesis in organic and polymer synthesis is comprehensively described in excellent monographs –6. HowevVinylsilanes show a specific reactivity towards alkylidene ruthenium complexes because of a strong effect of the silyl group on the properties of the double bond. In general, the substituents at the silicon atom determine the regioselectivity of the vinylsilane cycloaddition to the Ru=C bond. The knowledge of this untypical reactivity is pivotal for the application of metathesis for the modification of vinylsilanes, vinyl-substituted siloxanes, spherosilicates and silsesquioxanes. The appropriate choice of substituents permits the control of the process to a certain degree. The reactivity of vinylsilanes with different substituents at silicon towards alkylidene ruthenium complexes is illustrated in A), the active methylene complex B and the corresponding (E)-1-phenyl-2-(silyl)ethene are formed. The methylene complex B in the presence of styrene undergoes metathetic conversion to benzylidene complex A and ethene. When dichloro-substituted vinylsilanes are used, the pathway shown in A leads to styrene and (silyl)methylidene complex C. Formation of (silyl)methylidene complex C has not been confirmed by spectroscopic methods. The reaction of the postulated complex C with vinylsilane gives the corresponding (E)-1,2-bis(silyl)ethenes and the methylene complex B. The methylene complex B may react with vinylsilane to form ethene and regenerate complex C. In the presence of vinylsilanes containing alkyl substituents the Grubbs catalyst undergoes fast decomposition as a result of β-transfer of the silyl group in the appropriate β-(silyl)rutenacyclobutane complex to ruthenium followed by reductive elimination of the corresponding propene derivative  of vinyl-substituted POSS with terminal olefins, acyclic diene metathesis (ADMET) copolymerization of divinyl-substituted POSS with α,ω-dienes and ring-opening metathesis polymerization (ROMP) of POSS-substituted norbornene  underwent cross metathesis of terminal and internal olefins, functionalized olefins  and styrene.The first metathetic transformations of vinyl-substituted silsesquioxanes and spherosilicates  were repRu-1) was demonstrated in CM of OVS with pent-4-en-1-ol and 5-bromopentene. It has been found that terminal alkenes undergo cross metathesis much more readily and are clearly better than internal alkenes from the cost perspective. However, internal alkenes are less volatile and cannot produce any ethene, which makes them interesting starting materials. A slight vacuum had to be applied to reactions with terminal alkenes in order to remove ethene, because ethene would strongly slow down the desired cross metathesis and inactivate Schrock-type metathesis catalysts. CM of OVS with styrenes proceeded stereoselectively. A mixture of cis- and trans-isomers was obtained in the transformations of other olefins tested. Spherosilicate was shown to undergo CM with pent-1-ene and styrene in the presence of Mo-1. No data on the activity of Ru-1 in metathesis transformation of spherosilicates was provided.Moreover, the catalytic activity of the first generation Grubbs’ catalyst  occurring in the presence of first generation Grubbs’ catalyst Ru-1,  9]..Ru-1, 9]E-stereochemistry around the newly formed C=C double bond, while aliphatic α-alkenes  gave a mixture of stereoisomers (E/Z = 94:6). Additionally, when 1-hexene was used as reacting partner, the product of the cross metathesis was accompanied by considerable amounts of those of olefin homometathesis. Under optimized conditions, CM of OVS with styrene proceeds quantitatively despite the low loading of the catalyst . The product was obtained in 91% isolated yield, however, the process required a temperature elevation to 60 °C and the use of a catalyst amount of 4 mol % ..10].Ru-1. The reaction mixtures were stirred for 72 h to ensure complete conversion of the silsesquioxane. The quantitative conversion of the substrate can be achieved by blowing a gentle stream of nitrogen above the reaction mixture to remove the ethylene formed. The resulting 4-bromostyryl derivatives were subsequently modified via Heck coupling with a set of 4-substituted styrenes to give the next generation of functionalized derivatives. The authors also demonstrated the possibility of further functionalization of an amino-substituted derivative via the reaction with 3,5-dibromo or dinitrobenzoyl chloride. The proposed synthetic method based on the gradual development of the organic part can be used for the synthesis of new star polymers, dendrimers or hyperbranched molecules. Further examples of the use of cross metathesis of OVS with styrenes in order to form functionalizable dendrimer cores have been reported by Cole-Hamilton  ethyl]  and octal)ethyl] . The abiRu-1 (Cross metathesis of monovinyl-substituted POSS with olefins has been reported for the first time by Marciniec . It was Ru-1 .Ru-1) and led to the formation of the expected products with isolated yields ranging from 85% to 97%. In all cases the exclusive formation of E-isomers was detected and the formation of competitive olefin homometathesis was not observed. The reactions were carried out using a small excess of olefin (1.5–3 equiv) to ensure complete conversion of the reactants. In the reaction of monovinyl-POSS with allylbenzene, CM was accompanied by double bond migration, which results in reduction of the isolated yield of the CM product (85%) and the formation of minor amounts (15%) of 1-propenylbenzene. No similar isomerization was observed in the reaction of POSS with allyltrimethylsilane. Further research enabled Marciniec to extend the scope of the reaction by reporting efficient CM of monovinyl-POSS with a series of substituted styrenes. The reported procedures permit efficient and selective functionalization of mono- and octavinylsilsesquioxanes with π-conjugated substituents via cross metathesis  anE-arrangement around the C=C double bonds was observed.In 2016 Marciniec reported the synthesis of a series of new cubic POSS in which one vertex silicon atom was replaced by a germanium atom bearing a vinyl group . MonovinRu-1, in whose presence no undesirable competitive reaction of olefin homometathesis occurred. Full conversion of monovinylgermasilsesquioxane required the use of 1 mol % of the catalyst. The reactions described are the first examples of metathesis activity of vinylgermanium compounds.The most suitable catalyst for CM was found to be More than a decade ago Yoshida developed a new class of silsesquioxyl compounds containing rigid Si–O–Si bonds, called double-decker silsesquioxanes –21. ThisE-isomers and was not accompanied by competitive homometathesis. This selectivity was obtained thanks to the use of the Ru-1 catalyst, moderately active in homometathesis of the olefins studied. Effective transformation was observed for substituted styrenes. Expected products were isolated with yields in the range of 88–95%. When allyl derivatives  were tested as olefinic partners, incomplete conversions of reactants (55–60%) were observed, despite the increased catalyst loading (2 mol %). Effective metathesis transformation was observed also in the presence of Ru-2 but then considerable amounts of olefin homometathesis product were formed. The presence of a methyl group at the vinylsilyl moiety was responsible for the lack of activity, which was consistent with earlier studies  and proved effective functionalization of such compounds by cross metathesis with a series of 4-substituted styrenes and allylbenzene, in the presence of Ru-1  ..24].Mn in the range from 9100 to 18300 Da and Mw in the range from 13600 to 46100 Da. Thermogravimetric analyses indicate a high level of thermal resistance of the obtained systems, reaching the temperature values over 550 °C. Analogous ADMET copolymerization of divinylgermasilsesquioxanes with 4,4'-divinylbiphenyl or 4,4''-divinylterphenyl can be