Atualmente, o vício em heroína é um problema de saúde preocupante, e as informações sobre os efeitos eletrocardiográficos da heroína são limitadas. O objetivo do presente estudo é investigar os efeitos da dependência de heroína em parâmetros eletrocardiográficos. Um total de 136 indivíduos, incluindo 66 indivíduos que fumam heroína como grupo de estudo e 70 indivíduos saudáveis sem dependência de drogas como grupo de controle, foram incluídos no estudo. Indivíduos que injetam heroína foram excluídos. A avaliação eletrocardiográfica (ECG) dos usuários de heroína foi realizada e comparada com as do grupo controle. Além disso, os ECGs pré e pós-tratamento do grupo usuário de heroína foram comparados. Um valor de p<0,05 foi aceito como estatisticamente significativo.versus71,4±11,2; p=0,02) foi maior no grupo usuário de heroína em comparação com o grupo controle. Os intervalos QT , QTc  e o intervalo do pico ao fim da onda T (Tpe)  foram significativamente menores no grupo usuário de heroína. Nenhuma diferença foi observada entre os grupos com respeito às razões Tpe/QT e Tpe/QTc. Na análise de subgrupo do grupo usuário de heroína, os intervalos QT  e QTc  foram significativamente mais curtos no período pré-tratamento. A frequência cardíaca :1135-1141) Com a perda de tolerância, a overdose de heroína pode ser letal após um período de abstinência. As outras complicações da dependência da heroína, como edema pulmonar,3 choque,4 lesão miocárdica, insuficiência renal aguda,5 rabdomiólise6 e leucoencefalopatia,7 são descritas na literatura. Além disso, a heroína tem se mostrado eficaz na modulação vagal e regulação autonômica.8 No entanto, nosso conhecimento sobre os efeitos cardíacos da dependência de heroína é limitado, o que é um problema de saúde pública importante nesse aspecto. Além disso, há alguns estudos na literatura que mostram a relação entre heroína, toxicidade miocárdica e arritmias.10 Portanto, é essencial compreender as alterações no ECG dependentes de heroína. O objetivo do presente estudo é investigar os efeitos da dependência de heroína sobre os parâmetros eletrocardiográficos.A heroína, um depressor do sistema nervoso central (diacetilmorfina), é um opiáceo semissintético. A heroína é um opioide altamente utilizado, e o vício em heroína causa um prejuízo significativo para a sociedade em todo o mundo. A prevalência do uso de heroína aumentou nos últimos anos. A mortalidade entre os usuários de heroína varia entre 1 e 3%, e o método de tratamento mais eficaz para a dependência de heroína é a terapia de reposição de opioides.Após a aprovação do Comitê de Ética, um total de 136 indivíduos foram incluídos no estudo, que incluiu 66 pacientes usuários de heroína via tabagismo e em terapia no Centro de Tratamento e Treinamento para Dependência de Álcool e Drogas, entre 2014 e 2017, como grupo de estudo, além de 70 indivíduos saudáveis sem dependência de drogas que não seja o tabaco como grupo controle. O grupo controle foi selecionado consecutivamente entre os pacientes que visitavam a clínica de cardiologia. A avaliação de ECG daqueles que usam heroína foi realizada e comparada com aqueles do grupo controle. Além disso, foi avaliado o ECG pré e pós-tratamento do grupo usuário de heroína. As características clínicas e demográficas dos pacientes, o estado e a duração da dependência da heroína foram coletados junto aos pacientes e seus prontuários no hospital. Apenas aqueles que fumavam heroína foram incluídos no estudo. Os registros eletrocardiográficos (ECG) dos pacientes foram obtidos com o dispositivo Schiller Cardiovit AT-102 plus, usando a derivação 12 padrão  na primeira admissão no hospital. Somente os pacientes que recebiam heroína em até 12 horas após os registros de ECG obtidos foram incluídos no estudo. As medições do ECG dos intervalos QT e Tpe foram realizadas manualmente por dois cardiologistas especialistas, usando uma lupa para diminuir os erros de medição. As derivações 2 e V5 foram selecionadas para medir os intervalos QT e Tpe, respectivamente. A média dos três batimentos em cada derivação de ECG foi calculada. O intervalo QT é calculado como o intervalo do início do QRS ao fim da onda T. O intervalo Tpe é definido como o intervalo do pico da onda T ao fim da onda T. Os intervalos QTc foram calculados usando a fórmula de Bazett. O hemograma completo (HC) e os exames bioquímicos foram realizados com Beckman Coulter LH-750 e Beckman Coulter L × 20, respectivamente. Os resultados de cada paciente foram registrados. As avaliações ecocardiográficas de todos os pacientes foram feitas na primeira admissão ao hospital. Todos os participantes foram submetidos à avaliação ecocardiográfica 2D e Doppler  e a fração de ejeção do ventrículo esquerdo foi calculada usando as regras de Simpson modificadas. Os indivíduos que usaram heroína por via intravenosa, dependentes de álcool, aqueles com doenças das artérias coronárias, insuficiência cardíaca, distúrbios das válvulas cardíacas, arritmias conhecidas, hipertensão, doenças cardíacas congênitas, diabetes, insuficiência hepática ou renal, doença pulmonar obstrutiva crônica, doenças endócrinas, distúrbios metabólicos ou eletrolíticos, infecções agudas ou crônicas ou pacientes que tomaram medicamentos que podem afetar os intervalos QT e QTc foram excluídos do estudo.Statistical Package for the Social Sciences(SPSS) para Windows 20  e Medcalc 11.4.2 . A conformidade dos dados com a distribuição normal foi testada com o teste de Kolmogorov-Smirnov. As variáveis numéricas normalmente distribuídas foram expressas como média ± desvio padrão. Variáveis categóricas foram expressas em números e porcentagens. Para as comparações entre o grupo usuário de heroína e os grupos de controle, o teste-t não pareado foi usado. Os testes de qui-quadrado e exato de Fisher foram realizados para comparar variáveis categóricas. Para as comparações de ambos os períodos pré-tratamento e pós-tratamento, o teste de McNemar e amostras pareadas de teste t foram usados. Um valor de p <0,05 foi aceito como estatisticamente significativo.A análise estatística foi realizada com os programasA distribuição da população estudada  foi a seguinte: 66  Heroína (+) e 70  Heroína (-). O sexo feminino correspondeu a 8,82% da população estudada. Não houve diferença significativa na distribuição da média de idade entre os grupos. No grupo usuário de heroína, a duração média do uso da heroína foi de cinco anos. Estatisticamente, não há diferenças significativas entre os grupos em relação ao sexo, tabagismo, fração de ejeção do ventrículo esquerdo e doença cerebrovascular. Nenhuma diferença foi determinada nas outras características demográficas e laboratoriais entre os dois grupos .versus379,11±45,23, p<0,01) e QTc  os intervalos foram significativamente mais curtos do que o grupo usuário de heroína (-). O tempo T-pico até o T-final foi significativamente menor no grupo heroína (+), em comparação com o grupo heroína (-) . Nenhuma diferença significativa foi observada entre os dois grupos em termos de razões Tpe/QT e Tpe/QTc , os intervalos QT  e QTc  foram significativamente mais curtos no período pré-tratamento. Nenhuma diferença foi determinada nos outros parâmetros eletrocardiográficos entre os períodos pré e pós-tratamento  é uma medida da dispersão miocárdica da repolarização. Evidências crescentes sugerem que o intervalo Tpe pode predizer a suscetibilidade à arritmia em pacientes com várias doenças cardiovasculares.22 Marjamaa et al.,23 concluíram que o alelo menor da variante comum rs7219669 está associado ao encurtamento do intervalo Tpe em duas populações de estudo independentes, sendo, portanto, um candidato a modular a suscetibilidade à arritmia em nível populacional.23 No presente estudo, o uso de heroína teve um efeito significativo sobre esses parâmetros importantes. Usuários de heroína apresentam redução da modulação vagal cardíaca, e a terapia com metadona aumentou a atividade vagal diretamente em indivíduos que tiveram recidiva recente de uso de heroína.8 Além dos achados inflamatórios aumentados do miocárdio, também acreditamos que essa diminuição na atividade vagal possa ser responsável por alterações no ECG. A maioria dos estudos sobre dependência de heroína e ECG dizem respeito à metadona. A metadona é usada no tratamento da dependência de heroína, e um de seus efeitos conhecidos mais importantes é o prolongamento das durações QT e QTc.25 A metadona é um inibidor do canal iônico cardíaco KCNH226 e causa o prolongamento do intervalo QT de maneira relacionada à dose consumida.26 Por outro lado, pode aumentar a atividade vagal e prolongar a duração do intervalo QT.8 Após o presente estudo, acreditamos que parte dos efeitos da metadona pode estar relacionada à neutralização do efeito da heroína. Nosso estudo demonstrou que o vício em heroína mudou significativamente os intervalos QT, QTc e Tpe, independentemente do efeito dos adulterantes e da metadona. Além de indivíduos dependentes de heroína, a metadona também é conhecida por prolongar o intervalo QT. No entanto, no grupo de usuários de heroína, esse resultado deve ser levado em consideração ao discutir o efeito de prolongamento do intervalo QT da metadona em pessoas dependentes de heroína. Embora o efeito da heroína nos canais de potássio seja desconhecido, o efeito na atividade vagal foi analisado.8 Com esses resultados, embora ela não seja responsável por todos os mecanismos de prolongamento do intervalo QT, acreditamos que a neutralização dos efeitos da heroína contribui significativamente para o prolongamento do QT. Com o presente estudo, não podemos explicar exatamente se o prolongamento QT é efeito direto da metadona ou o resultado da neutralização do efeito da heroína em estudos anteriores. Quando consideramos o assunto um novo ponto de vista, é um tema importante para orientar os demais estudos. Ainda assim, a metadona é um agonista opioide completo, e as mortes por overdose são um grande problema. A buprenorfina, um agonista opioide parcial, tem se tornado uma opção cada vez mais popular na prática clínica em nosso país e em todo o mundo. A buprenorfina é, provavelmente, o agente mais seguro devido à sua ação farmacológica única e foi declarada uma nova opção para o tratamento da dependência de heroína com menor potencial de abuso e baixo risco de overdose.27 Doses terapêuticas de buprenorfina mostraram não ter efeitos sobre a duração de QT e QTc,29 e a buprenorfina em doses comumente utilizadas é uma alternativa adequada à metadona, no que diz respeito ao risco de prolongamento do intervalo QTc.30 Portanto, os indivíduos do presente estudo foram tratados com buprenorfina em vez de metadona. A buprenorfina não teve efeitos significativos nas durações de QT e QTc, o que foi uma das vantagens do presente estudo. Visto isso, observamos os efeitos da heroína no ECG com mais clareza. Quando olhamos para o subgrupo de tratamento, há uma mudança significativa nas durações de QT e QTc devido à interrupção do uso da heroína. Se esses indivíduos fossem tratados com metadona, seria muito difícil entender se esse efeito era devido à heroína ou à metadona. Infelizmente, assim como em outros países, a desvantagem mais importante nesse subgrupo é a proporção de pacientes que podem concluir o tratamento médico para dependência de heroína, insuficiente em nosso estudo.31 A maioria dos pacientes não conseguiu completar o tratamento, e é por isso que o número de indivíduos no subgrupo do estudo diminuiu significativamente. Embora tenha havido uma mudança significativa nos intervalos QT e QTc no grupo pós-tratamento, houve apenas um aumento numérico na duração do Tpe, o qual não atingiu significância estatística. Presumimos que isso seja devido ao número insuficiente de indivíduos que concluíram o tratamento. Porém, estudos com maior número de participantes são necessários para uma decisão definitiva sobre o assunto.Em um dos primeiros estudos sobre os efeitos eletrocardiográficos da dependência de heroína, Glauser et al.,32 Na urina, o metabólito ativo 6-AM pode ser detectado por um período mais longo, possivelmente até 12 horas.33 Portanto, foram incluídos no presente estudo pacientes que usaram heroína apenas nas últimas 12 horas. Por outro lado, quando a heroína é usada por via intravenosa, é administrada em conjunto com substâncias químicas adicionais, denominadas adulterantes , e os potenciais efeitos cardíacos dessas substâncias complicam a avaliação dos efeitos eletrocardiográficos da heroína.34 Desa forma, para investigar os efeitos cardíacos da heroína apenas, excluímos aqueles que usaram heroína por injeção intravenosa. O presente estudo demonstrou que o uso de heroína diminuiu significativamente os intervalos QT, QTc e Tpe independentemente do efeito dos adulterantes.A heroína tem uma meia-vida extremamente curta no sangue (menos de cinco minutos) e é imediatamente convertida no metabólito ativo 6-acetilmorfina (6-AM), que é posteriormente metabolizado em morfina.As principais limitações do presente estudo foram o desenho unicêntrico e o número relativamente menor de indivíduos.O vício em heroína afeta significativamente os intervalos de tempo QT, QTc e Tpe. Os efeitos de arritmia desses parâmetros já são conhecidos. Os parâmetros eletrocardiográficos desses indivíduos merecem mais atenção. Tendo em vista que o conhecimento atual sobre os efeitos do uso de heroína nas funções cardíacas é limitado, o estudo do assunto é imprescindível para sua contribuição para a literatura. Ainda assim, estudos futuros com uma amostra maior são necessários para alcançar consenso e resultados concretos. 