Lost to follow-up (LTFU) negatively affects the treatment success of Anti-Retroviral Therapy (ART) and thus, increases Tuberculosis-Human Immunodeficiency Virus (TB/HIV) related morbidity, mortality and hospitalization. However, the incidence and predictors of loss to follow up (LTFU) among adults with TB/HIV co-infection have not yet well-investigated in Ethiopia. Therefore, this study was aimed at investigating the incidence and predictors of LTFU in the study setting in particular.A facility based retrospective cohort study was employed among 305  TB/HIV co-infected adults in two governmental hospitals , Mekelle, Ethiopia from 2009 to 2016 and data were collected using checklist. Besides to descriptive statistics, a cox regression analysis was applied to identify statistically significant predictors of LTFU at 5% level of significance. Eventually, the Adjusted Hazard Ratio (AHR) and 95% Confidence Interval (CI) were estimated and interpreted for predictors of LTFU in the final cox model.Generally, 45 of 305 (14.8%) of TB/HIV co-infected adults were LTFU with an incidence rate of 4.5 new LTFUs per 100 Person Years (PYs) and a median follow up time of 3.1 years (Interquartile Range (IQR): 0.8–5.3 Years). Hemoglobin level ≤ 11.0 g/dl , and any history of OI/s  were risk factors of LTFU. While, adverse drug events , TB treatment completion , and being on Isoniazid Preventive Therapy (IPT)  had protective effect against LTFU.One in approximately seven TB/HIV co-infected adults had experienced of LTFU with an incidence rate 4.5 LTFUs per 100 PYs. The LTFU rate was higher among adults with low baseline hemoglobin level, no adverse drug events, presence of OI/s, failure to complete TB treatment, and being not on IPT. Therefore, it is advisable to treat anemia and active TB, and preventing the occurrence of OIs including TB using IPT to reduce the incidence of LTFU among TB/HIV co-infected adults. From infectious diseases’ statistics, Tuberculosis (TB) and Human Immunodeficiency Virus (HIV) are the leading causes of death globally. Despite many efforts have been made to control and prevent the dual occurrences of the diseases, yet TB is the leading cause of mortality and morbidity in People Living with HIV (PLHIV) .Above one third of the world population is infected with Tuberculosis (TB) and 5–10% of them develop active TB. The reactivation of Latent TB infection (LTBI) to active TB diseases is pronounced among PLHIV by approximately 20 times on average. Globally, approximately one million PLHIV co-infected with TB in 2017. TB is the leading cause of death among PLHIV attributable to 300,000 HIV-associated TB deaths in 2017 in which Africa accounted for the largest shares (84%). Moreover, PLHIV faces the threat of drug-resistant TB. If diagnosis is delayed there is increased risk of mortality from multi-drug resistant and extensively drug-resistant TB [Studies from SSA have also shown that the cumulative incidence of attrition after 3 years of follow-up can be up to 35%. A declined from 86% at 6 months to 77% by 36 months after ART initiation in SSA . Thus, aCurrently, quality of HIV health care services and Anti-Retroviral Therapy (ART) coverage are significantly increasing from time to time. However, LTFU rate is still high and many people drop out at various phases of the treatment pathway. Because of the reasons why people LTFU are not clearly investigated, a poor adherence to treatment is achieved that results in undesired treatment outcomes .LTFU minimizes the benefits of ART in PLHIV which can enhance the occurrence of Opportunistic Infections (OI/s) including TB as a result of immune suppression. Therefore, it intensifies the TB/HIV or Acquired Immune Deficiency Syndrome (AIDS) related negative consequences – morbidity, mortality, and hospitalizations – as a result of dual burdens of the two infections. Moreover, LTFU in HIV or TB/HIV co-infected adults has also adverse consequences such as drug toxicity, treatment failure, and drug resistance that ultimately shorten the survival of patients –10.Different literatures have been shown that LTFU attributes to an increased mortality globally and approximately 1 death every 3 LTFUs (40%) in the SSA region particularly –14. In aEarly screening and treatment of TB are on the other hand important to reduce the dual burdens of TB and HIV, reactivation from Latent TB infection (LTBI) to active TB disease, and prevent the transmissions of HIV and TB. Ethiopia has adopted the World Health Organization (WHO) Stop TB strategy which currently is being used as a guideline for the anti TB treatment regimen in almost all health facilities throughout the country since Directly Observed Therapy Short Course (DOTS) full implementation coverage in 2000. Nationally, however, the utilization was yet restricted to approximately 92% due to the low health coverage and poor health access .Generally, LTFU is more common in resource poor setting countries as a result of poor adherence and low health care seeking behavior of