used in the synthesis of stereoregular trans-germasilsesquioxyl–vinylene–phenylene oligomers (The products were polymers characterized by ligomers  19]..Mn in thMw in the range from 9057 to 11033 Da and polydispersity index (PDI) = 1.5.This method permitted obtaining a polymer with The chemistry of inorganic–organic hybrid materials has emerged as a fascinating new field of modern nanotechnology. The inclusion of POSS cages into the polymeric material can significantly improve such properties of the polymer as thermal and oxidative resistance, surface properties, improvement of mechanical properties as well as reduced flammability, heat release and viscosity during processing . SynthesRing-opening metathesis polymerization (ROMP) is the type of olefin metathesis chain-growth polymerization that uses metathesis catalysts to generate polymers from cyclic olefins –41. To oThe aim of this section is to indicate the applications of ROMP in the synthesis of hybrid materials containing the POSS moiety covalently bonded to organic polymeric chains rather than the discussion of the properties of the obtained materials.Ru-1–Ru-6 because of their tolerance to moisture, atmospheric oxygen and most functional groups as well as commercial availability. The choice of solvents is determined by the solubility of monomers, with methylene chloride, chloroform, and toluene being the most commonly used. Polymerization is terminated by addition of ethyl vinyl ether to the reaction mixture. Ruthenium residues from the obtained copolymer are removed on a short alumina plug.The synthesis of polymers by ROMP is carried out almost exclusively in the presence of ruthenium-based catalysts Mo-2  substituents with dicyclopentadiene (DCPD) [Tg up to 15 °C in relation to that of polyDCPD. Similar examinations were reported by Coughlin who used first generation Grubbs catalyst (Ru-1) for copolymerization of POSS-functionalized norbornene with DCPD  . Moreoveith DCPD  46]. Du. DuRu-5 Dicyclopentadiene and norbornenylethyl-POSS or tris(norbornenylethyl)-POSS  have beeN-(propyl-POSS)-7-oxanorbornene-5,6-dicarboximide was tested in ring-opening metathesis copolymerization with 3-(trifluoromethyl)phenyl-7-oxanorbornene-5,6-dicarboximide in the presence of second generation Grubbs catalyst phenyl-7-oxanorbornene-5,6-dicarboximide  48]. It. It48]. polymers . A serieperature  49]..Tg valueIt has been demonstrated that the length of the spacer affects the crystallizability of POSS groups so that the use of a reasonably long spacer to link the POSS groups to the main chain can make POSS groups crystallizable.Ru-1) is a practical route to the synthesis of block copolymers containing POSS nanoparticles  and narrow molecular weight distributions (in the range Mn/Mw = 1.19–1.29). Copolymerization was employed by a sequential monomer addition. At first, the POSS-NBE was introduced into the reaction system containing the catalyst and after its complete conversion methyltetracyclododecene was added. The reaction was terminated with ethyl vinyl ether as soon as the second monomer was fully converted. A series of copolymers with different POSS-NBE content were obtained. The PDI values were in the range of 1.32–1.53 with average molecular weights of ca. 48000–63000 Da.Kim and Kwon have shown that ring-opening metathesis copolymerization of norbornenylethyl-POSS with methyltetracyclododecene in the presence of first generation Grubbs catalyst (articles  50]. RO. RORu-1)endo-3-exo-5-norbornene-2,3-dicaboxylic acid trimethylsilyl ester was performed in the presence of Ru-1. The block copolymer was obtained via sequential monomer addition  containing polyhedral oligomeric silsesquioxane (POSS) in the side chain was demonstrated by Shen and Fan  52]. Th. Th52]. Ru-6. It was shown that the polymers obtained can self-assemble in THF solution into aggregates, when water was added.Wang has reported living ROMP of a series of monomers bearing a polymerizable norbornene dicarboxyimide group attached via an appropriate linker to 1–4 POSS units . Copolymexo-2,3-dicarboximido)dodecanoylamino)propylheptaisobutyl-POSS and exo-5-norbornene-2-carbonyl-end poly(benzyl methacrylate, Lee has performed a series of sequential ring-opening metathesis copolymerization of norbornene-Surface-initiated ROMP was used to grow an organic corona phase on the surface of CdSe/ZnS quantum dots . FunctioOlefin metathesis, a universal tool in organic and polymer synthesis, offers numerous advantages for the synthesis of POSS-based materials. Ruthenium-based olefin metathesis catalysts tolerate the presence of water, air and nearly all functional groups. Commercially available vinylsilsesquioxanes can be easily modified and/or functionalized by cross metathesis. According to the CM product-selectivity model , vinylsi


Introduction: Immunoglobulin G4-related disease (IgG4-RD) represents an immune-mediated fibro-inflammatory condition resulting in mass forming lesions with characteristic histopathological appearances. The disease can affect multiple different organs, with a range of clinical presentations to different medical and surgical specialties.Case description: We describe a case of a 71-year old female who presented with refractory facial pain following a dental procedure. She was treated with repeated courses of antibiotics by the dentist and was subsequently referred for an ENT opinion. A CT scan revealed complete opacification of the left maxillary sinus with bony erosion of the floor, medial, anterior and posterior aspects of the maxillary antrum. Histological biopsies were reported as showing eosinophilic angiocentric fibrosis with an IgG4:IgG ratio of > 40%, consistent with a diagnosis of IgG4-RD. Peripheral blood IgG4 levels were normal and extended autoantibody screening, including ANCA, viral and TB screening were negative. Past medical history was significant for an unexplained episode of bronchus intermedius stenosis, ten years earlier. Thoracotomy yielded multiple biopsies which were reported at the time as showing non-specific granulomatous inflammation. Analysis of the historic samples revealed elevated levels of IgG4 staining. The patient was referred to rheumatology and commenced prednisolone 30mg orally with some improvement in facial pain. The dose was subsequently weaned to a maintenance dose of 7.5mg daily. Mycophenolate mofetil (MMF) was commenced as a steroid sparing agent (1 gram twice daily). Unfortunately, there was radiological evidence of disease progression with ongoing bone loss, despite this therapy and the decision was made in the IgG4-RD MDT, to proceed with Rituximab with cyclophosphamide induction. There has been some resolution of the inflammatory changes and treatment is ongoing.Discussion: With an estimated incidence of 60m per million populations, IgG4-RD is a rare, but increasingly recognized condition. First line therapy remains glucocorticoids. Rituximab has been commissioned by NHS England for use in refractory or organ threatening IgG4-RD. In this case rituximab has shown superiority to MMF in improving the symptoms and resolution of inflammation. Arguably the trial of MMF delayed the more effective treatment and resulted in more bony loss. The extent to which the addition of iv cyclophosphamide potentiated the effects Rituximab is not known and more research is needed. The episode of bronchus intermedius stenosis suffered almost a decade earlier, coupled with the multiple courses of antibiotics and delayed contemporary diagnosis, highlights the diagnostic challenges of IgG4-RD.Key Learning Points: Consider further review and IgG4 staining of any available previous histopathology samples if a diagnosis of IgG4-RD is made. Consider the addition of cyclophosphamide in organ threatening disease as an induction agent. Consider Rituximab early in organ threatening disease to prevent progression to irreversible damage.Disclosure: F. Osman: None. J. Vila: None.