2Its main adverse effect is respiratory distress, which can lead to death. With loss of tolerance, heroin overdose can be lethal after a period of abstinence. The other various complications of heroin addiction, such as pulmonary edema,3shock,4myocardial damage, acute renal failure,5rhabdomyolysis,6and leukoencephalopathy7have been described in the literature. Besides that, heroin has proved to be effective on vagal modulation and autonomic regulation.8However, our knowledge of the cardiac effects of heroin addiction is limited, which is an important public health problem of this extent. There are also some studies in the literature showing the relation between heroin, myocardial toxicity, and arrhythmias.10Therefore, understanding heroin dependent ECG changes is essential. The aim of the present study is to investigate effects of heroin addiction on electrocardiographic parameters.Heroin, which is a central nervous system depressant (diacetylmorphine), is a semi-synthetic opiate. Heroin is a highly abused opioid, and heroin addiction incurs a significant detriment to society worldwide. The prevalence of heroin use has increased in recent years. Mortality among heroin users varies between 1 and 3%, and the most effective treatment method of heroin addiction is opioid replacement therapy.After approval by the Ethics Committee, a total of 136 individuals were included in the study, which included 66 patients who use heroin via smoking and undergoing therapy in the Alcohol and Drug Addiction Treatment and Training Center, between 2014 and 2017, as the study group; and 70 healthy individuals with no drug addiction other than smoking as the control group. Control group was selected consecutively from the patients visiting the cardiology clinic. ECG evaluation of those using heroin was performed and compared to those of the control group. In addition, pre-treatment and post-treatment ECG of the heroin group was evaluated. The clinical and demographic characteristics of patients, status and duration of heroin addiction were collected from patients and their files in the hospital. Only those with heroin use via smoking were included in the study. Electrocardiography (ECG) records of patients were obtained with Schiller Cardiovit AT-102 plus, using the standard 12 derivation  at first admission to the hospital. Only the patients on heroin which was taken within 12 hours of the ECG records obtained were included the study. ECG measurements of QT and Tpe intervals were performed manually by two expert cardiologists, using a magnifying glass to decrease measurement errors. Leads 2 and V5 were selected for measuring QT and Tpe intervals, respectively. The average of the three beats in each ECG leads was calculated. QT interval is calculated as the interval from the beginning of the QRS to the end of the T wave. Tpe interval is defined as the interval from the peak of T wave to the end of T wave. QTc intervals were calculated using the Bazett formula. Complete blood count (CBC) and biochemical tests were performed using a Beckman Coulter LH-750 and a Beckman Coulter L × 20, respectively; the results of each patient were recorded. Echocardiographic evaluations of all patients were made at the first admission to the hospital. All participants underwent 2D and Doppler echocardiographic evaluation  and the left ventricular ejection fraction was calculated using modified Simpson rules. Those who used heroin via the intravenous route, alcohol-dependent, those with coronary artery diseases, cardiac failure, cardiac valve disorders, known arrhythmias, hypertension, congenital cardiac diseases, diabetes, hepatic or renal failure, chronic obstructive pulmonary disease, endocrine diseases, metabolic or electrolyte disorders, acute or chronic infections, or patients that took medications which can affect QT and QTc intervals were excluded from the study.p-value of <0.05 was accepted as statistically significant.Statistical analysis was performed with Statistical Package for Social Sciences (SPSS) for Windows 20  and Medcalc 11.4.2  programs. Data compliance with the normal distribution was tested using the Kolmogorov-Smirnov test. The normally distributed numeric variables were expressed as mean ± standard deviation. Categorical variables were expressed as numbers and percentages. For the comparisons between the heroin and the control groups, unpaired student’s t test was used. Chi-square and Fisher’s exact tests were carried out to compare categorical variables. For the comparisons of both the pre-treatment and post-treatment periods, McNemar’s test and t-test paired samples were used. AThe distribution of the study population  was as following: 66 (48.52%) Heroin (+) and 70 (51.47%) Heroin (-). Females corresponded to 8.82% of the study population. There was no significant difference in the distribution of mean age between groups. In the heroin group, the mean duration of heroin use was five years. Statistically, there are not significant differences between the groups, related to the gender, smoking, left ventricular ejection fraction, and cerebrovascular disease. No difference was determined in the other demographic and laboratory characteristics between both groups .versus379.11±45.23, p<0.01) and QTc  intervals were significantly shorter than heroin (-) group. T-peak to T-end time was significantly shorter in the heroin (+) group, compared to the heroin (-) group . No significant difference was observed between both groups in terms of Tpe/QT and Tpe/QTc ratios  group, QT  and QTc  intervals were significantly shorter in the pre-treatment period. No difference was determined in the other electrocardiographic parameters between both the pre-treatment and posttreatment periods  is a measure of myocardial dispersion of repolarization. Increasing evidence suggests that Tpe interval may predict arrhythmia susceptibility in patients with various cardiovascular diseases.22Marjamaa et al.23concluded that the minor allele of common variant rs7219669 is associated to Tpe interval shortening in two independent study populations, thus being a candidate to modulate arrhythmia susceptibility at population level.23In the present study, heroin use was found to have a significant effect on these important parameters. Heroin users show reduced cardiac vagal modulation, and methadone therapy raised vagal activity directly in individuals who had recently relapsed into heroin use.8In addition to the increased myocardium inflammatory findings, we also believe that this decrease in vagal activity may be responsible for ECG changes. Most studies on heroin addiction and ECG are about methadone. Methadone is used in the treatment of heroin addiction, and one of its most important known effects is the prolongation of QT and QTc durations.25Methadone is an inhibitor of the cardiac ion channel KCNH226 and causes QT prolongation in a dose-dependent manner.26On the other hand, it can increase vagal activity and prolong QT duration.8After the present study, we believe that a part of methadone’s effects may be related to the neutralization of the heroin effect. Our study demonstrated that heroin addiction significantly changed QT, QTc, and Tpe intervals independent from the effect of adulterants and methadone. In addition to heroin dependent individuals, methadone is also known to prolong QT. However, in heroin user group, this result should be taken into consideration when discussing the QT prolonging effect of methadone in those who depend on heroin. Although the effect of heroin on potassium channels is unknown, the effect on vagal activity has been shown.8With these results, even though it is not responsible for all QT prolongation mechanism, we think that neutralizing heroin effects significantly contributes to QT prolongation. With the present study, we cannot explain exactly whether QT prolongation is the direct effect of methadone or the result of the neutralization of the heroin effect in previous studies. In terms of bringing a new point of view, it is an important subject in guiding the other studies. On the other hand, methadone is a complete opioid agonist, and overdose deaths are a major problem. Buprenorphine, a partial opioid agonist, has become an increasingly popular option in clinical practice in our country and all over the world. Buprenorphine is probably the safer agent due to its unique pharmacological action and has been declared a new dawn for treating heroin dependence with less abuse potential and low overdose risk.27Therapeutic doses of buprenorphine were shown to have no effects on QT and QTc duration,29and buprenorphine in commonly used doses is a suitable alternative to methadone, with regard to the risk of QTc prolongation.30Therefore, individuals from the present study were treated with buprenorphine instead of methadone. Buprenorphine had no significant effects on QT and QTc durations, which was one of our study advantages. Seen that, we observed the effects of heroin on ECG more clearly. When we look at the treatment subgroup, there is a significant change in QT and QTc durations because of heroin discontinuation. If these individuals were treated with methadone, understanding whether this effect was due to heroin or methadone would be very difficult. Unfortunately, like in other countries, the most important disadvantage in this subgroup is the proportion of patients who can complete medical heroin-addiction treatment, which is insufficient in our study.31Most patients could not complete the treatment, and that is why the number of individuals in the study subgroup decreased significantly. Although there was a significant change in QT and QTc in the post-treatment group, there was only a numerical increase in Tpe duration, and this increase did not reach statistical significance. We assume this is due to insufficient number of individuals who have completed the treatment. However, studies with a larger number of participants are needed to make a definite decision on this issue.In one of the first studies on the electrocardiographic effects of heroin dependence, Glauser et al.32In urine, active metabolite 6-AM can be detected for a longer period, possibly up to 12 hours.33Therefore, patients who had used heroin only in the last 12 hours were included in the present study. On the other hand, when heroin is used via the intravenous route, it is administered together with additional chemical substances, named as adulterants , and the potential cardiac effects of these substances complicate the evaluation of heroin electrocardiographic effects.34Thus, in order to investigate the cardiac effects of only heroin, we excluded those who used heroin by intravenous injection. The present study demonstrated that heroin use significantly decreased the QT, QTc and Tpe intervals independent from the effect of adulterants.Heroin has an extremely short half-life in blood (less than five minutes), and is immediately converted to the active metabolite 6-acetylmorphine (6-AM), which is further metabolized to morphine.Our study had some limitations: the single-center design and the relatively lower number of individuals are the most important ones.Heroin use is a serious public health issue, which significantly affects the QT, QTc, and Tpe time intervals. The arrhythmia effects of these parameters are well known, and we should be more alert to the electrocardiographic parameters of these individuals. Given that the present knowledge on the effects of heroin use on cardiac functions is limited, studying the matter is imperative for its contribution to the literature. Nonetheless, further studies with larger sample sizes are needed for a consensus and clear results.