people. In an ART in LMICs study, the LTFU after 1 year of ART initiation is greater than 40% that is mainly associated with advanced WHO clinical stage (AIDS) and very low baseline CD4 as a result of late initiation. Though the first 6 months of ART initiation are very important to reduce the LTFU rate, the health care seeking behavior of people remains a major challenge yet to increase retention rate , 19.In Ethiopia, ART was begun in 2003 and free ART was launched in 2005. An estimated 739, 000 Ethiopians are currently living with HIV and all of them eligible to initiate ART irrespective of their baseline CD4 cell count. However, the ART coverage is approximately 76% in adult HIV patients [A facility based retrospective cohort study was employed among adults with TB/HIV co-infection who have been initiated ART from 2009 to 2016 at two governmental hospitals , Mekelle, Ethiopia to determine the incidence and predictors of LTFU. Mekelle is the capital city of the national regional state of Tigray which is 783 km far from Addis Ababa, the capital of Ethiopia.Moreover, Mekelle Hospital has been begun ART services delivery since September, 2004 and up to now, more than 10,000 HIV patients have been followed their ART services. All TB/HIV co-infected patients who have been started ART from January, 2009 to December, 2016 were included in the study for the final analysis. However, TB/HIV patients with unknown ART follow up status and short follow up time (less than 1 month) were excluded from the study.1 = proportion exposed group and qo = proportion of non-exposed), 80% statistical power, 95% confidence level, and probability of LTFU from the previous study on LTFU. The number of events (LTFUs) needed to keep the optimum statistical power are estimated to be 62 as follows.In this study, the study subjects were selected from ART registration book based on their TB and HIV status. Furthermore, a minimum sample size was also estimated from previous study that was focused on LTFU conducted in Northern Ethiopia  by consiZα/2=1.96,Zβ = 0.84,Hazard Ratio (HR) = Where: qo = proportion of non-exposed HIV adults (> 11.0 g/dl) = 57.1% ≈ 0.57q1 = proportion of exposed HIV adults (<= 11.0 g/dl) = 42.9% ≈0.43Probability of LTFU = 9.8% ≈0.10.However, 305 TB/HIV co-infected adults were included retrospectively from January, 2009 to December, 2016 using all consecutive sampling in the study setting.2), Functional Status, WHO clinical stage, Hemoglobin level (g/dl), CD4 Cell Count (cells/mm3), Adverse events (Drug Side Effects), IPT prophylaxis, Co-trimoxazole Prophylactic Therapy (CPT), Opportunistic Infections (OI/s) diagnosis, TB Category, Isoniazid Preventive Therapy (IPT), and TB treatment completion – Four trained data collectors were participated in data collection. In addition, data collectors were supervised and data collection tool (checklist) was assessed for completeness, consistency and accuracy by the principal investigator through daily supervision.A standardized data collection checklist was prepared by the principal investigator to extract data from the ART registration book and patient medical records (charts). Prior to data collection a pilot test was done and thus, the data collection tool was modified accordingly. The checklist consisted of variables registered during ART initiation and follow up are – Sex, Age, Marital Status, Educational Level, Weight (kg), Body Mass Index  was labeled as 1 and 0 else considered as censored.p-value of 0.25 in univariate analysis for potential candidate variables selection to be considered in the final model. Variables with a p-value of less than 0.05 (5%) were considered as statistically significant predictors of LTFU after interaction effects and model diagnostics checked. Finally, the Adjusted Hazard Ratio and 95% CI were interpreted for the statistically significant predictors of LTFU in the final cox model.First, data were entered, and then exported to STATA version 12.0. Data were cleaned and categorical variables were labeled prior to data analysis and data were also declared to be survival-time data. Survival data analysis was done using cox regression analysis besides to the descriptive statistics. Kaplan Meier (KM) curves were drawn to estimate the probability of LTFU and proportionality hazards assumption was checked for covariates intended to be included in the final cox model. Log rank test was used to select categorical predictors. Decision was based on a A total of 305 TB/HIV co-infected adults were involved in the study that contributes 944.4 PYs. Above half (51.5%) of the study subjects were females, and above two –fifth (42.3%) of them had also an age above their median (> 35 years), 129 (42.3%) had married marital status, 119 (39.0%) had secondary educational status.Regarding LTFU, 45 of 305 14.8%) of adults co-infected with TB/HIV experienced LTFU throughout the entire follow up time and the overall LTFU rate was found to be approximately 4.5 LTFU cases per 100 PYs with a median follow up time of 3.1 