Anchored steel bars have been widely used in retrofitting of existing concrete structures. The bonding strength between the anchored steel bar and the concrete is critical to the integrity of the strengthened concrete structure. This paper presents a method to monitor epoxy-grouted bonding strength development by using a piezoceramic-enabled active sensing technique. One concrete beam with an anchored steel bar was involved in the monitoring test, and two concrete beams with six anchored steel bars were used in the pull-out test. To enable the active sensing, a Lead Zirconate Titanate (PZT) patch was bonded to the surface of the exposed end, and piezoceramic smart aggregates were embedded in each concrete specimen. During the monitoring experiment, signals from PZT sensors and smart aggregates were acquired at intervals of 0, 20, 40, 60, 80, and 100 min. In addition, a pull-out test was performed on each of the remaining six anchored steel bars in the two concrete beams, while the signal was recorded in the test. Furthermore, a wavelet packet analysis was applied to analyze the received signal energies to investigate the bonding strength development between the concrete and the anchored steel bar during the epoxy solidification process. The test results demonstrate the effectiveness of the proposed method in monitoring the bonding strength development between the anchored steel bar and the concrete, using the PZT-enabled active sensing. The retrofit, reinforcement, and strengthening of existing structures have been widely practiced and researched ,2, and vDue to its wide application, the anchored steel bar technology has received much attention in the literature. In the 1990s, Cook  and CookPresently, epoxy is one of the commonly used adhesives in the anchored steel bar technology. In addition, the epoxy can repair concrete cracks to prevent or reduce further corrosion ,21,22,23Piezoceramic materials, exhibiting piezoelectricity, have been one of the popular materials used in structural health monitoring ,28,29,30The PZT-enabled active sensing method has been found to be effective in detecting de-bonding between a steel plate and concrete ,72,73, aDue to the piezoelectricity, PZT can be either used as an actuator or a sensor ,39,46. TRemark: In some applications, the exposed end of the steel bar may not be available to be bonded with the PZT patch. In such cases, we will design and try re-usable PZT transducers with integrated magnets. These re-usable PZT transducers can be attached to the end surface or the side surface of the anchored steel rod by using magnetic force. Furthermore, the re-usable PZT transducers will help to broaden the potential applications of the proposed approach., where The wavelet-packet-based energy index (WPEI) analysis has been demonstrated to be an effective technique to quantitatively estimate the differences between the stress wave responses due to structural changes, such as damage ,68,72,73The total energy of the signal can be computed by the summation of all the decomposed signals. The computed energy of the signal The received signal can be characterized by using the WPEI analysis. The WPEI has been used to monitor or evaluate the structural health condition in various concrete structure applications ,83. In tThe dimensions of the specimen for the monitoring test and the location of PZTs and SAs are presented in The dimensions of concrete beams CB1–CB3 is 550 mm × 150 mm × 150 mm and each specimen was made with concrete of class C40. The size of steel bars SB1–SB7 is Φ12 mm × 50 mm and the type is HRB335. The material properties of the test specimens are shown in The experimental setup of the monitoring test includes the concrete beam (CB1) with an anchored steel bar (SB1), PZTs and SAs, a power amplifier (Trek 2100HF) for piezoceramic load, and a data acquisition system (NI USB6363) hosted by a laptop computer, as shown in The pull-out test involved two concrete beams (CB2 and CB3) with anchored steel bars (SB2–SB7), as shown in The results of the monitoring test are shown in It can be seen from Furthermore, the WPEI was applied to quantify the energy of the received signal, as shown in Remark: In field implementations, it is important to know when the epoxy reaches its full bonding strength so that the next step of the retrofit or reinforcement, that requires the application of a programmed load through the anchored steel bar, can be carried out. The WPEI results  clearly In this paper, a straightforward piezoceramic-based active sensing approach was utilized to monitor epoxy-grouted bonding strength development between an anchored steel bar and the concrete in real time. To verify the proposed methods, experiments were carried out on specimens with anchored steel bars. A PZT patch was bonded to the exposed end of each of the anchored steel bars, and piezoceramic smart aggregates were embedded in the concrete specimens to realize the active sensing approach. In the monitoring test, the time domain signal amplitude and WPEI demonstrate that, during the first 20 min, since the epoxy was liquid, the stress wave can hardly travel through it. During the next 40 min, the epoxy began to solidify and to bond to the steel bar and the concrete beam. Therefore, the stress wave can easily transmit from the steel to the concrete beam through the gradually cured epoxy. During the 60–80 min period, the time domain signal amplitude and WPEI increased but at a much slower rate, which suggests that the curing process of the epoxy was almost completed. In addition, both the received time-domain signals and signal energy reached a steady-state value from 80 to 100 min. It directly indicated that the curing process of the epoxy was completely done. Comparing the WPEI with the maximum tensile force from the pull-out tests clearly shows that the maximum pull-out force and the energy indices are closely correlated. The results of monitoring test and pull-out tests demonstrate that the piezoceramic-based active sensing approach is capable of monitoring the epoxy-grouted bonding strength development in real time.In further work, the different installation methods of transducers and the sensing range of the PZTs and SAs will be further studied. In addition, aspects of a real-life situation, including external loading and, especially, ambient temperature, will also be investigated. Furthermore, numerical or theoretical models will be developed to compare with the current experimental results.