Immunoglobulin G4-related disease (IgG4-RD) of the biliary tract and pancreas is a fibroinflammatory disease of unknown origin with striking male predominance. We aimed to investigate whether blue-collar work and occupational contaminant exposure are risk factors for IgG4-RD of the biliary tract and pancreas.ALOHA and DOM were utilized to assess the years individuals were exposed to compounds. The disease control cohort consisted of patients from 6 equally sized groups. Conditional logistic regression was used to assess effects of blue-collar work and exposure to occupational contaminants on developing IgG4-RD of the biliary tract and pancreas.We performed an age-/sex-matched case-control study in the largest academic medical centers of the Netherlands. Occupational history was surveyed by questionnaires. The International Standard Classification of Occupations (ISCO88) was used to classify jobs. Job exposure matrices p = 0.001). Individuals who at least once performed blue-collar work (>1 year), had higher odds of developing IgG4-RD than individuals that only performed white-collar work . Being ever exposed (>1 year) to industrial ALOHA  and DOM compounds (e.g. asbestos) resulted in higher odds of IgG4-RD .Overall, 101 patients with IgG4-RD of the biliary tract and pancreas were matched 1:3 to 303 controls. Patients with IgG4-RD had a lower level of education  causes tumor-like lesions and typically affects middle-aged to elderly men. The background and cause of this disease remain relatively unknown. In this study, we identified blue-collar work as a risk factor for developing IgG4-RD of the biliary tract and pancreas, which may explain the striking male predominance among patients. Furthermore, these results suggest that toxic exposure to occupational contaminants may drive autoimmunity in IgG4-RD of the biliary tract and pancreas.  •Blue-collar work in 68% of people with biliary/pancreatic IgG4-RD vs. 39% of disease controls•Toxic exposure to selected occupational contaminants may drive autoimmunity in IgG4-RD•Blue-collar work may explain the striking male predominance in biliary/pancreatic IgG4-RD Auto-reactive B cells are thought to be key players in the disease process, given the fact that selective depletion of CD20+ B cells with rituximab completely reverses signs and symptoms of IgG4-RD.We previously reported that blue-collar jobs were highly prevalent among an early cohort of our patients with IgG4-RD of the biliary tract and pancreas.,We performed an age- and sex-matched case-control study in the 2 largest academic medical centers and national reference centers for hepatopancreatobiliary diseases in the Netherlands: the Amsterdam University Medical Centers  and the Erasmus Medical Center (EMC) in Rotterdam. Participants were included between June 2017 and December 2018. Cases comprised patients with IRC and/or AIP meeting the widely used HISORt diagnostic criteria.We developed a questionnaire with the main objective to collect participants’ complete job history. For every job that lasted more than 1 year, information was asked about job title, dates of employment, average working hours per week, sector of occupation and company name. Furthermore, questions on level of education and lifestyle  were posed. The questionnaire also included questions on allergies, malignancies, autoimmune disease and family history. The questionnaire was available in 2 versions, a paper questionnaire and a web-based survey supported by Castor Electronic Data Capture.Paper surveys were digitalized in Castor EDC, using the unique study ID as identifier and blinded for case-control status.The International Standard Classification of Occupations (ISCO88)ALOHADOM,ALOHA assesses exposure to biological dust, mineral dust, gas fumes, VDGF , all pesticides, herbicides, insecticides, fungicides, aromatic solvents, chlorinated solvents, other solvents, and metals. DOM assesses exposure to asbestos, chromium, diesel motor exhaust, nickel, polycyclic aromatic hydrocarbons, silica, animals, biological dust, and endotoxin. ALOHA uses the ISCO88 codes whilst the DOM uses the ISCO68 codes. To calculate the duration of each job employment and thereby the duration of the occupational exposure, both the number of years worked and the hours worked per week were taken into account, yielding a full-time equivalent. Exposures were censored at the time of diagnosis of the patient with IgG4-RD, for both cases and their corresponding controls. In other words, only jobs reported before the date of diagnosis of the cases, and their matched controls, were included in the analyses. To assess exposure to the compounds of interest over time, we calculated the number of years exposed per contaminant for each individual, regardless of the intensity of the exposure.Occupational exposures to a variety of compounds were assigned using job exposure matrices (JEMs). Applied JEMs are semi-quantitative and assign zero, low or high exposure to job codes. For this study, we utilized the JEMs Prior to the study, a sample size calculation for 2-sample chi-squared test was performed in nQuery based on the pilot study where 74% of all patients with IgG4-RD had a history of blue-collar work.Case to control matching was performed using the MatchIt packageT test and chi-square tests were used to test for group differences for parametric data, whereas Wilcoxon signed-rank test was used for non-parametric data. Risk was expressed as odds ratio (OR) and 95% CI. Conditional logistic regression was used to assess the effect of blue-collar work and being exposed to occupational contaminants on developing IgG4-RD. In a post hoc analysis, comparing cases and controls among blue-collar workers only, we tested whether duration of blue-collar work and duration of exposures to ALOHA and DOM compounds were related to the risk of IgG4-RD. Furthermore, we analyzed exposures over time to the compounds of interest among cases and controls performing blue-collar work.Descriptive data are expressed as the mean and standard deviation for continuous variables, and frequency (n) and percentage for categorical variables. The main blue-collar work analysis was stratified by sex and hospital. To correct for potential confounding that may have been introduced by the matching process, we performed a sensitivity analysis by correcting the results for the matching variables (age and sex). Additionally, 1 disease control group was omitted at a time to assess whether it unduly influenced results.This study was considered exempt from further review (W16_221) by the Institutional Review Board of the Academic Medical Center, University of Amsterdam and was performed according to the principles of the declaration of Helsinki. All authors had access to the study data and reviewed and approved the final manuscript.Before the start of the study, the questionnaire was piloted on a small group of patients to verify that questions were clearly formulated and resulted in useful answers. Furthermore, patients were asked to give feedback on the time and effort to complete the questionnaire, which was not seen as a major burden. Patients were not involved in the recruitment and conduct of the study.a priori and optimal pairs regarding age and sex were formed, resulting in improved standardized mean differences per matching variable  in 32.0% vs. 18.9%, and high  in 21.0% vs. 40.1% of cases and matched controls, respectively. Numbers of smoking pack years and regular alcohol consumption, defined as ≥1 alcoholic drinks per week, were not different between cases and controls. The geographical distribution of cases and controls showed clear overlap .Of cases with IgG4-RD, 68% (n = 69) ever performed a blue-collar job, compared to 39% of controls n = 117, , corresperistics . There wTaken together, a history of blue-collar work is strongly associated with IgG4-RD of the biliary tract and pancreas, whereas duration of blue-collar work might be related to this risk of developing IgG4-RD.ALOHA and DOM scores, we investigated whether occupational exposure to industrial compounds differed between cases and controls. In cases, 78% ever had a relevant exposure for at least 1 year to any of the ALOHA compounds and 73% to any of the DOM compounds, compared to 62% and 48% of the controls, respectively. Conditional logistic regression indicated that individuals who were exposed to any of the ALOHA and DOM compounds, at least once in their working life, had 2.14-  and 2.95-  fold higher odds of developing IgG4-related disease compared to individuals who were never exposed to industrial compounds in their professional life. Furthermore, cases with blue-collar work  showed a trend towards longer exposure to ALOHA compounds and were exposed to DOM compounds for significantly longer during their career compared to control blue-collar workers  and 14% , respectively. Thus, occupational exposure to industrial compounds might be driving the increased risk of developing IgG4-RD among blue-collar workers, and the duration of exposure is a significant factor herein.Using the ALOHA compounds , and the DOM compound asbestos. In summary, a number of industrial compounds (dusts and fumes), mostly taken up through inhalation, could be associated with the attributable risk of blue-collar work on the development of IgG4-RD of the biliary tract and pancreas.Finally, we investigated whether it was possible to pinpoint specific compounds that could lie beneath this increase in risk of developing IgG4-RD. To this end, we compared exposure to several compounds over time among cases and controls in the blue-collar work group. We found that exposure to several compounds led to an increased OR for developing IgG4-RD , includiIn this occupational case-control study in 404 patients, we showed that blue-collar work is a significant risk factor for developing IgG4-RD of the biliary tract and pancreas, putatively driven by occupational exposure to specific industrial compounds.p <0.001). Among blue-collar workers, the duration of blue-collar work tended to increase the risk of IgG4-RD and the duration of exposure to both ALOHA and DOM compounds clearly increased the risk of IgG4-RD. We found different exposures over time to mineral dusts, VDGF and asbestos between patients with IgG4-RD and disease controls even among blue-collar workers, underlining a putative driving role for occupational exposures in the attributable risk of blue-collar work for developing IgG4-RD.Overall, a history of blue-collar work was reported in 68% of patients with IgG4-RD of the biliary tract and pancreas, while only 39% of matched disease controls ever performed blue-collar work for more than 1 year. In our case-control study, the odds of developing IgG4-RD among blue-collar workers were 3.66-fold higher than among white-collar workers  and Rotterdam EMC (63%). However, blue-collar work was more common in the EMC (52%) than in the AUMC (33%) among controls. This difference can be explained by differences in the composition of the population. In general terms, Rotterdam houses the largest continental European harbor with a large industrial zone, attracting a working population of variable skills, whereas Amsterdam as capital of the Netherlands (Den Hague being the seat of the government) is mostly a knowledge, finance and business center drawing highly educated professionals. National statistics performed by the Central Bureau of Statistics (CBS) of the Netherlands in 2011 support this distinction, showing that among the adult population in Rotterdam 42% had a low education level, 35% an intermediate level and 23% a high level, compared to 29%, 31% and 40%, respectively, in Amsterdam.We aimed to reduce several biases occupational epidemiology studies are prone to.post hoc analysis among cases and controls with blue-collar work only. Although matching was broken inevitably, both groups were comparable in terms of age and sex. We found that exposure to mineral dusts, VDGF and asbestos led to increased ORs of developing IgG4-RD. Notably, asbestos was found to be a risk factor for idiopathic retroperitoneal fibrosis, which is nowadays known as a manifestation of IgG4-RD, in 2 large case-control studies.,mineral dusts and VDGF have not been related to IgG4-RD yet. How occupational exposure to these industrial compounds contributes to developing IgG4-RD remains subject for further research. The delay between occupational exposure and the development of disease/symptoms in IgG4-RD suggests that effects outlast exposure. Both biochemical and immunological pathogenetic processes are most probably involved.,,,,In this study, we also performed a Collectively, we report that blue-collar work is a risk factor for developing IgG4-RD of the biliary tract and pancreas, possibly driven by long-term occupational exposure to a variety of industrial compounds. These findings challenge the current paradigm of IgG4-RD, possibly explaining the striking male predominance among patients with IgG4-RD of the biliary tract and pancreas. This study opens up new avenues for further research on the pathophysiology of this disease. Future collaborative research efforts should try to further unravel the possible relationship between long-term toxic exposure and autoimmunity on the molecular level in IgG4-RD.10.13039/501100008985German Morbus Crohn & Ulcerative colitis patient organization (DCCV)’, section PSC (to UB), and the Netherlands Gastroenterology & Hepatology Foundation (MLDS) (to UB).The study was supported by grants from the ‘L. Hubers: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Project administration, Visualization, Writing – original draft. A. Schuurman: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Project administration, Visualization, Writing – original draft. J. Buijs: Data curation, Investigation, Project administration, Writing – review & editing. N. Mostafavi: Formal Analysis (Statistician), Methodology, Validation, Writing – review & editing. M. Bruno: Resources, Writing – review & editing. R. Vermeulen: Methodology (Risk assessment), Software, Writing – review & editing. A. Huss: Conceptualization, Methodology (Risk assessment), Software, Writing – review & editing. H. van Buuren: Resources, Writing – review & editing. U. Beuers: Conceptualization, Acquisition of the financial support for the project leading to this publication, Methodology, Project administration, Resources, Validation, Supervision, Writing – review & editing.The anonymized data that support the findings of this study are available on reasonable request from the corresponding author.The authors have declared that no conflict of interest exists related to this work.Please refer to the accompanying ICMJE disclosure forms for further details.