years (Interquartile Range (IQR): 0.8–5.3 Years). Moreover, 26 of 45 (57.8%) of the LTFU cases were experienced among females with an incidence rate of 5.1 new cases per 100 PYs. On the other hand, majority (68.9%) of the total LTFUs were also seen among adults who had an age above the median (> 35 years) and approximately 89.0% of the event of interest was observed among orthodox followers (Table 4.8% of a2). In addition, 54 of 305 (17.7%), 142 of 305 (46.6%), 250 of 305 (82.0%), and 114 of 305 (37.4%) of adults had baseline: bedridden functional status, clinical stage IV, CD4 cell count (<=200 cells/mm3), and anemic status, respectively.Based on their baseline clinical characteristics, above half (51.5%) of the TB/HIV adults had a baseline BMI above the median  of TB/HIV co-infected adults were not reported any adverse drug events with a LTFU rate of approximately twice those we had been experienced with adverse events , adverse drug events , history of OI/s , TB Treatment Completion , and being on IPT . Thus, TB/HIV co-infected patients with anemia status, and presence of at least one OI were approximately 2.66, and 3.80 times more likely to LTFU than their respective comparative groups, respectively. However, TB/HIV co-infected patients who: reported adverse drug events, completed TB treatment phase, and being on IPT were protected by nearly – 55%, 88%, and 92% - against LTFU than their counter reference categories, respectively (Table ).The current study was investigated to determine the incidence and predictors of LTFU among adults with TB/HIV co-infection in the study setting. Thus, the incidence rate was found to be 4.5 new LTFU cases per 100 PYs with a median follow up time of 3.1 years (IQR: 0.8–5.3 years). Up on running our final cox model, statistically significant predictors of LTFU are: baseline hemoglobin level, adverse drug events, history of OI/s, TB treatment completion, and IPT  TB/HIV co-infected patients developed the event of interest (LTFU). The incidence rate was found to be 4.5 per 100 PYs collectively among TB/HIV co-infected adults. The incidence of LTFU was lower as compared to other studies conducted in India, Vietnam, South Africa (Johannesburg), and SSA region , 22, 23.). Finding of the current study is consistent with a study conducted in Myanmar, India though that has estimated Odds Ratio (OR) instead of HR and the study subjects were HIV patients than TB/HIV co-infected [In this study, LTFU was significantly affected by the baseline hemoglobin level where the incidence of LTFU is roughly twice among the anemic groups than their normal counterpart . Though literatures on this study variable were scarce, a facility based case control study has been revealed that presence of drug side effect among HIV/AIDS patient was significant risk factor of LTFU in which patients with adverse drug side effect had approximately 12 times the odds of developing LTFU than those in the control group . The unuAny history of OI/s was also statistically positively associated with LTFU among TB/HIV co-infected adults. Controlling the effect of other predictors, TB/HIV adults who have been diagnosed with at least one OI during the course of their treatment were approximately 4 times more likely to experience LTFU than those who did not . It is evidenced that both TB and HIV have bidirectional and synergistic relation-ships and thus, the presence of either infection has its own significant contribution for the occurrence of other. The strong association between the two infections will be broken up on the successful completion of TB treatment that may reduce the dual burdens proportionally through TB treatment success rate. Therefore, the health status would be improved, and the incidence of LTFU on the other hand will be decreased as a result of treatment success for TB.In the current study, majority 84.3%) of the patients were off IPT prophylaxis (Table % of the The cause and effect relationship of TB and HIV (was TB or HIV come first) is in fact unclear and considered as TB/HIV co-infected regardless of their temporal relationship. Literatures are also scarce and majority of the literatures are LTFU about HIV patients than TB/HIV co-infected. Moreover, some HIV adults who were on IPT initially tend to develop active TB and thus, started anti TB regimen that may mislead to that IPT was given to active TB co-infected adults.Overall, 1 in approximately seven of TB/HIV co-infected adults had experienced of LTFU with an incidence rate 4.5 LTFUs per 100 PYs. TB/HIV co-infected adults diagnosed with anemia at base line, and diagnosed with any OI/s have been strengthened the occurrence of LTFU. Conversely, being on IPT, Completing TB treatment, and developing adverse drug events had protective effect against LTFU among TB/HIV co-infected adults. Therefore, it is advisable to treat anemia and active TB, and preventing the occurrence of OIs including reactivation of latent TB using IPT to reduce the incidence of LTFU among TB/HIV co-infected adults.