The long-term performance of externally-bonded reinforcements (EBR) on reinforced concrete (RC) structures highly depends on the behavior of constituent materials and their interfaces to various environmental loads, such as temperature and humidity exposure. Although significant efforts have been devoted to understanding the effect of such conditions on the anchorage resistance of unstressed EBR, with or without sustained loading, the effect of a released prestressing has not been thoroughly investigated. For this purpose, a series of experiments has been carried out herein, with concrete blocks strengthened with carbon fiber-reinforced polymer (CFRP) strips, both unstressed, as well as prestressed using the gradient anchorage. The gradient anchorage is a non-mechanical technique to anchor prestressed CFRP by exploiting the accelerated curing property of epoxy under higher temperatures and segment-wise prestress-force releasing. Subsequently, strengthened blocks are transferred into a chamber for exposure in dry freeze-thaw cycles (FTC). Following FTC exposure, the blocks are tested in a conventional lap-shear test setup to determine their residual anchorage resistance and then compared with reference specimens. Blocks were monitored during FTC by conventional and Fabry–Pérot-based fiber optic strain (FOS) sensors and a 3D-digital image correlation (3D-DIC) system during gradient application and lap-shear testing. Results indicate a reduction of residual anchorage resistance, stiffness and deformation capacity of the system after FTC and a change in the failure mode from concrete substrate to epoxy-concrete interface failure. It was further observed that all of these properties experienced a more significant reduction for prestressed specimens. These findings are presented with a complementary finite element model to shed more light onto the durability of such systems. To this day, the most common strengthening approach is the application of either fabrics or pultruded strips on the tensile face of the member, often referred to as externally-bonded reinforcement (EBR). CFRP has excellent mechanical properties  with gradient anchorage solely relies on its constituents, that is concrete, epoxy and CFRP. The long-term behavior of these constituents and their interactions need a thorough investigation to guarantee a safe performance over the design life. At Empa, the long-term behavior of a CFRP-strengthened concrete structure is currently a core research topic ,10. It wIn this study, an experimental campaign was launched to investigate the effect of common environmental deterioration phenomena, such as carbonation and freeze-thaw cycles (FTC). Results have shown that carbonation bears a positive effect through the increased strength of concrete , yet FTCAlthough the effect of FTC on the residual anchorage resistance of gradient anchorage was quantified within the previous study, the underlying mechanisms causing the deterioration and shift in failure mode remain unknown. With the current knowledge, it is assumed that the deterioration initiates and propagates due to chemical and mechanical processes ,17. AnotIn order to investigate the effect of prestressing and FTC on the residual anchorage resistance, four scenarios were considered for the experimental campaign. Two reference scenarios, one unstressed (Ref-US) and one prestressed (Ref-PS), provide the necessary benchmark behavior of the anchorage in the short-term time scale. Subsequently, identically-constructed blocks were subjected to freeze-thaw cycles, henceforth referred to analogously as FTC-US and FTC-PS. Three specimens were prepared for each scenario, and their key steps for each scenario are summarized and provided in A Portland-limestone cement (CEM II/A-LL 42.5 N) was used with well-rounded alluvial gravel (S&P 220 ) was use  was useFollowing the preparation of both unstressed and prestressed blocks, a set of strain sensors was installed on the blocks that are undergoing freeze-thaw cycles, as presented in Furthermore, 5 unstrengthened concrete blocks from the same batch were deployed with deformeter bolts to measure the shrinkage/swelling behavior of concrete itself in ambient and varying RH conditions. For this purpose, blocks were taken out of storage (90% RH) and stored for two weeks (approximate time the strengthened blocks have spent before initiating FTC) at ambient conditions . Due to technical difficulties, the CFRP strains are not measured after the end of the cycles, so swelling data might provide some insights for the strains existing in the system just before lap-shear testing.Conventional single-sided lap-shear tests are conducted at the end of each case to compare their bond behavior. The specimens were tested between the ages of 9–32 months after casting, where concrete is assumed to be mostly cured and no significant developments in material properties are expected to occur. The concrete block is supported at various locations to restrain the movement of the block while the CFRP strip is pulled. The aforementioned clamping system at the end of the strip is concentrically connected to a load cell and a hydraulic cylinder via a threaded rod. The hydraulic cylinder is connected to a pump, which is operated manually to increase the loading. The force is measured by a 150-kN load cell  and recorded with a 1-Hz sampling rate by a data acquisition system (HBM GmbH). A complete schematic description of the test setup is provided in Full-field displacements of the strengthened surface were measured with a 3D-DIC system  . The meaThe images were then processed using the ARAMIS software, and raw displacements were extracted from sections of interest. The slip, obtained by subtracting average concrete displacements from CFRP displacements, was used for comparison purposes. The definition of slip is demonstrated in A finite element (FE) model was constructed in Abaqus to support experimental findings and infer more information about the behavior of the anchorage during and after FTC, at locations and instances where no experimental measurements were available.Due to the symmetric nature of the problem, only half of the system was modeled. Aside from the obvious constituents, all secondary components of the strengthened blocks were also modeled to capture the behavior as accurately as possible. These secondary components were namely the clamping system, modeled as a simplified set of discrete rigid blocks, and the steel tubes that were embedded in the concrete to attach the clamping system to the block. A screenshot of the constructed model is provided in The volumetric changes experienced by concrete were introduced by a relatively simple method, i.e., by setting the coefficient of thermal expansion (CTE) to zero for every material except concrete, since only concrete was assumed to undergo swelling and shrinkage, and subsequently applying a temperature-time history to recreate the deformations induced by swelling under 100% RH exposure. Since no material degradation was modeled, even with the aforementioned contact definitions, the implicit (standard) solver of Abaqus with non-linear geometry (Nlgeom) enabled was sufficient to successfully calculate the structural response.The loads were applied in two distinct steps, a step where the prestressing force was released from one end , and a second step where the previously explained temperature-time-history was applied. The concrete block was restrained from moving during prestress-force-releasing by appropriate boundary conditions (BC) at the block base, as well as the top; however these BC’s were removed for the swelling/shrinkage simulations to allow for unconstrained volume change. The response of the system was obtained every 86,400 s, corresponding to daily measurements.The initial comparison to observe the effects of FTC was conducted based on the lap-shear behavior of strengthened blocks. Load-slip curves of each experiment are shown in The stiffness of the bond within the elastic range (defined by 02 mm in ) can be The residual load carrying capacity, denoted by zaderski , the prezaderski ,28,29.In addition to the lap-shear test results, measurements from strain sensors at the free-length  assumed CTE of concrete, 10 Measured shrinkage data following humidity exposure were further employed within the