ABSTRACT IMPACT: By identifying clear gaps in our knowledge of racial and ethnic disparities in antibiotic-resistant infections, this research is informing the design of (a) community-based interventions and (b) patient-centered research studies that we are currently leading to address these disparities and improve human health. OBJECTIVES/GOALS: Antibiotic resistance (AR) is widely considered to be the next global pandemic. As with COVID-19, the potential for AR to disproportionately impact racial/ethnic minorities is a major concern. Our goal was to identify gaps in knowledge of AR disparities in order to inform the types of interventions that might be most appropriate to address this. METHODS/STUDY POPULATION: We reviewed the literature to examine evidence of racial/ethnic disparities in (a) infections with the most concerning drug-resistant bacteria in the United States, and (b) underlying social-economic or behavioral factors that could contribute to such infections. We searched PubMed and Google Scholar to identify studies published in English between August 1973 - August 2020. We used keywords that included: antibiotic resistance, antibiotic-resistant infections, antibiotic-seeking behavior, prescription/non-prescription antibiotic use, antibiotic education, or health literacy AND race, ethnicity, or socioeconomic status. We screened all abstracts to identify US-based studies that assessed (a) or (b) above. RESULTS/ANTICIPATED RESULTS: We identified 11 studies investigating racial/ethnic disparities for 5 of the 17 drug-resistant bacteria flagged in the CDC’s 2019 Antibiotic Resistance Threats Report. Black, Hispanic, and lower-income individuals were found to be at higher risk of some community-acquired antibiotic-resistant infections. We identified multiple factors that may contribute to disparities in AR-related morbidity and mortality, including reported differences in antibiotic use, higher likelihood of living in crowded/multigenerational homes, more frequent employment in potentially high exposure settings (e.g. slaughterhouses), lower health literacy, and more frequent underlying comorbidities, which increases risks for hospitalization and subsequent acquisition of drug-resistant infections. DISCUSSION/SIGNIFICANCE OF FINDINGS: Given the small number of studies on this topic, educational interventions that aim to raise awareness of this issue must target not only the public but also researchers. Community-based interventions that seek to address disparities in ‘antibiotic resistance literacy’ among minority and underserved groups could be particularly impactful.


This paper aims to analyse the consumption of antibiotics in the Slovak health care system from 2011 to 2020. The data source on the consumption of antibiotics is sales data from SUKL and NCZI. The study employed the ATC/DDD Index and focused on the consumption of antibiotics in the primary care sector. Total antibiotic consumption decreased from 19.21 DID in 2011 to 13.16 DID in 2020. Consumption of beta-lactamase-sensitive penicillins, expressed as a percentage of the total consumption of antibiotics, decreased from 8.4% in 2011 to 4.2% in 2020. Consumption of the combination of penicillins, including beta-lactamase inhibitor, expressed as a percentage of the total consumption of antibiotics, increased from 16.2% in 2011 to 17.9% in 2020. Consumption of third- and fourth-generation cephalosporins, expressed as the percentage of the total consumption of antibiotics, increased from 2.0% in 2011 to 4.6% in 2020. Consumption of fluoroquinolones, expressed as the percentage of the total consumption of antibiotics, decreased from 10.7% in 2011 to 8.6% in 2020. Overall, antibiotic consumption significantly changed in Slovakia from 2011 to 2020. The ratio of the consumption of broad-spectrum to the consumption of narrow-spectrum penicillins, cephalosporins and macrolides decreased from 14.98 in 2011 to 13.38 in 2020. The basic concept of the Slovak health care system includes mandatory public insurance and a general benefits package for citizens .There is a pluralistic system of health insurance companies, with three health insurance companies operating: the state-owned Všeobecná zdravotná poisťovňa  and the private companies Dôvera (Trust) and Union, which covered 56.25%, 31.98% and 11.76% of the Slovak population, respectively, in 2021.Act No. 363/2011 sets the regulations for pharmacoeconomic evaluation and external price referencing in the reimbursement of antibiotics for the primary care sector from health insurance funds [The “maximum retail price” of antibacterials for systemic use may not rise above the average of the three lowest prices of the same antibiotic across the European Union. With the aim of facilitating price erosion after the patent expiry of original antibiotics, the 2018 legislation came out with mandatory discounts for generic antibiotics. According to the legislation, the first off-patent antibiotic entering the Slovak market must achieve a 45% initial price reduction compared to the original antibiotic, the second off-patent antibiotic must achieve an additional 10% price reduction compared to the first and the third off-patent antibiotic must achieve an additional 5% price reduction compared to the second .Market access standards for second and third off-patent antibiotics have developed into rigorous criteria, which could limit some manufacturers from launching generic antibiotics in Slovakia.The three-step approach to reducing prices has also led to new packaging forms of generic antibiotics. For example, when the package size was changed from 14 to 28 tablets, it was also considered a new generic antibiotic, which complicated the launch of off-patent antibiotics with new package sizes . Consumption of antibiotics was considerably higher in Slovakia than in several other EU Member States ,5, with The European Centre for Disease Prevention and Control (ECDC) estimated that more than 33,000 patients die each year in the EU/EEA as a consequence of infection with antimicrobial-resistant bacteria . The OrgHaving valid information on antibiotic consumption is crucial to fighting public health issues related to antimicrobial resistance .A very limited number of new antimicrobial medicines are available; therefore, infection prevention, control measures and the prudent use of existing antimicrobial medicines remain vitally important in the effort to prevent and control antimicrobial resistance ,12. SincThe revision of current pharmaceutical legislation is recommended along with non-legislative measures, improvements in existing regulatory approaches to solve antimicrobial resistance, the promotion of the reasonable use of antimicrobial medicines and the dissemination of information to health care professionals and patients .This paper aims to analyse the consumption of antibacterials for systemic use in the Slovak health care system from 2011 to 2020 and to calculate related quality indicators for outpatient antibiotic use. The high prescription of antibiotics is connected to medical risks and needless financial expenditures; therefore, valid data about the consumption of antibiotics, as discussed in this paper, are needed for appropriate decision making concerning antibiotic policy in Slovakia. We found that the consumption of antibacterials for systemic use in the primary care sector fell by31.29% between 2011 and 2020. In Slovakia, a 47.17% drop can be seen in the consumption of beta-lactam and penicillin antibacterials from 2011 to 2020. It is important to mention the 84.82% increase in the consumption of other antibacterial beta-lactam antibiotics from 2011 to 2018, but there was also a 46.86% decrease for this group of antibiotics from 2018 to 2020. From 2011 to 2020, macrolide, lincosamide and streptogramin use significantly fell by 41.70%. The consumption of quinolone antibacterials declined 47.22% between 2011 and 2020. From 2011 to 2020, tetracycline consumption rose only 16.08% and there was only a 10.81% rise in the consumption of sulphonamides and trimethoprim. In Slovakia, the 133.33% increase in other antibacterial usage from 2011 to 2020 can be seen. An overview of antibiotic consumption on the level of the Anatomical Therapeutic Chemical classification system (ATC) in the primary care sector, expressed in DDD per 1000 inhabitants per day, for Slovakia from 2011 to 2020, is available in The composition of beta-lactam antibacterials, penicillins; other beta-lactam antibacterials; and macrolides, lincosamides and streptogramins was analysed on the level of ATC-4 groups in the community sector.The overview presented in The composition of beta-lactam antibacterial penicillins in the community (3.11 DID in 2011 compared to 2.36 DID in 2020) for combinations of penicillins, including beta-lactamase inhibitors; (1.61 DID in 2011 compared to 0.55 DID in 2020) for beta-lactamase-sensitive penicillins; and (1.47 DID in 2011 compared to 0.36 DID in 2020) for the rest of the beta-lactam antibacterial penicillins can be seen.The overview presented in The composition of other beta-lactam antibacterials in the community (2.78 DID in 2011 compared to 2.58 DID in 2020) for second-generation cephalosporins; (0.39 DID in 2011 compared to 0.61 DID in 2020) for third-generation cephalosporins; and (0.18 DID in 2011 compared to 0.12 DID in 2020) for other beta-lactam antibacterials can be seen.The overview presented in The composition of macrolides, lincosamides and streptogramins (MLS) in the community (5.30 DID in 2011 compared to 2.81 DID in 2020) for macrolides and (0.36 DID in 2011 compared 0.49 DID in 2020) for lincosamides and streptogramins can be seen.The quality indicators for outpatient antibiotic use, to better describe the Slovak antibiotic prescribing patterns, are calculated in Consumption of antibacterials for systemic use, expressed in DDD per 1000 inhabitants per day, decreased from 19.21 DID in 2011 to 13.17 DID in 2020. Consumption of penicillins, expressed in DDD per 1000 inhabitants per day, decreased from 6.19 DID in 2011 to 3.27 DID in 2020. Consumption of cephalosporins, expressed in DDD per 1000 inhabitants per day, decreased from 3.36 DID in 2011 to 3.30 DID in 2020. Consumption of macrolides, lincosamides and streptogramins, expressed in DDD per 1000 inhabitants per day, decreased from 5.66 DID in 2011 to 3.30 DID in 2020. Consumption of quinolones, expressed in DDD per 1000 inhabitants per day, decreased from 2.16 DID in 2011 to 1.14 DID in 2020.Consumption of beta-lactamase-sensitive penicillins, expressed as a percentage of the total consumption of antibacterials for systemic use, decreased from 8.4% in 2011 to 4.2% in 2020. Consumption of the combination of penicillins, including beta-lactamase inhibitor, expressed as a percentage of the total consumption of antibacterials for systemic use, increased from 16.2% in 2011 to 17.9% in 2020. Consumption of third- and fourth-generation cephalosporins, expressed as the percentage of the total consumption of antibacterials for systemic use, increased from 2.0% in 2011 to 4.6% in 2020. Consumption of fluoroquinolones, expressed as the percentage of the total consumption of antibacterials for systemic use, decreased from 10.7% in 2011 to 8.6% in 2020.The ratio of the consumption of broad-spectrum to the consumption of narrow-spectrum penicillins, cephalosporins and macrolides decreased from 14.98 in 2011 to 13.38 in 2020.High antibiotic consumption and the misuse of antibacterials for systemic use have led to antimicrobial resistance, resulting in a failure of pharmacotherapy, increased financial expenditures for the health care system and an elevated risk of mortality . AddressValid data are required to analyse patterns of AMC and to monitor the issue of AMR. Quantitative figures need be