In current epidemiology of tuberculosis (TB), heterogeneity in infectiousness among TB patients is a challenge, which is not well studied. We aimed to quantify this heterogeneity and the presence of “super-spreading” events that can assist in designing optimal public health interventions.k, of the NBD measures the level of heterogeneity, where low values of k (e.g. k < 1) indicate over-dispersion. Super-spreading was defined as patients causing as many or more secondary infections as the 99th centile of an equivalent homogeneous distribution. Contact infection was determined based on a tuberculin skin test (TST) result of ≥10 mm. A NBD model was fitted to identify index characteristics that were associated with the number of contacts infected and risk ratios (RRs) were used to quantify the strength of this association.TB epidemiologic investigation data notified between 1 January 2005 and 31 December 2015 from Victoria, Australia were used to quantify TB patients’ heterogeneity in infectiousness and super-spreading events. We fitted a negative binomial offspring distribution (NBD) for the number of secondary infections and secondary active TB disease each TB patient produced. The dispersion parameter, k for secondary infections was estimated at 0.16 (95%CI 0.14–0.17) and there were 414 (9.9%) super-spreading events. From the 3213 secondary infections, 2415 (75.2%) were due to super-spreading events. There were 226 contacts who developed active TB disease and a higher level of heterogeneity was found for this outcome than for secondary infection, with k estimated at 0.036 (95%CI 0.025–0.046). In regression analyses, we found that infectiousness was greater among index patients found by clinical presentation and those with bacteriological confirmation.There were 4190 (2312 pulmonary and 1878 extrapulmonary) index TB patients and 18,030 contacts. A total of 15,522 contacts were tested with TST, of whom 3213 had a result of ≥10 mm. The dispersion parameter, TB transmission is highly over dispersed and super-spreading events are responsible for a substantial majority of secondary infections. Heterogeneity of transmission and super-spreading are critical issues to consider in the design of interventions and models of TB transmission dynamics.The online version of this article (10.1186/s12879-019-3870-1) contains supplementary material, which is available to authorized users. The Global Tuberculosis (TB) Strategy looks towards the ultimate vision of elimination of the TB epidemic, although the disease still causes more than 10 million cases and 1.8 million deaths each year . The epiSeveral studies have reported heterogeneity in the capacity of individual source patients to transmit various pathogens to their contacts –7. VariaMycobacterium tuberculosis (M. tb) infection [M. tb transmission will help to understand better how its transmission is sustained. A study in the Netherlands using genotypic clustering data quantified M. tb transmission heterogeneity and reported signs of super-spreading [TB patients are diverse in their capacity to transmit infection to their contacts, with a systematic review that included several contact tracing studies showing that clinical, demographic and behavioural characteristics of TB patients were associated with their ability to transmit nfection . Quantifpreading . Howeverpreading , understM. tb is routine in this setting. While hospitalisation of cases is not mandatory, those with pulmonary disease are typically maintained in isolation until they are considered non-infectious (> 2 weeks of effective therapy and/or smear-negativity) [Victoria is a state of Australia with approximately 5.6 million people and a single centralised tuberculosis program . Notification of all confirmed or suspected cases of TB disease is mandatory for both laboratories and clinicians and culture confirmation of ativity) , 13. On M. tb infection. Testing of contacts is conducted by either tuberculin skin testing (TST) using the Mantoux procedure or an interferon gamma release assay (IGRA), although during the study period the large majority of testing was undertaken with TST. Those negative on initial testing are tested again 8–12 weeks following exposure. Contacts with either symptoms suggestive of active disease or a positive test for TB infection undergo chest x-ray (CXR) and further clinical assessment, with isoniazid preventive treatment offered for those where active disease is excluded [Contact investigation initially consists of clinical assessment and serial testing for M. tb infection, with cases of subsequent active TB disease linked to these contact episodes now extending to March 2017  were used for the following analysis. Index patients were classified as confirmed cases of TB notified from 1 January 2005 to 31 December 2015 in