simulation to obtain the CFRP strain after FTC exposure until the lap-shear test. It can be observed in Lastly, a comparison of stresses at the epoxy-concrete interface is made for both unstressed and prestressed samples, computed by the FE simulations and presented in A similar observation was also made experimentally from the FOS sensors attached on the CFRP strip along the bonded area (Denoted as S1–S5 in Freeze-thaw cycles in combination with humidity provoked a reduction in the residual anchorage resistance, maximum slip capacity, as well as elastic bond stiffness.Degradation became more significant in the case of prestressed blocks, especially for the residual load carrying capacity, indicating that synergistic exposure to mechanical and environmental loading led to a more severe deterioration.A shift in failure mode from concrete substrate to an epoxy-concrete interface failure was observed both in unstressed and prestressed blocks that were subjected to FTC.A trend towards tensioning was observed at the free-length of the strips during FTC. This was later explained via the swelling of concrete due to exposure to humidity. This effect was reversed after the blocks were transferred to the laboratory, in which the strains returned back to initial state via shrinkage.The average difference between minimum and maximum strain values during FTC revealed that the strip exerted a considerably high cyclic loading on the bond, with values ranging 6.6–9.2 kN for unstressed and 2.7–3.3 kN for the prestressed blocks.An FE model was constructed to observe the effect of swelling during FTC on the bond and further derive the prestressing force after FTC. An agreeable match was observed with the use of a temperature field as the input to emulate the volumetric swelling behavior. An insignificant amount of loss was computed for the prestressing force after FTC. The effect of asymmetric stress states, both due to prestressing, as well as swelling was visualized.An experimental and numerical investigation on the behavior of a non-mechanical unstressed/prestressed CFRP anchorage during and after FTC was conducted. The obtained results indicated a deteriorative effect of FTC on the bond, and the main findings of the study are summarized below.In light of these findings, future research focus could be steered towards two distinct directions. The first path would be methodologies that embrace the deterioration and involve the development of techniques that incorporate such effects during the design process. It needs to be kept in mind that the results presented herein were from a single type of a commercially available epoxy; hence, a similar experimental campaign would be needed to obtain the deterioration effect quantitatively of another epoxy resin. A second path would be to investigate ways to enhance the bonding properties of epoxy against humidity exposure. In recent studies, silane coupling agents have been successfully utilized to decrease the effect of bond degradation after hygrothermal aging ,31. Inco


MET-mutated/amplified), H596 (MET-mutated), and H1993 (MET-amplified) cells, as well as BEAS-2B bronchial epithelial cells, we investigated whether MET is involved in the regulation of immune checkpoint pathways. In a microarray analysis, MET suppression using a MET inhibitor or siRNAs up-regulated co-stimulatory molecules, including 4-1BBL, OX40L, and CD70, and down-regulated co-inhibitory molecules, especially PD-L1, as validated by measuring total/surface protein levels in Hs746T and H1993 cells. MET activation by HGF consistently increased PD-L1 expression in H596 and BEAS-2B cells. Co-culture of human peripheral blood mononuclear cells with Hs746T cells suppressed interferon-γ production by the immune cells, which was restored by MET inhibition or PD-L1 blockade. A significant positive correlation between MET and PD-L1 expression in lung cancer was determined in an analysis based on The Cancer Genome Atlas (TCGA) and in an immunohistochemistry study. The former also showed an association of MET overexpression in a PD-L1high tumor with the decreased expressions of T-cell effector molecules. In summary, our results point to a role for MET overexpression/activation in the immune escape of tumors by PD-L1 up-regulation. MET-targeted-therapy combined with immunotherapy may therefore be an effective treatment strategy in patients with MET-dependent cancer.The MET tyrosine receptor kinase is essential for embryonic development and tissue regeneration by promoting cell survival, proliferation, migration, and angiogenesis. It also contributes to tumor development and progression through diverse mechanisms. Using human cancer cell lines, including Hs746T ( MET amplification [MET mutations described thus far, a somatic mutation at or around the exon 14 splice junction that leads to deletion of the exon and, thus, to a protein lacking the juxtamembrane domain has been implicated in oncogenic MET activation [MET exon 14 skipping mutation causes impaired c-Cbl-mediated degradation of MET and therefore sustains MET activation [MET exon 14 skipping mutation is frequently observed in pulmonary sarcomatoid carcinoma (the most aggressive entity of NSCLC) [MET is a receptor tyrosine kinase that plays an important role in cancer development and progression . MET genfication . MET profication ,5. Amongtivation ,7. The jtivation ,7. Despitivation ,7. The Mf NSCLC) ,9,10,11.f NSCLC) .Immune checkpoint pathways refer to a variety of inhibitory interactions between T cells and target cells, including antigen-presenting cells and cancer cells. Examples include the programmed cell death-1 (PD-1)/programmed cell death-1-ligands (PD-Ls) and CTLA-4/CD80 pathways . Engagemhigh NSCLC. Taken together, these results suggest that MET may be involved in tumor immune evasion. Combined MET-targeted therapy and immunotherapy may therefore be an effective strategy in the treatment of several forms of cancer.PD-L1 expression in tumor cells and immune cells is associated with the efficacy of PD-1/PD-L1 blockades in cancer patients and thus serves as a predictive biomarker ,16. The MET exon 14 skipping mutation) and H1993 (harboring a MET amplification), and a gastric carcinoma cell line, Hs746T (harboring both a MET exon 14 skipping mutation and MET amplification), were used in this study. In H1993 cells and Hs746T cells, p-MET expression was up-regulated in the absence of HGF , CD70, and ICAM1, and the down-regulation of co-inhibitory molecules, including CD274 (PD-L1), PDCD1LG2 (PD-L2), PVRL1, and CD276 (B7-H3) following MET suppression , TNFSF4 , and CD70 were significantly increased in Hs746T cells treated with the MET inhibitors PHA665752 or crizotinib (TNFSF9 (4-1BBL), TNFSF4 (OX40L), and CD70 and to the up-regulation of CD274 (PD-L1), at both the mRNA and surface protein levels  and PDCD1LG2 (PD-L2) were the genes most significantly down-regulated by MET suppression. To determine whether a causal relationship existed between MET signaling and PD-L1 expression, p-METlow H596 cells were stimulated with rhHGF and p-METhigh Hs746T and H1993 cells were treated with MET inhibitors or transfected with MET-specific siRNAs. MET activation of H596 cells by rhHGF enhanced PD-L1 expression at the mRNA, total protein, and surface expression levels  and METp < 0.001 for both), with a more consistent positive correlation of MET with PD-L1 than with PD-L2 (n = 5470) from the TCGA showed that the correlations between MET and PD-L1 or PD-L2 varied depending on the cancer type  and between MET and PD-L2 in breast cancer and bladder cancer . In stomach cancer, MET correlated weakly with PD-L1 but not PD-L2 . In particular, a significant positive correlation between MET and PD-L1 expression was consistently observed in NSCLC tissues with either a KRAS mutation (n = 93)  or without KRAS mutation/EGFR mutation/ALK translocation (n = 152) , although a statistically significant positive correlation was not seen in those with an EGFR mutation (n = 41)  for pan-cancer cell lines and the TCGA for pan-cancers. In NSCLC tissues, IHC showed a significant positive correlation between MET and PD-L1 or PD-L2 expression by tumor cells (th PD-L2 A–D. An acer type . Of note(n = 41) E–G. The (n = 41) , but notPDCD1), granzyme A (GZMA), granzyme B (GZMB), perforin (PRF1), IFNγ (IFNG), CXCL9, and