considered a starting point for further analysis to better recognize the use of antibiotics in clinical practice . We demonstrated that the consumption of antibacterials for systemic use in the primary care sector fell by 31.29% in Slovakia between 2011 and 2020. An international study by Bruyndonckx et al.  describeIn Slovakia, a 47.17% drop can be seen in the consumption of beta-lactam and penicillin antibacterials (J01C) from 2011 to 2020, even though they were the most consumed antibiotic group in the country during the six-year period ending 2017. The study by Bruyndonckx et al. [A study by Versporten et al.  that focFrom 2011 to 2020, macrolide, lincosamide and streptogramin (J01F) use signifi-cantly fell by 41.70% in Slovakia. This is consistent with the outcome of an international study by Adriaenssens et al.  describiIn Slovakia, the consumption of quinolone antibacterials (J01M) declined 47.22% between 2011 and 2020, while an international study by Adriaenssens et al.  of serioFrom 2011 to 2020, tetracycline consumption rose only 16.08% for (J01A) and only 10.81% for sulphonamides and trimethoprim (J01E). The trends are in line with the study published by Versporten et al. [This can be seen in the 133.33% increase in other antibacterial usage (J01X) in Slovakia from 2011 to 2020. According to Versporten et al. [There are different categories of interventions that may support general practitioners (GPs) in reducing the prescribing of antibiotics, namely regulatory, externally administered interventions that GPs can implement individually, supporting GPs’ access to near-patient diagnostic testing . From a regulatory point of view, the pricing and reimbursement process, including copayment settings, can be considered as the crucial factor in regard to the impact on antibiotic consumption during the analysed time period in Slovakia. An internal reference pricing system for antibiotics can be seen in Slovakia. Slovakia has implemented a system in which a maximum price is set for a standard daily dose of an antibiotic in each internal reference group (based on ATC) concerning the same active substance and which is administered in the same galenic form. A standard daily dose of a medicine with the particular active substance is reimbursed only at the level of the least expensive antibiotic in the given internal reference group. The difference between the price of antibiotics and the reimbursement levels based on the least expensive antibiotics in a given internal reference group represents the copayment of patients. However, there is also a mandatory copayment for the least expensive antibiotic in a given internal reference group prescribed by general practitioners at the level of 25%. The regulatory impact of copayments on consumption is used to prevent the wasting of antibiotics by patients , which was often seen in the past when all antibiotics were fully covered by the health insurance fund. On the other hand, the copayment does not represent a significant financial burden for patients, which could limit their access to antibiotics. It is important to emphasise that specific groups of antibiotics with restrictive access for prescriptions through physician specialties are fully covered by health insurance funds.The project Slovak Medical Dialog , which was established with international cooperation between the General Health Insurance Fund, the Brussels Gentile Insurance Companies Association, the Municipality of Bratislava, the Pharmaceutical Faculty and the Civil Society Health City, was a very important step from an externally administered point of view and has influenced the consumption of antibiotics. The target was to initiate communication between the health insurance fund and general practitioners with the aim of supporting effective antibiotic prescriptions and improving mutual cooperation. The S-MedDial project helped to increase the quality of antibiotic prescriptions through the education of prescribing practitioners. The study of the prescription habits of paediatricians was based on (a) prospective data collection according to treatment registers during a one-month period, and (b) retrospective analysis of health insurance data. Particular regions were studied based on antibiotic resistance trends in cooperation with microbiologists from selected regions ,23.From the point of view of supporting GPs’ access to patient diagnostic testing, a study published in 2012 by the Union Health Insurance Fund had a significant impact. The study on the impact of the availability of quick C-reactive protein testing, as a service furnished by medical care providers, and antibiotic treatments involved a population sample of 365,690 health insurance clients from Slovakia over a period of 22 months . Based oIn terms of interventions that GPs can implement individually, a significant impact can be seen on projects in which the Trust health insurance company agreed with general practitioners for children and adolescents on an evaluation parameter concerning the consumption of antibiotics in order to prevent their excessive use. This evaluation parameter is also supported financially by the health insurance company.Patient pressure. It is the patients who often demand that doctors prescribe antibiotics, even when there is no reason to do so. A significant number of patients who request a prescription for antibiotics will also receive them.Emergency rooms. Patients often go to emergency rooms for a prescription for antibiotics, even if their GP refuses to prescribe them.Effectiveness of prescriptions and availability of doctors in the region.Economic power of the region. The number of doctor visits is also related to the strength of the economy or the unemployment rate in the region. In the case of economically more developed regions, patients with influenza or other common diseases seek out doctors less often  than in regions with higher unemployment .Through interviews between doctors and representatives of the Trust health insurance company, the following causes of regional differences in antibiotic consumption in outpatient practice were defined.It is important to emphasise that Slovakia used rapid antigen tests to target the whole population in an effort to identify SARS-CoV-2 infections in 2020 . A totalExperts have challenged the use of antigen tests, which the WHO declared unsuitable for mass testing unless used alongside PCR tests. There was the possibility of many false-positive and false-negative results, which may have allowed infected people to spread the disease while keeping others under meaningless quarantine. Infectious disease scientists warned that the plan could put people’s health at risk and erode public trust towards testing and all other pandemic containment approaches .Quality indicators for outpatient antibiotic use provide valuable data on the appropriateness of antibiotic consumption . The study by Adriaenssens et al.  describiAlthough the ratio in the consumption of broad-spectrum (J01(CR + DC + DD + (F-FA01))) to narrow-spectrum penicillin, cephalosporin and macrolides (J01(CE + DB + FA01)) fell from 14.98 in 2011 to 7.80 in 2012, it increased to 13.38 in 2020.Large variations in antibiotic consumption occur between countries, as described by global analyses. Significant differences in the percentage of AMR in key pathogens have been published for both EU/EEA countries and non-EU/EEA European countries ,33,34. RA long-term integrated educational programme with financial incentives from health insurance funds for better quality of antibiotic pharmacotherapy shows that prescription habits changed in Slovakia during the analysed time period. It is likely that the concepts of social distancing and restrictions on movement, which were implemented as non-pharmaceutical infection prevention to decrease contact between those who are infected with a disease-causing pathogen and those who are not, are among the reasons for the significant changes in antibiotic consumption in 2020. Other measures implemented during the COVID-19 pandemic in 2020 within the Slovak health care system most likely also played an important role in influencing the consumption of antibacterials for systemic use, and as a result, the related quality indicators for outpatient antibiotic use in 2020, as well.The data source on the consumption of antibiotics, i.e., antibacterials, for systemic use (ATC group J01) is sales data from the State Institute for Drug Control and the National Health Information Centre. The State Institute for Drug Control (SUKL) represents the Slovak national authority in the area of human pharmacy ; there iAccording to the WHO , “The deAdditionally, we calculated the quality indicators for outpatient antibiotic use, as proposed by Coenen , to bettData from the State Institute for Drug Control and the Slovak National Health Information Centre were reported to the European Surveillance of Antimicrobial Consumption Network, which is a Europe-wide network of national surveillance systems providing European reference data on antimicrobial consumption . ESAC-Ne


The multi-cell composite T-shaped concrete-filled steel tubular (MT-CFST) element is an innovative structural form. It has great potential for construction applications because of favorable advantages over traditional composite elements. The flexural strength of MT-CFST beams was investigated in this study to provide recommendations in line with existing design codes. First, formulations to evaluate the flexural strength of MT-CFST beams were derived based on the Unified Theory and plastic stress distribution method (PSDM). For the Unified Theory-based formula, a modified confinement effect factor that considers the shape of a cross-section was proposed. An experimental study on the flexural behavior of six MT-CFST beams as well as two hollow section counterparts was conducted. The influence of bending moment direction, concrete infill, wall thickness, and cross-section sizes were investigated. The accuracy of the proposed formulations was verified against the test results and numerical results from finite element modeling. The comparisons showed that the formula in line with the Unified Theory provided more accurate predictions with reasonable conservatism for the studied MT-CFST beams. Concrete-filled steel tubular (CFST) elements can reduce the materials costs and the construction time effectively in comparison to equivalent steel or reinforced concrete elements, since CFST cross-sections make full use of the advantages of both steel and concrete materials ,2,3,4,5.Design provisions for normal CFST members have been established in a series of design codes such as AISC 360-16 , EC4 1414 and GByf) in both compression and tension regions, and the concrete compressive stress is equal to a shape factor times the compressive strength (c’f) of concrete. The tensile strength of concrete is neglected in establishing the internal force equilibrium of the section. Differently from AISC 360-16 ; cσ and cε are the stress and strain of the concrete, respectively; c’f is the compressive strength of the concrete, and ξ is the confinement effect factor.The concrete damaged plasticity model  combinedξ) in the above equations was replaced by the modified confinement effect factor (MTξ) proposed in the present paper. For the validation of created FE models, stress–strain relationships obtained from material tests were used.To appropriately consider the confinement effect of the multi-cell T-shaped section, the confinement effect factor , yield stresses (yf) and compressive strengths (cf’) were studied. The height-to-width ratio (a + b/2c) of the composite T-shaped section varied from 0.8 to 2.5, and t ranged from 3 to 7mm. Yield stress (yf) of 345–500 MPa and compressive strength (c’f) of 30–85 MPa were considered. Both positive and negative bending moments were considered for two identical FE models. A total of 52 FE models were created. A summary of the FE models used for verification is shown in A series of FE models with a wider range of cross-section sizes, wall thicknesses  for a few FE models. From the perspective of practice, the unconservative errors were still acceptable since safety factors of no less than 1.10 are commonly used in design. The maximum error of underestimation of Mu-FE was 39% for PSDM and 21% for the Unified Theory-based formula. The results indicate that the Unified Theory-based formula provided improved estimations in comparison to PSDM.From (1)All the MT-CFST beams exhibited very noticeable ductile behavior. The flexural strength of MT-CFST specimens under positive bending was higher than the counterparts under negative bending. This was aligned with the Unified Theory in which the T-shaped cross section was taken as one new composite material whose compressive strength was greater than its tensile strength. Increasing in-filled concrete and cross-section sizes had an appreciable influence on the flexural strength of the studied MT-CFST specimens.