residents of Victoria. The data set includes contact tracing information and results of testing for 017  and permission was given by the VTP and DHHS.Mycobacterium tuberculosis, while clinical/radiological diagnosis may also be made by a medical practitioner experienced in TB management. Approximately 90% of TB cases in Victoria are bacteriologically confirmed [A microbiologically confirmed case of TB requires culture or polymerase chain reaction (PCR) diagnosis of onfirmed . All casContacts were individuals identified as having had personal contact with index patients by the VTP, through school, workplace, household and other settings. Contact latent TB infection (LTBI) was defined as a TST result of ≥10 mm in an identified contact. Where contacts had had multiple TSTs, we used the value of the latest TST result performed within three months of exposure. Any contact developing active TB during the stated period until 21 March 2017 was considered to have secondary TB. We define a super-spreading event as the number of secondary infections per index case that was greater than the 99th centile of the equivalent Poisson distribution .We were primarily interested in the distribution of the number of secondarily infected contacts from each index patient. Although super-spreading is usually defined in terms of the number of secondary cases of active disease produced by each index patient, we wished to estimate parameters in the absence of preventive therapy. As isoniazid prophylaxis is used widely in our setting and its use may be clustered according to index patients (e.g. family members electing together to undertake preventive treatment), we anticipated this could artefactually inflate our estimates of over-dispersion. Although there are contact factors that are likely to affect progression to active disease after infection, these factors may not be differentially distributed by index patient and the distribution of infections would be unaffected by use of preventive therapy. Therefore, the number of secondarily infected cases is our primary analysis throughout the remainder of the paper, although analogous analyses are presented for the distribution of contacts  and secondary cases of active disease to facilitate epidemiological interpretation.Our primary outcome can be described using a probability distribution termed an offspring distribution, defined as the probability of the number of transmission events across the range of index TB patients. This process can be modelled by a negative binomial distribution (NBD), which has the advantage of being able to accommodate over-dispersed count data –18. The v and, the distribution of individual reproductive numbers (offspring distribution) by Z. To incorporate individual infectious histories, v follows a negative binomial offspring distribution with dispersion parameter k and mean m, such that Z~NegB. The dispersion parameter k quantifies the extent of over-dispersion in the count data. If there is extra-heterogeneity between index patients in the number of secondary infections produced, dispersion increases and the parameter k approaches zero (k → 0). In the absence of over-dispersion, k → ∞ and the mean and the variance approach parity, with the negative binomial distribution reducing to the Poisson distribution. If k = 1, the negative binomial distribution reduces to the geometric distribution, such that the negative binomial model can accommodate Poisson, geometric and over-dispersed distributions [We denote the individual reproductive number by ibutions .v number of infected contacts is given by:The probability of observing index patients with m), over-dispersion decreases, i.e. k → ∞.As the variance m(1 + (m/k)) approaches the mean (k and m were estimated by maximum likelihood estimation (MLE), which provides unbiased estimates, especially for large sample sizes [m is the sample mean of Z or the mean number of secondary infections. The dispersion parameter, k, was estimated after fitting the data to the negative binomial distribution using the MASS package [k less than one interpreted as evidence of super-spreading [The parameters, le sizes . The MLE package  of the R package , with a preading .Rn; second, we constructed a Poisson distribution with mean Rn, representing the range of Z (offspring distribution) due to stochasticity without individual variation; third, we define a super-spreading event as any patient who infected more than Z(i) contacts, where Z(i) is ith centile of the offspring Poisson distribution. Arbitrarily but as in this previous study for SARS, we considered the 99th percentile of this distribution as the cut-point to determine super-spreading events. For prediction of the expected proportion of super-spreading event, we produce a negative binomial distribution with dispersion