CXCL10, correlated inversely with MET expression, especially in the PD-L1high vs. PD-L1low group , but not EGFR-mutated tumors, there was a strong positive correlation between MET and PD-L1.Microarray analysis revealed that, in response to MET suppression, PD-L1 and PD-L2 were the most strongly down-regulated co-inhibitory molecules. MET and PD-L1 levels correlated positively in lung cancer, gastric cancer, and renal cell carcinoma as determined by IHC ,18,21,22 studies ,24. Howe studies ,25,26. I pathway . Martin  pathway ,28,29, t pathway ,25, as wMET alterations were variable and highest in Hs746T cells, harboring both MET mutation and amplification. In H1993 cells harboring only MET amplification, p-MET levels were comparable to those of Hs746T cells  cells suppressed immune cell function, which was partially recovered by anti-PD-L1 blocking antibody and the clinically administered MET inhibitor crizotinib. Consistent with this result, TCGA analysis revealed an inverse correlation between MET overexpression and T-cell effector gene expression in PD-L1high NSCLC, which suggested an immunosuppressive role of MET in the microenvironment of this tumor. This correlation was not observed in patients with gastric cancer, indicating a biologically different role for, or significance of, MET in immune evasion across different tumor types. Clinical trials for MET-targeted therapies are ongoing for patients with lung cancer and colorectal cancer based on the pro-tumorigenic role of MET . Thus, MET may be an effective therapeutic target in combination therapy with PD-1/PD-L1 blockade, based on its pro-tumorigenic and immune suppressive role in cancer. The utility of MET-targeted therapy in combination with immune targets within the PD-1/PD-L1 pathway or other immune checkpoint molecules remained to be determined in preclinical and clinical studies.Although a positive and causal relationship between MET and PD-L1 expression has been reported for in some cancer types ,20,21,22high lung cancer. Combined MET-targeted therapy and immunotherapy should therefore be considered for patients with MET-dependent cancer. However, the therapeutic strategies likely to be most effective in a combined approach and the patients most likely to benefit from such approach must still be clarified.In summary, this study demonstrated that MET has a role in tumor immune evasion via up-regulating the expression of co-inhibitory molecules, particularly PD-L1, and down-regulating the expression of co-stimulatory molecules. In vitro, MET overexpression in tumor cells contributed to the suppression of immune cell function, which was partially restored by treatment with a MET inhibitor or PD-L1 blockade. Our results also showed that MET overexpression was related to immunosuppressive features in the tumor microenvironment of PD-L1MET exon 14 skipping mutation) and H1993 (MET amplification), the gastric carcinoma cell line Hs746T (both the MET exon 14 skipping mutation and amplification), and the non-neoplastic human bronchial epithelial cell line BEAS-2B were used in this study. H596 and Hs746T cells were kindly provided by James G. Christensen , and H1993 cells by Sukjoon Yoon . BEAS-2B (ATCC CRL-9609) cells were purchased from the American Type Culture Collection . Hs746T cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics, while H596 and H1993 cells were maintained in RPMI-1640 medium supplemented with 10% FBS and 1% antibiotics, and BEAS-2B cells were in DMEM/F12 (1:1) with 5% FBS and 1% antibiotics. All cell lines were kept in a humidified atmosphere of 5% CO2 at 37 °C. PHA665752 (a MET inhibitor) was purchased from Sigma-Aldrich , crizotinib came from Selleckchem , and rhHGF was from ProSpec-Tany TechnoGene Ltd. .The lung adenocarcinoma cell lines H596 (harboring the Three types of specific siRNAs targeting human MET and a scrambled (sc) siRNA were synthesized by Bioneer  (sequences in http://www.geneontology.org/index.shtml) by GeneSpringGX 7.3.1.Hs746T cells were treated with MET inhibitor or transfected with MET siRNA, after which total RNA was extracted using the TRIzol reagent (Invitrogen). Microarray analysis and data acquisition were performed using an Agilent human 44K (V2) gene expression microarray by E-BIOGEN  according to the manufacturer’s instructions. Briefly, target cRNA probes and hybridization were synthesized using the LowInput QuickAmp labeling kit . After the labeling efficiency was confirmed, 1650 ng of cyanine 3-labeled cRNA target was subject to fragmentation by the addition of 10× blocking agent and 25× fragmentation buffer. The fragmented cRNA was resuspended in 2× hybridization buffer and used directly in the assembled Agilent human 44K (V2) gene expression microarray. The arrays were hybridized at 65 °C for 17 h in an Agilent hybridization oven and then washed. The hybridization images were captured and quantitated using an Agilent DNA microarray scanner and Agilent feature extraction software 10.7. The average fluorescence intensity for each spot was calculated after subtracting the local background intensity. All data were normalized and fold-changed genes were selected using GeneSpringGX 7.3.1 . Functional annotation of the genes was performed according to the Gene Ontology Consortium  − .Total RNA (1 μg) was reverse-transcribed using a cDNA synthesis kit . The qRT-PCR was performed using SYBR green , primers , and a SCells were stained with the phycoerythrin (PE)-conjugated anti-PD-L1 antibody , PE-conjugated anti-CD70 antibody , PE-conjugated anti-OX40L antibody , and APC-conjugated anti-4-1BBL antibody (Biolegend) or the isotype control  and subjected to flow cytometry using a FACS Canto .Total cellular protein was extracted using RIPA lysis buffer supplemented with phosphatase and protease inhibitor cocktail and EDTA . The protein samples (20–40 µg each) were subjected to SDS-PAGE and transferred to a PVDF membrane . Immunoblotting was performed using antibodies against MET , phospho-MET (p-MET) (Tyr1234/1235) , PD-L1 , PD-L2 , or β-actin . The bound antibody was visualized using a chemiluminescence kit .Human PBMCs obtained from the heparinized peripheral blood of healthy volunteers were isolated using Ficoll-Paque Plus  density gradient separation. The cells were activated for 8 h with 50 ng phorbol 12-myristate 13-acetate (PMA)/mL and 1 µg ionomycin/mL. Surface expression of PD-1 on T cells was determined by flow cytometry using PE-conjugated anti-human PD-1 (CD279) antibody . Hs746T tumor cells were seeded in 6-well plates and cultured in RPMI-1640 medium before their co-culture with activated PBMCs. Alternatively, Hs746T cells were pretreated for 24 h with crizotinib or anti-PD-L1 blocking antibody. PBMCs and Hs746T cells were co-cultured at a ratio of 2:1 and for 16 h, after which the PMBCs and culture medium were harvested for qRT-PCR and ELISA for IFNγ, respectively. This study was performed in accordance with the World Medical Association Declaration of Helsinki and was approved by the Institutional Review Board (IRB) of Seoul National University Hospital (SNUH) (H-1408-007-598).IFNγ levels were measured in the cell-free supernatants from the co-culture system using a human IFNγ ELISA kit  according to the manufacturer’s protocol.EGFR and KRAS mutations were examined using direct sequencing or peptide nucleic acid-clamping PCR. ALK translocation and MET gene copy number were evaluated using fluorescence in situ hybridization (FISH) as previously reported [Tissue microarrays (2-mm-diameter cores) were constructed from the formalin-fixed paraffin-embedded tumor tissues of 789 patients with NSCLC who underwent surgical resection of the tumor and were then followed-up at SNUH. IHC was performed using rabbit anti-PD-L1 (clone E1L3N) and anti-MET , and mouse anti-PD-L2  antibodies together with the Ventana Benchmark XT autostainer. MET IHC was scored by the criteria used in the clinical trials of a MET inhibitor as follows: Zero (0), absence of staining or any staining in <50% of the tumor cells; 1, weak to moderate intensity staining in ≥50% of the tumor cells; 2, moderate to strong intensity staining in ≥50% of the tumor cells; 3, strong intensity staining in ≥50% of the tumor cells. PD-L1 IHC was evaluated based on the intensity and proportion of membranous and/or cytoplasmic staining in tumor cells, and scored as follows: Zero (0), negative; 1, weak or moderate in <10% of tumor cells; 2, moderate in ≥10% of tumor cells; 3, strong in ≥10% of tumor cells. The clinicopathological features of the patients are summarized in https://portals.broadinstitute.org/ccle). Level 3 data of the TCGA, downloaded from the UCSC Cancer Browser (https://genome-cancer.ucsc.edu) on 3 June 2015, were used. TCGA data included clinical information and mRNA expression data obtained by RNAseq . Samples of 14 cancer types (n = 5470) were included in our analysis of the association between MET and PD-L1. Data from 1015 patients with NSCLC were analyzed to explore the expression pattern of immune-related molecules according to the expression status of PD-L1 and MET.The relationship between MET and PD-L1 expression in human cancer cell lines was explored in a CCLE analysis . For patient sample data and in vitro studies, the expression levels of several continuous variables, including MET and PD-L1, were compared using Student