(2)Both the Unified Theory-based formula and PSDM generally gave conservative predictions. Predicted results from the Unified Theory-based formula were in good agreement with both experimental results and numerical results. The Unified Theory-based formula produced reasonably conservative predictions for most MT-CFST beams and gave acceptable unconservative errors for few MT-CFST beams. PSDM provided safe predictions but produced large conservative errors in some cases.The flexural strength of MT-CFST beams was investigated in this paper. A Unified Theory-based formula applicable to MT-CFST beams was developed by means of introducing a modified confinement effect factor that accounts for the geometry effects on confinement to in-filled concrete. A series of experimental and numerical studies were carried out. The accuracy of the Unified Theory-based formula and the plastic stress distribution method (PSDM) was verified against the test results and numerical results. Based on the experimental observations and numerical analysis, the following conclusions were made:


The effect of the thermal properties of aggregates on the mechanical properties of high-strength concrete was evaluated under loading and high-temperature conditions. For the concrete, granite was selected as a natural aggregate, and ash-clay and clay as lightweight aggregates. The mechanical properties of the concrete  were evaluated experimentally under uniform heating from 20 to 700 °C while maintaining the load at 0, 20, and 40% of the compressive strength at room temperature. Experimental results showed that the concrete containing lightweight aggregates had better mechanical properties, such as compressive strength and elastic modulus, than that of the concrete with a granite aggregate at high temperature. In particular, the concrete containing lightweight aggregates exhibited high compressive strength (60–80% of that at room temperature) even at 700 °C. Moreover, the concrete containing granite exhibited a higher thermal strain than that containing lightweight aggregates. The influence of the binding force under loaded conditions, however, was found to be larger for the latter type. The transient creep caused by the loading was constant regardless of the aggregate type below 500 °C but increased more rapidly when the coefficient of the thermal expansion of the aggregate was above 500 °C. Concrete is a non-combustible composite composed of inorganic materials, such as cement and mineral admixtures, as well as water and fine and coarse aggregates. Therefore, it has been widely used to secure the fire-resistance of structures and various experiments have been performed to predict its structural behavior more accurately in the event of fire ,2,3,4.To predict the behavior of concrete structures at high temperatures, the thermal properties of various materials, such as aggregates, cement paste, and admixture, must be sufficiently reflected. In general, the mechanical behavior of concrete at room temperature is explained using the elastic theory. At high temperatures, concrete exhibits a shrinking or expansion under the influence of the thermal properties of its components. It also exhibits non-linear behavior due to the influences of spalling, compressive strength and elastic modulus degradation, and short-term high-temperature creep ,7,8,9,10Among the materials that constitute concrete, coarse aggregate accounts for 30–50% of the concrete volume, and its thermal properties significantly affect concrete. Therefore, the Comite Europeen de Normalisation (CEN) ,12 and CThe data used in these models present criteria based on the data identified by researchers, such as Abrams , CastillThese models are mainly used for the design of fire-resistance performance because of the excessive design that considers a sufficient safety factor, and because the thermal behavior of the concrete of actual structures can differ owing to the presence of design loads. Therefore, there is an increasing demand for data of the thermal properties of concrete under loaded conditions, and researchers such as Harada, Anderberg, Sullivan, and Kodur have studied the short-term high-temperature creep  generated under load ,19,20,21For concrete that uses lightweight aggregates, the CEB has suggested a concrete model that utilizes expanded clay under unloaded conditions. Because lightweight aggregates have different thermal properties depending on their production method or components, more data are required to understand the behavior of lightweight aggregate concrete. In addition, research on concrete behavior under loaded conditions is required because lightweight aggregates have a low strength.Therefore, the effects of high temperatures and loading on the mechanical properties  of concrete containing natural and lightweight aggregates with different thermal properties were studied here. Granite aggregate was used for the natural aggregate, and aggregates with ash-clay and clay as the main components were used for the lightweight aggregates. In addition, the effects of the thermal strain of the aggregate and the shrinkage deformation under load on the mechanical properties of concrete were analyzed.2/g and a density of 3150 kg/m3 was used. SiO2 represented more than 90% of the silica fume (SF), which had a specific surface area of 200,000 m2/g and a density of 2200 kg/m3. For the coarse aggregates, granite was selected as the natural aggregate, and ash-clay and clay as the lightweight aggregates. The granite had a density of 2670 kg/m3, a maximum size of 20 mm, and an absorption of 1.0%. Furthermore, sea sand  was used as the fine aggregate. The corresponding values of the ash-clay were 1680 kg/m3, 13 mm, and 15.2%, while those of the clay were 1790 kg/m3, 13 mm, and 17.4%.Ordinary Portland cement (OPC) having a specific surface area of 3160 m−6/°C, 4–6 × 10−6/°C, and 5–8 × 10−6/°C for granite, ash-clay, and clay, respectively, as the temperature increased from 20 to 700 °C.The loading conditions of the concrete were set to 0.0, 0.2, and 0.4× the compressive strength. The specimens were heated at temperatures of 100–700 °C during preloading. The stress–strain, compressive strength, elastic modulus, thermal strain, and transient creep were then evaluated.3, a slump of 190 mm, and an air content of 3.3%. The corresponding values of the AC were 1958 kg/m3, 180 mm, and 3.5%, while those of the CC were 2031 kg/m3, 175 mm, and 3.6%.As for the properties of the fresh concrete, the GC exhibited a unit weight of 2410 kg/mThe compressive strength of the concrete was evaluated at 28 and 180 d. Finally, the compressive strengths before the fire-resistance test were found to be 60.2 MPa for the GC, 65.3 MPa for the AC, and 52.0 MPa for the CC.The concrete specimens were heated using a combined electric heater, as shown in As for the heating rate of the specimen, a preliminary test was conducted at a temperature below 1 °C, based on the recommendations of previous studies and RILEM TC 129-MHT ,28,29. CThe concrete specimen was heated at a rate of 1 °C/min, but it was heated at a rate of 0.77 °C/min for the initial 50 °C section before reaching the target temperature, and the temperature was maintained for 30 min for heating the inside and outside uniformly. At ±50 °C of the target temperature, the heating rate was slowed to 0.77 °C/min, and the temperature was maintained for 60 min when the final temperature was reached. As a result, the temperature difference of the concrete specimen became equal to ±5 °C from the target temperature.For the concrete specimens, the loads of 0.0, 0.2, and 0.4 fcu compared to the strength at room temperature were maintained for three hours before heating to ensure a stable application of the loads. In addition, the same loading conditions were maintained through an automatic program during heating.The specimen displacement due to heating under load was measured using the LVDT strain gauges  installed in the upper and lower parts after installing quartz pipes by drilling ∅15 mm holes in the center of the top and bottom pressure jigs. The maximum capacity of the LVDT strain gauges was 5 mm, and a data logger was used for recording during the experiment.Lc,ε is the thermal strain of concrete; ΔL2 and ΔL1 are the displacements of the upper and lower strains gauge, respectively; and iniitalL is the initial length of the specimen before heating.tr↋ is the transient creep, tot↋ is the total strain , th↋ is the thermal strain (0.0 fcu loaded), and el↋ is the initial elastic deformation.r↋ is the ratio of strain, c,t↋ is the concrete strain at temperature t, and a,t↋ is the thermal strain of the aggregate at temperature t.The relationships were analyzed considering the thermal properties of the aggregate and loading conditions. Equation (3) was used to analyze the relationship between the thermal strains of the aggregate and those of concrete:In this study, three concrete specimens were used, and the results were calculated as an average value.−3).