parameter, k, and the mean number of secondary infections per index, Rn [We used the protocol proposed by Lloyd-Smith et al  in whichndex, Rn , 21.The outcome variable was the number of secondary infections per index patient, which was found to be over-dispersed as described below. Therefore, we fitted a negative binomial regression model with both bivariate and multivariate regression with the MASS package  of the RWe evaluated the need for a negative binomial regression model  with the likelihood ratio test. We also evaluated the predictive accuracy of the model with rootograms .The Victorian TB program data had a total of 4190 confirmed TB index patients and 18,030 contacts within the period from 1 January 2005 to 31 December 2015. The mean age of index patients was 33.0 years and 54.9% were male. Among index patients, 1878 (44.8%) were extrapulmonary, while 1757 (42.0%) were pulmonary and 555 (13.2%) had both pulmonary plus other site involvement.The average age of contacts was 28.4 years, 9276 (51.4%) were female and 8510 (47.2%) were male (with 244 (1.4%) contacts sex not stated). The majority of contacts  were contacts of pulmonary only TB patients, while  were contacts of patients of TB at pulmonary plus other sites and only 11 contacts were identified from EPTB patients . Henceforward we use the term “pulmonary” to refer to any patient with pulmonary involvement, i.e. both the “pulmonary only” and the “pulmonary plus other sites” categories. There were five categories for the types of contacts, 8059 close contacts, 5484 school contacts, 2366 work contacts, 1286 casual contacts and 824 contacts from other congregate setting such as hospitals, nursing homes, airlines and childcare facilities.k = 0.16, 95%CI 0.14–0.17) for all types of contacts.A total of 15,522 of 18,019 contacts of PTB index patients were tested with TST. Based on our cut-off for diagnosing infection as those with a TST result of ≥10 mm as positive, 3213 of contacts were infected and 12,309 were not. Of 3213 infected contacts 2050 (63.8%) were close contacts. Of the 4190 index patients (1878 extrapulmonary and 2312 pulmonary) 3166 (75.6%) did not produce any secondary infection, with all extrapulmonary patients assumed not to have produced any secondary infections  .Restricting our analysis to index patients with pulmonary involvement only, the mean and variance of the number of infections per index was 1.4 and 8.3 infections per index, respectively with evidence of over-dispersion .We constructed a Poisson distribution with the mean number of infections per index (i.e. 0.77) to establish a cut-off number of secondary infections per index for defining super-spreading events. The 99th centile was three infections per index. Therefore, we classified transmission events where index patients produced three or more secondary infections as super-spreading events. Accordingly, there were 414 (9.9% index patients) associated with super-spreading events, which accounted for a total of 2415 (75.2%) of the 3213 secondary infections. For predicting the number of super-spreading events in TB transmission, we estimated the expected proportion of index patients, with confidence intervals considering the dispersion parameter, k = 0.036 (95%CI 0.025–0.046)  were the sole secondary case identified among the contacts of a specific index patient. Among 4190 index patients, only 137 (3.3%) were responsible for all 226 secondary TB cases. Two of the secondary cases were contacts of extrapulmonary index patients . The largest cluster of secondary TB disease was 12 cases. The distribution of secondary TB cases per index, was over dispersed with the dispersion parameter estimated at k= 0.38, 95%CI 0.36–0.41) for the distribution all contact types  after restricting our analysis to close contacts only  was independently associated with a 42% decrease in the number of secondary infections. Identification through contact tracing and the Australian post-migration follow up program  compar.Table 1AP-value < 0.001), whereas model evaluation indicated that the negative binomial model fitted well to the observed data. Compared to the equivalent Poisson model, the negative binomial model was well-fitted and predicted counts well with minimal residuals  [M. tb transmission than genotypic clusters, our approach may even underestimate heterogeneity (overestimate k) due to dilution of differences between patients from more homogeneously distributed distant past infection (i.e. prior to the index exposure identified). Our estimate of k was slightly higher (i.e. less heterogeneous) than an estimate for SARS transmission heterogeneity (k=0.1) [k< 0.04) than secondary infections. This finding was also more heterogeneous compared to the previous TB estimate from genotypic data (k = 0.1) [M. tb transmission dispersion