The goal of the study was to evaluate the application of biochar (BC) to the sewage sludge (SL) on the adsorption and desorption capacity of Cd(II), Cu(II), Ni(II) and Zn(II). The effect of biochar contribution in the sewage sludge  was investigated. The isotherms data were fitted to the Langmiur (LM), Freundlich (FM) and Temkin (TM) models. The best fitting for kinetic study was obtained for the pseudo-second-order equation. The best fitting of the experimental data was observed for the LM in the case of SL and BC, and for the FM in the case of SL- and SL/BC-amended soil. SL was characterized by even four-order higher sorption capacity than BC. The addition of the BC to the SL and next to the soil increased the adsorption capacity of the soil and the SL-amended soil. In the case of all investigated potentially toxic elements (PTEs), the highest adsorption capacity was achieved for SL-amended soil in comparison with the control soil. In the case of other experimental variants, the adsorption capacity of metal ions was as follows: 2.5% BC > 5.0% BC > 10% BC. The negative correlation between hydrated radius of metal ions and the kinetics of sorption was observed. However, the desorption of PTEs from BC/SL-amended soil was significantly lower than for SL-amended soil (except of Cd) and non-amended soil. It can be concluded that the addition of the biochar enhanced the immobilization of PTEs and reduced their bioavailability and mobility in the soil amended by the sewage sludge.The online version of this article (doi:10.1007/s10653-017-0036-1) contains supplementary material, which is available to authorized users. Sewage sludge (SL) as a product of the wastewater treatment can be considered as one of the most difficult material to manage. This difficulty is connected not only with constantly raising amount of the produced sewage sludge, but also with sewage sludge composition, especially contaminants content. Sewage sludge contains a lot of different contaminants, including potentially toxic elements (PTEs), polycyclic aromatic hydrocarbons (PAHs), personal care products (PCPs) as well as pathogens (Devi and Saroha Lolium perene)  addition to the sewage sludge (SL) and subsequently to soil on the sorption capacity of SL or SL/BC-amended soil. The adsorption capacity of four different metal ions , very common in sewage sludge, was investigated. Furthermore, the desorption of investigated metal ions from studied materials was also investigated to evaluate the strength of bonding between PTE and fertilized soil.The soil (S) used in the experiment was collected from Bezek near Chełm city (N: 51°12′02″ E: 23°17′37″). It was a podzolic soil (S), classified as a V class of soil and poor rye soil complex. Sewage sludge (SL) was collected during spring 2014 from municipal sewage treatment plant localized in Chełm (51°07′56″N 23°28′40″E). Sewage treatment plants were located on the agricultural areas and used mainly municipal wastewater without the significant influence of the industry. BC was a commercial available biochar and was produced from willow at 600 °C in limited oxygen conditions (1–2%) and provided by Fluid SA . Physicochemical properties of control soil, sewage sludge, biochar and their mixtures are presented in Tables dw/ha) and soil (S + SL); (3) sewage sludge mixed with 2.5% of biochar and with soil (S + SL + BC2.5), (4) sewage sludge mixed with 5% of biochar and with soil (S + SL + BC5.0) and (5) sewage sludge mixed with 10% of biochar and with soil (S + SL + BC10). Methods of materials characterization and description of material properties are presented in Supporting Information (SI).Following experimental variants were prepared: (1) control soil without amendments (S); (2) sewage sludge 2, Ni(OH)2 and Zn(OH)2 (Charlot and Bezier The adsorption process of Cd(II), Cu(II), Ni(II) and Zn(II) was conducted under pH of 5.5, to eliminate the possibility of the precipitation of metal hydroxides in the volumetric phase. The PTEs hydroxides precipitate under following pHs 8, 6, 7 and 6.5, for Cd(OH)The effect of time on adsorption of PTEs onto adsorbent materials was carried out versus time intervals up to 24 h. The initial concentration of Cd(II), Cu(II), Ni(II) and Zn(II) ions in the single element solutions was 100 mg/L (each component separately). The optimal pH was adjusted to be 5.5 for all metal ions. Kinetics solutions were agitated on a shaker  at 120 rpm constant speed at room temperature 22 ± 2 °C. Next, mixtures were separated by centrifugation , and the filtrates were analyzed for the PTEs concentration.The adsorption isotherms of Cd(II), Cu(II), Ni(II) and Zn(II) were performed in the single component systems at initial pH of 5.5. The 0.2 g ± 0.03 g of each sorbent was mixed with 50 mL solutions of either Cd(II), Cu(II), Ni(II) or Zn(II) in the range of concentrations from 5 to 600 mg/L. The solutions were agitated on a shaker (120 rpm)  with temperature of 22 ± 2 °C for a specific period of contact time determined on the basis of kinetics study. After shaking, the samples were filtered and the concentration of Cd(II), Cu(II), Ni(II) and Zn(II) was measured in the supernatant.Desorption study with distilled water versus time interval was also investigated. All studied materials with known amount of loaded Cd(II), Cu(II), Ni(II) or Zn(II) ions were applied. To obtain the materials with loaded heavy metal ions, the 0.3 g ± 0.05 g of each sorbent was mixed with 50 mL solutions of either Cd(II), Cu(II), Ni(II) or Zn(II) at the concentration of 150 mg/L. The solutions were mixed for 24 h. After 24 h, solid phase was separated from solution by filtration and the concentrations of PTEs in solution were measured and the adsorbed concentration of PTEs was calculated. Solid phase was dried before using. Materials loaded with Cd(II), Cu(II), Ni(II) or Zn(II) about 0.2 g ± 0.03 g were placed into Erlenmeyer flask and mixed with 50 mL of distilled water. Suspensions were agitated in shaker (120 rpm)  at room temperature of 22 ± 2 °C for 160 h.Final suspensions after particular experiments  were centrifuged, filtered, and the supernatant solution was analyzed regarding to Cd(II), Cu(II), Ni(II) and Zn(II) ions using the flame atomic absorption spectrometer Varian Spectra AA-880  with hollow cathode lamp HCL (Varian). Detailed information about metal ions determination and kinetics and isotherm data calculations is presented in SI.Figure 2 values of the PSO model, it can be concluded that the adsorption equilibrium rate for the individual metals, regardless of the type of matrix , was as follows: Zn < Cd < Cu < Ni. Such an order can be explained based on the hydrated radius of the metal ions (Table S5). When the radius increases, the adsorption decreases , the best fit of the experimental data was obtained for the PSO model Table . Based oFigure R2 > 0.918). In the case of SL and BC, on the other hand, LM best reflected the experimental data (R2 > 0.975). This observation was confirmed for all PTEs studied. The best fit to the FM model demonstrates that in the soil the process of adsorption occurred on a heterogeneous surface, which is also indicated by the irregular shape of the adsorption isotherms . For the soil, the best fit of the experimental data was observed for FM , with these molecules blocking a part of adsorption sites and thus