−3).When loads of 0.2 and 0.4 fcu were applied, a linear behavior was observed as the temperature increased. The stress decreased, while the slope of the stress–strain curve did not change significantly; the strain ranged from 2.3–4.5 .For the AC, the slope of the stress–strain curve decreased at 300 °C under a load of 0.0 fcu, and brittle failure was observed even at a relatively high temperature compared to the GC. When loads of 0.2 and 0.4 fcu were applied, the slope of the stress–strain curve became linear with approximately 90% of the stress remaining, and the strain ranged from 2.3–6.0  under a load of 0.0 fcu and from 2.3–4.0  at 0.2 and 04 fcu.For the CC, a brittle behavior similar to that of the AC was observed, but the residual strength tended to be somewhat lower. In addition, the strength showed a tendency to decrease in the same pattern as the GC at temperatures higher than 500 °C. Therefore, the strain of the CC was not significantly different from that of the GC even though it ranged from 2.3–7.3  at 700 °C. A comparison with the CEN and CEB codes shows that the actual measurements were slightly different between 600 and 700 °C. In addition, the thermal strains of concrete presented by the CEN are those of the concrete that used silica and calcium aggregates , and val2), the main component of quartz, has a higher composition ratio than that of calcium (Ca).The main components of the granite aggregate used in this study were quartz and plagioclase. It is estimated that slight differences occurred because silica , which was 50% of that of the GC and more than twice as high as that of the aggregate ). This appears to be because 7% SF was substituted to develop a certain strength, instead of cement.At 700 °C, the thermal strain of the AC was 6  after reaching 300 °C under a load of 0.2 fcu. Under the loading condition of 0.4 fcu, the strain slightly increased or remained constant until 400 °C but sharply dropped above 500 °C, which led to failure at 700 °C.Under the loading conditions of 0.2 and 0.4 fcu, the thermal strains of all the concrete specimens were significantly under control. For the GC, the strain continuously increased until reaching 300 °C and converged to 3 (ε, ×10Unlike the GC, the AC and CC maintained their specimen geometry before heating without any thermal strain under the loading condition of 0.2 fcu. However, under a load of 0.4 fcu, they rapidly contracted and exhibited increased deformation after reaching 300 °C. Above 700 °C, the GC did not fail.2 occurs at 538 °C or higher, and secondary decomposition occurs up to 710 °C, thereby causing matrix collapse [When unloaded, concrete shows the formation of macro-cracks between the aggregate and paste at 100 °C or higher ,30,31. Icollapse .In contrast, the loading condition can control the thermal expansion of concrete and delay the matrix collapse. Hertz  reportedHowever, above 500 °C, the thermal expansion of granite significantly increases, thereby increasing the thermal stress at the aggregate-paste interface. This accelerates the collapse of the concrete matrix owing to the increase in internal cracking, and a rapid increase in shrinkage leads to the failure of the GC.It was confirmed that AC and CC have small initial expansion due to the small expansion stress of the aggregates, and they are significantly affected by shrinkage due to their low aggregate strength. The concrete containing lightweight aggregates did not fail due to shrinkage under load because the aggregate and paste exhibited integrated behavior owing to the small thermal stress at their interface.The results of this study indicate that the strength of concrete can be secured by controlling its deformation under loaded conditions.As the applied load increased, the strain ratio decreased. However, the GC expanded owing to the high thermal expansion of the aggregate itself despite the shrinkage and offset effect caused by the applied load. When a load of 0.4 fcu was applied, rapid shrinkage occurred at temperatures higher than 540 °C, which was found to be large despite the continuous thermal strain of the aggregate.For the AC, the ratio of strain continuously converged to two even under the condition of 0.0 fcu. The AC exhibited a high shrinkage tendency under loading conditions of 0.2 and 0.4 fcu. This is because the strength of the aggregate was relatively low.The CC exhibited a behavior that was slightly higher than the thermal expansion of the aggregate even under the condition of 0.0 fcu. As the loads of 0.2 and 0.4 fcu were applied, the ratio of strain exhibited a strong shrinkage, and an approximately 1.5 to 2.5 times higher shrinkage tendency compared to the thermal strain of the aggregate was observed at 700 °C.Deformation at high temperatures can be inhibited under loading conditions of 0.2 to 0.4 fcu, and it was confirmed that the residual strength and deformation control effect were larger as the thermal strain of the aggregate decreased. Therefore, it can be said that the residual strength of concrete increases if the thermal strain of the aggregate is small, and the thermal expansion stress can be controlled according to the load.The CC containing lightweight aggregate, however, exhibited a tendency similar to that of the GC. Thus, it may have somewhat uncertain aspects that cannot be explained by the thermal strain control effect of loading. Therefore, further analysis is required based on various factors, such as cracks at the interface between the aggregate and paste, deformation under the maximum load, and shrinkage under load.The loaded conditions were confirmed to be effective in maintaining the elastic behavior of concrete specimens by inhibiting the reduction in the stress–strain slope, and lightweight aggregates with a small coefficient of thermal expansion are more affected by such conditions.The residual compressive strength of the concrete tended to increase as the thermal strain of the aggregate decreased. In the case of lightweight aggregates, the residual compressive strength may vary depending on the chemical composition, despite the small thermal strain. In addition, the residual compressive strength under loaded conditions was higher compared to that at 0.0 fcu, but there was no significant difference at 0.2 fcu or higher.The elastic modulus of the concrete showed a large reduction, unlike the residual compressive strength, but it was approximately 20% higher under loaded conditions. Therefore, the control of deformation by loading decreases the reduction rate of the elastic modulus.Under the loading conditions of 0.2 and 0.4 fcu, the thermal strain of all concrete specimens was significantly under control. Under loaded conditions, however, deformation is significantly inhibited because lightweight aggregates have a strong tendency to shrink, which may have a significant influence on the residual strength of concrete.The main conclusions of this study are as follows:In the scope of this study, the effect of the thermal properties of aggregates on the mechanical properties of concrete was investigated under loading and heating conditions. The experimental results confirmed that the concrete containing lightweight aggregates with a relatively small thermal strain had a higher residual compressive strength and residual elastic modulus than that containing natural aggregate. However, lightweight aggregates may exhibit behaviors similar to those of natural aggregates, depending on their chemical composition. Therefore, further analysis of the state of the interface between the aggregate and paste and behavior of the aggregate in future studies is necessary.


Ln-CDs) is designed to induce pseudo-contact shifts in the 19F-NMR spectrum of Ln-CD-bound guest. Then, capitalizing on reversible host-guest binding dynamics and using magnetization-transfer 19F-MRI, pseudo-colored maps of complexed arrays are acquired and applied in molecular-steganography scenarios, showing CODE-HD’s ability to generate versatile outputs for information encoding. By exploiting the widely shifted resonances induced by Ln-CDs, the guest versatility and supramolecular systems' reversibility, CODE-HD provides a switchable, polychromatic palette, as an advanced strategy for light-free, multicolor-mapping.Multicolor luminescent portrayal of complexed arrays is indispensable for many aspects of science and technology. Nevertheless, challenges such as inaccessible readouts from opaque objects, a limited visible-light spectrum and restricted spectral resolution call for alternative approaches for multicolor representation. Here, we present a strategy for spatial COlor Display by Exploiting Host-guest Dynamics (CODE-HD), comprising a paramagnetic cavitand library and various guests. First, a set of lanthanide-cradled α-cyclodextrins ( Host-guest supramolecular chemistry can be used as a tool to develop multicolor displays. Here, the authors present a system based on lanthanide-cradled cyclodextrins that allows to construct MRI-readable and erasable artificial non-luminescent color palettes. Indeed, illuminating  molecules5, polymers6, nanoparticles7, and proteins8 are all channels for the design of a broad palette of orthogonal and spectrally resolved colored materials, which are employed widely—from a better understanding of complex systems9 to sensing10 and logic gates11, through molecular steganography12, imaging of live organisms13, and up to electronic devices14. Nevertheless, several challenges remain for luminescent materials, including photobleaching, light penetration through nontransparent media, a limited number of spectrally resolved colors, color palette transformability , through-object tomography-based mapping capabilities, and more. Therefore, nonluminescent strategies that can capture the polychromatic capabilities of light could expand the material-based designs for multiplexing detection and multispectral display in scenarios beyond those that are applicable for luminescence-based colors.The palette of spectrally resolved luminescent colors provides us the ability to distinguish between dyed objects, and therefore, to observe and study complex milieus, where multiple components are indistinguishable in their “monochromatic” appearance. Thus, numerous color signatures and multiplexed readout features stimulated the development of advanced materials, used not only to tackle challenges in basic sciences, but also for advanced applications17 has opened numerous possibilities for designing noncovalent dynamic assemblies, resulting in the establishment of synthetic supramolecular systems as accessible tools for both fundamental research and advanced applications. Among the developed supramolecular architectures, those that are composed of large hosts and small molecular guests were developed and used in many disciplines24. Taking advantage of the dynamic nature of host–guest systems and exploiting the chemical shift difference in NMR spectra, a unique method for magnetization transfer between a host-bound and free guest molecules was proposed25. This method, based on chemical exchange saturation transfer (CEST), which is frequently used for MRI mapping of solutes through a proton exchange process26, was shown to be applicable for host–guest systems that are composed of Xe gas and a variety of large hosts through an advanced hyperpolarized 129Xe-NMR setup 32. The extension of CEST into 19F-NMR33, together with the diversity of potential fluorinated guests, to obtain the host–guest variant of CEST, termed GEST36 (guest exchange saturation transfer), created the opportunity to design large number of innovative supramolecular platforms.The introduction of supramolecular chemistry1H-CEST-MRI maps for biomedical applications38. The ability to induce pseudo-contact shifts (PCSs) to exchangeable protons and introducing the paramagnetic CEST (paraCEST)40 approach, has further expanded the spectrally resolved pseudo-colors for MRI applications41. The multicolor features shown by hyperCEST27 and paraCEST MRI40, together with the ability to implement the PCS principles to 19F-NMR42 and 19F-MRI43 inspired us to develop a supramolecular palette for COlor Display by Exploiting Host–guest Dynamics (CODE-HD).Depending on the different chemical shift offsets of exchangeable protons of different solutes and their fast exchange with water protons, artificial colors were produced in 19F-MRI framework. This method enables multiplexed information encoding and multicolor displays of supramolecular systems without the need for a light source. As a proof-of-concept, we show the potential use of CODE-HD for molecular steganography applications, demonstrating its high level of security, versatility , and eraseability. Finally, based on the principles of tomography-based techniques, MRI among them, CODE-HD is utilized to simultaneously encode different layers of information from a single studied object.Here, we show the design, development, characterization, and implementation of a supramolecular system that generates multispectral pseudo-colors based on host–guest exchange kinetics, paramagnetic-induced PCS, and CEST contrast in a ω) of the bound guest depends on the paramagnetic element in the host  and two-dimensional  NMR experiments  to obtain the desired set of lanthanide-cyclodextrins  approach, in which the obtained chemical shift , which share the same benzyl amine backbone  of the paraGEST effects can be attributed to the distance (and angle) between the affected nucleus (fluorine) and the lanthanide (Dy3+)50, but may also be the result of steric hindrances that mediate exchange rate and guest inclusion. Importantly, an analog of guest 1, which is substituted with a carboxylic acid instead of a primary amine group, also yielded a pronounced paraGEST effect  for a successful magnetization transfer effect  analysis , relatively small Δω values were obtained in the paraGEST spectra  from which only the one included in the plate will generate the related pseudo-color. Given nine artificial colors and nine wells, CODE-HD can be used to encode nine factorial  different colored barcodes, like those shown in Fig. 4a,Ce-CD and Sm-CD, which induced relatively small Δω values in 2 . To demonstrate this, we capitalized on the ability to generate a 3 × 3 typeface of all Latin alphabet letters, 0–9 digits and additional symbols , the encrypted information was decrypted. Figure Ln-CD hosts in each well cannot be revealed by either 1H-MRI or 19F-MRI, applying CODE-HD at the Δω values of −18.8 ppm (Dy-CD:2), +7.3 ppm (Tm-CD:2), −8.8 ppm (Ho-CD:2), and −2.3 ppm (Pr-CD:2), i.e., the code “Dy–Tm–Ho–Pr,” revealed the hidden word (CEST). In the same manner, the word “color” was encoded by “Tb–Yb–Er–Yb–Ho”, as shown in Fig. Ln-CD library, identity of the 19F-guest and 19F-paraGEST principles). Compared to other MRI-based multicolor approaches, either CEST-based or 19F-MRI based, where only a single component of the encoding system (the used agent) can be controlled, in CODE-HD, the higher level of security is reflected by the fact that the hidden code is composed of two components (host and guest).To this end, each well of the 3 × 3 multiwell plate was first loaded with several 2) with another one, which generates significant 19F-paraGEST effects in different Δω values in the presence of the same members of the Ln-CDs library  object. For that purpose, we designed and manufactured a sealed three-dimensional (3D) apparatus that is composed of three layers of 3 × 3 multiwell plates, thereby gaining 27 different compartments, which were loaded with different D:2 Fig.  to creatD:2 Fig. . AssemblD:2 Fig. , benefitD:2 Fig. . If used19F-MRI, the principles by which CODE-HD is operated are established. By inducing PCSs to the chemical shift of dynamically exchanging fluorinated guests in a 19F-NMR framework, which is controlled by the lanthanide element of the paramagnetic host, spectrally resolved artificial colors are obtained. Following its implementation in 19F-MRI to allow the spatial display of the artificial colors, the potential use of CODE-HD is demonstrated in several setups. The number of color display permutations resulting from one library of nine pseudo-colors is 387,420,489 (99), when arranged in a 3 × 3 well plate. However, it can be readily extended by adapting an alternative guest, generating different spectrally resolved pseudo-colors, or by using compartmentalized arrays that generate additional color combinations. While adopting the principles of paraCEST provides CODE-HD with a high spectral resolution and well-resolved colors, its host–guest landscape affords versatility (using a variety of hosts and multiple guests) for color-code switch ability and eraseability. Relying on MRI basics for data acquisition and spatial display of the artificial colors, CODE-HD is exemplified for molecular steganography applications, demonstrating its 3D potential by virtually decoding a multilayered object. While colors that are based on luminescence  are still the obvious choice for distinguishing between objects, spectrally resolved, light-alternative “colors” can extend the available “multicolor” toolbox for scenarios that are not amenable to the use of light . Thus, we envision that CODE-HD will be utilized towards multiplexed displays in various fields in basic and applicative sciences.In summary, we show here the design, development, principles, and implementation of a nonluminescent supramolecular system, CODE-HD, which enables 1D (NMR), 2D, and 3D displays of artificial colors. By synthesizing a library of paramagnetic cavitands that are based on a lanthanide-cradled α-CD, identifying putative fluorinated guests, and combining host–guest-binding kinetics with paraCEST and Supplementary InformationPeer Review File


Panax ginseng C.A. Mey (ginseng) is a classic medicinal plant which is well known for enhancing immune capacity. Polysaccharides are one of the main active components of ginseng. We isolated water-soluble ginseng polysaccharides (WGP) and analyzed the physicochemical properties of WGP including molecular weight, monosaccharide composition, and structural characteristics. WGP had minimal effect on the growth of hepatocytes. Interestingly, WGP significantly increased the mRNA and protein levels of complement component 4 (C4), one of the core components of the complement system. Promoter reporter gene assays revealed that WGP significantly enhanced activity of the C4 gene promoter. Deletion analyses determined that the E-box1 and Sp1 regions play key roles in WGP-induced C4 transcription. Taken together, our results suggest that WGP promotes C4 biosynthesis through upregulation of transcription. These results provide new explanation for the intrinsic mechanism by which ginseng boosts human immune capacity. Panax ginseng C.A. Mey (ginseng), a perennial plant belonging to genus Panax, is one of the most appreciated medicinal plants  have been proved playing an important role in the modulation of immunity . Human iC4 gene product, fourth component of human complement (C4), is an important component of the complement system  was boiled in deionized water (4 L) for 3 h. After filtration, the solid phase was boiled in deionized water at the same ratio (g/ml) two more times. The filtrate was combined, centrifuged, and concentrated. The concentrated solution was mixed with anhydrous ethanol at a volume ratio of 1:3 and left overnight at 4°C. Discard the supernatant by centrifugation and the precipitate was successively washed with 75% ethanol, 95% ethanol, anhydrous ethanol, and ether. The crude ginseng polysaccharides were obtained after drying and subsequently redissolved in deionized water. After removal of proteins using a Sevage reagent, a 3 KD molecular sieve was used to remove small molecules to obtain WGP. The content of sugar was tracked and monitored by the phenol sulfuric acid method .via 0.45 μm microfiltration membrane. The filtered sample (20 μL) was loaded into a TSK-Gel G4000PWXL column  controlled by LC-10Avp system . High performance gel permeation chromatography (HPGPC) was performed using 0.2 M NaCl as mobile phase at flow rate of 0.5 ml/min. T-series Dextran standards were used for reference standards.WGP (5 mg/ml) was filtered via 0.22 μm organic membrane. Conversion of monosaccharides with PMP were detected via high-performance liquid chromatography (HPLC). The sample (10 μL) was injected into a 4.6 mm × 250 mm COSMOSIL 5C18-PAQ column  controlled by an LC-20AT system . HPLC was performed using a mobile phase composed of 19.5% Acetonitrile and 80.5% 0.1 M PBS (pH 7.0) at flow rate of 1 ml/min. The absorbance values at wavelength 245 nm were compared with those of monosaccharide standards including arabinose, fucose, galactose, galacturonic acid, glucose, glucuronic acid, mannose, rhamnose, and xylose to determine the monosaccharide composition of WGP.Monosaccharide composition analysis was performed as previously described . Brieflyvia Tenor 27 spectrophotometer . WGP was ground with KBr powder at a mass ratio of 3:1 and compressed into a pellet. The FT-IR spectra were recorded in range of 400–4,000 cm−1.Fourier transform-infrared spectroscopy (FT-IR) spectra of WGP was acquired 2O. The 1H and 13C nuclear magnetic resonance (NMR) spectrum were performed on AV-500, 600, and 800 instruments  using tetramethylsilane as the internal standard.The lyophilized WGP was dissolved in Dmycoplasma monthly by the PCR method described by Uphoff and Drexler . Cells were cultured using DMEM media containing 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine  and 10% (V/V) fetal bovine serum  and incubated in HERAcell150i incubator (Thermo Fisher Scientific) set to 5%CO2/95% air and 37°C. Cells were tested for  Drexler .via a microplate reader.The L-O2 cells were treated with a series of equal ratio gradient concentrations of WGP for 72 h in a 96-well plate. MTT   was added at the final concentration of 0.5 mg/ml and incubated for 4 h in incubator. Then, the cells were lysed in 10% SDS containing 10 mM HCl overnight. The absorbance values at wavelength 590 nm were measured via ultrasonic cell disruption and subjected to SDS-polyacrylamide gel electrophoresis. Proteins were electrophoretically transferred onto PVDF (polyvinylidene difluoride) membrane (Thermo Fisher Scientific), and subsequently immunoblotted utilizing anti-β-actin  and -C4  antibodies. Visualization of immunoreactive proteins was conducted using the Odyssey Infrared Imaging System . Densitometry measurements were performed using Odyssey V3.0 software (LiCor).Whole cell lysates were obtained via reverse transcription PCR Kit (Thermo Fisher Scientific), as described previously .Total RNA was extracted by TRIZOL method and used to make cDNAs eviously . The Lig changes . C4 tranvia full length-forward and reverse primers (C4 promoter was identified and digested by XhoI and HindIII (Promega). The C4 promoter was then subcloned into reporter gene vector pGL4.19 basic (Promega) at the XhoI and HindIII restriction sites to generate pC4-1,007/+44. To generate the 5’-deletion constructs, pC4-119/+44, pC4-102/+44, pC4-92/+44, pC4-72/+44, and pC4-48/+44, genomic DNA fragments were PCR amplified from the pGL4.19 plasmid using forward (segment 1 to segment 5) and reverse primers . Differences were compared via hot-water extraction and alcohol precipitation from ginseng powder. The yield of WGP was 0.85%. Carbohydrate content of WGP was over 98% and protein impurity was less than 0.1%. HPGPC was used for molecular weight analysis of WGP. We obtained two overlapping peaks and a single symmetric peak indicating that the molecular weight range of the WGP was from 1 kD to 79.4 kD  was used to confirm the effect of WGP on C4 transcription. Transient transfection of pC4-1,007/+44 into L-O2 cells was performed first, then the cells were treated with WGP for 72 h. The p-C4-1,007/+44 construct showed significant reporter activity compared to pGL4.19 basic in the absence or presence of WGP. Interestingly, treatment of the cells with WGP for 72 h significantly enhanced the C4 promoter reporter gene activity , one NF1 binding site (positions −110 to −97), and one Sp1 (positions −57 to −49) binding element  site at −110 to −97, an Sp1 site at −57 to −49, and three basic helix-loop-helix -like transcription factor sites at −137 to −132, −98 to −93, and −78 to −73, respectively referred to as E-box1, E-box2, and E-box3. These regions appear to be critical for the transcript activity of C4 gene (C4 gene promoter. We found that E-box1 and Sp1 elements play key roles in WGP-regulated C4 transcription. A previous study has shown that the E-box motif of C4 promoter can be recognized by one of the many basic helix-loop-helix (bHLH) or basic helix-loop-helix-leucine zipper (bHLH-LZ) transcription factor family members  class III area on the sixth human chromosome.  closely . C4 promerase II . C4 prom C4 gene . Thus, w members . Moreove8 to −73 . Thus, aIn conclusion, our results demonstrate that polysaccharides derived from ginseng can significantly increase production of C4 through transcriptional mechanisms. These results provide a molecular explanation for the immunity enhancing function of ginseng extracts.