reducing the level of adsorption of the metals by BC, is also important information. Furthermore, considering the surface properties, it can be noted that the smaller pore size (PS) of BC made it impossible to capture large molecules of the adsorbates due to their charge and polarity . The adsorption equilibrium rate  than the LM model . This may be attributable to the complexity of the mechanism of adsorption of the PTEs studied on the mixed materials involving competition between physisorption and chemisorption, as indicated by the high regression coefficients both for LM and FM for the mixed materials , adding BC to SL caused an increase in the n coefficient. Moreover, with increasing contribution of BC in SL, the n value increased, indicating reduced adsorption heterogeneity. To determine the physicochemical properties that might potentially affect the sorption of PTEs in the treatment with sewage sludge amendment and sewage sludge with biochar addition, a statistical analysis of K and n was performed in relation to the physicochemical properties of the mixed materials (Table S8). But no relationships were found that would allow us to explain the specific observations.Adding SL to the soil resulted in a decrease in the Figure The largest range of desorption, depending on the type of metal from 3 to 25%, was observed for SL. This indicates the weakest binding of the metals by this material. Although SL was characterized by the best adsorption capacity, in its case the strength of these bonds is weakest. This shows that BC addition to SL may be an important factor that reduces the mobility and bioavailability of PTEs in sewage sludge-amended soils.SBET and Zn(II) desorption (Table S8). This may be related to the fact that with increasing specific surface area the hydrated Zn(II) ions were less tightly packed, and thereby access of the desorbing reagent was better, which was associated with increased desorption.Ni was bound weakest by SL and the other materials tested, whereas Zn was bound strongest Fig. . This waS) and BC were also characterized by high desorption of ions. Nevertheless, in all cases studied, mixing SL with the soil resulted in decreased desorption of these ions compared to the materials considered separately. Adding SL/BC to the soil had an even greater impact on reducing desorption of the metal ions studied. Cd was an exception because in its case it was difficult to observe any trend. It should also be stressed that only in the case of Cu a relationship was observed where an increase in biochar rate was accompanied by decreased desorption of this metal. In the other cases, biochar rate did not generally affect the desorption range. However, almost every time (except for Cd) much lower desorption was found for the treatments with biochar than for the sewage sludge-amended soil.Moreover, the control soil Below is the link to the electronic supplementary material.


Fewer than 200 cases have been reported in the literature. In some cases, γ-HCD occurs with other lymphoid neoplasms. This study reports clinical, biochemical, haematological, and histological findings in two cases of γ-HCD. We describe newer biochemical diagnostic tools  that can aid in the characterisation of γ-HCD. The first case is an 88-year-old woman with γ-HCD. The second case is an 81-year-old woman who developed γ-HCD during treatment for Waldenstrom's macroglobulinemia. In the second patient, histopathology identified a separate clone responsible for the secretion of the gamma heavy chain. Studies on the clonal evolution of the disease may provide insight into therapeutic implications and the genomic complexity of the disease.Gamma heavy chain disease ( The disease is defined as a neoplasm of lymphocytes, plasmacytoid lymphocytes, and plasma cells characterised by the production of an abnormally truncated gamma heavy chain protein that lacks associated light chains. Since Franklin first reported the case, fewer than 200 cases have been described in the literature. A wide variety of disorders have been associated with γ-HCD. In some cases, γ-HCD occurs with other lymphoid neoplasms or with a history of autoimmune disorder. γ-HCD shows a wide clinical spectrum ranging from completely asymptomatic to progressively malignant forms.Gamma heavy chain disease  that in addition harbor somatic point mutations. One possibility is that several types of oncogene translocations happen as a by-product of somatic hypermutation  repe (MGUS) .γ-HCD in serum. γ-Heavy chain was identified in the urine of both patients, suggesting that the lower-molecular weight of the truncated heavy chain allowed renal clearance. In a modification of the immunofixation method, Gulli et al. [In our patients, two methods, capillary zone electrophoresis and gel electrophoresis, identified a gamma heavy chain with no corresponding light chain. Results from this report and previous reports  suggest i et al.  preincubThe intact IgGkappa/IgGlambda (HevyLite) measurement confirmed the presence of gamma heavy chain in the absence of associated kappa or lambda light chains. In a previous study, HevyLite assays showed that in 10/15 patients, substantial portions of IgG were not quantitated by antibodies to intact IgGkappa and IgGlambda immunoglobulins .γ-HCD [Serum IgG subtypes showed an increase in IgG1 subclass in both patients. In a previous report, serum IgG measured by nephelometry on the Beckman Coulter Immage analyser was below the reference range in a patient with γ-HCD . Total Iγ-HCD . The disγ-HCD. Capillary electrophoresis with immunotyping is able to characterise free heavy chains in serum, demonstrating the absence of intact IgG immunoglobulins by the HevyLite assay and the absence of serum-free light chains by serum-free light analysis are further valuable tools for diagnosis. Serum/urine electrophoresis and immunofixation are analytical tools in the workup of γ-HCD patients.There have been several advances in the diagnosis of multiple myeloma. New techniques may further enhance the diagnosis of α), γ, and mu (µ) heavy chain diseases have been described. α-HCD, the most common of heavy chain diseases, appears to be a condition affecting primarily the secretory IgA system and mainly the digestive tract. α-HCD may present as a continuous sequence of events from an apparently reversible hyperplastic process to an overt neoplastic proliferation [µ-HCD is the least common of the heavy chain diseases. Patients present with lymphoplasma cell proliferative disorder, and index of suspicion is high in patients with vacuolated plasma cells in the bone marrow [Alpha (feration . µ-HCD ie marrow .γ-HCD is unclear. Bone marrow histology of the first patient with isolated γ-HCD suggested the presence of a clonal plasma cell which selectively secreted γ-heavy chain in the absence of kappa or lambda light chain. Genomic events that underlie the progression of MGUS to MM have been difficult to dissect. Both bone marrow microenvironment and genomic instability play a role in MM pathogenesis. Bone marrow biopsy in the second patient, with WM and γ-HCD, identified two possible malignant clones. It is worth noting that this patient's treatment included R-CVP, cladribine, and velcade. It is plausible that the treatment might have increased the transformation of the γ-HCD clone by increasing the instability of the MM cells. Clonal evolution in MM before and after therapy can follow several patterns. Branching clonal evolution involves one or more subclones that emerge at a later time point. Current therapeutic approaches have shortcomings, and understanding of MM tumorogenesis and progression may have implications for genomically guided precision treatment [The mechanism for the pathogenesis of reatment .γ-HCD. The findings also provide information for practical guidance for the laboratory evaluation of γ-HCD. In the first case, γ-HCD developed as the presenting lymphoid malignancy. Bone marrow biopsy revealed an IgG heavy chain secreting clone. The second case was a composite lymphoid neoplasm of WM and γ-HCD. γ-HCD developed during treatment for WM. Bone marrow biopsy revealed the presence of two neoplastic lymphoid cell lines. The review of the literature suggests further studies are indicated to develop an understanding of the clonal evolution of γ-HCD.In summary, we report two cases of