parameter is likely to fall somewhere between the k estimate for secondary infections and secondary active TB distributions.We found that  (k=0.1) . Althoug (k=0.1) , though k = 0.1)  and othek = 0.1) . The effM. tb infections occurred as a result of super-spreading events. Although the average number of secondary infections per index (Rn) was less than one (0.77), TB rates may not decline as expected in the population due to this high heterogeneity. Moreover, TB transmission goes well beyond the 20/80 rule-of-thumb for infectious disease transmission which states that 80% of transmission is due to only 20% of the population [Based on our definition , three-qpulation , since iOur regression analysis identified several important associations between index patients’ characteristics and the number of secondary infections they produced, rather than considering just the proportion of contacts infected as typically done in previous studies , 27. ComM. tb transmission in our setting and this definition could be used for future studies on the disease.The most important limitation of our study is that some secondary infections might be the result of distant past infections, rather than relating to the contact episode. Our definition of super-spreading events is based on contact infection rather than active disease in contacts, since there is no standard definition in the case of TB and the difficulty in interpreting active disease in a setting of widespread use of preventive therapy. However, we argue that contact infection is the best available measure of true M. tb transmission is a highly heterogeneous process in our population and super-spreading events are a major driver of transmission. Therefore, it is essential to consider this heterogeneity when modelling TB transmission dynamics and considering control strategies. Future observational studies should characterise super-spreading in different epidemiological settings to further characterise this phenomenon.We conclude that Additional file 1:Figure S1. Schematic presentation of number of index TB patient and contacts in Victoria, for the period 2005–2015. (DOCX 52 kb)Additional file 2:Figure S2. Distribution of number of contacts per index TB patient in Victoria, for the period 2005–2015. A. All contacts  strategies, the number of index patients with zero contacts was 639 . B. Subset of contacts (0–40 contacts per index only). (DOCX 34 kb)Additional file 3:Figure S3. Distribution of number of Close contacts per index TB patient in Victoria, for the period 2005–2015. A. All Close contacts . B. Subset of close contacts (0–40 close contacts per index), the number of index patients with zero close contacts was 653 . (DOCX 34 kb)Additional file 4:Figure S4. Hanging rootograms for a Poisson model (upper panel) and negative binomial model (lower panel) count data. (DOCX 37 kb)


APOE-ε4 allele has long been associated with increased risks of Alzheimer's disease and mortality, but the effect of the less prevalent APOE-ε2 allele on diseases in the elderly and survival remains elusive.Apolipoprotein E is a glycoprotein best known as a mediator and regulator of lipid transport and uptake. The APOE, and in particular APOE-ε2, with survival in the population.We aggregated data of 38,537 individuals of European ancestry  from six population-based cohort studies  to determine the association of APOE-ε3 carriers, APOE-ε2 carriers were at lower risk of death , whereas APOE-ε4 carriers were at increased risk of death . APOE was associated with mortality risk in a dose-dependent manner, with risk estimates lowest for homozygous APOE-ε2 , and highest for homozygous APOE-ε4 . After censoring for dementia, effect estimates remained similar for APOE-ε2 , but attenuated for APOE-ε4 . Results were broadly similar across cohorts, and did not differ by age or sex. APOE genotype was associated with baseline lipid fractions (e.g. mean difference(95%CI) in LDL(mg/dL) for ε2 versus ε33: -17.1(-18.1–16.0), and ε4 versus ε33: +5.7), but the association between APOE and mortality was unaltered after adjustment for baseline LDL or cardiovascular disease. Given the European ancestry of the study population, results may not apply to other ethnicities.During a mean follow-up of 11.7 years, 17,021 individuals died. Compared with homozygous APOE-ε3, APOE-ε2 is associated with prolonged survival, whereas mortality risk is increased for APOE-ε4 carriers. Further collaborative efforts are needed to unravel the role of APOE and in particular APOE-ε2 in health and disease.Compared with  APOE gene, on chromosome 19, contains four exons and codes for a 317 amino acid polypeptide that gives rise to a 299 amino acid long mature protein (34kD).[APOE isoforms designated APOE-ε4, -ε3, and -ε2, with corresponding allele frequencies of approximately 14%, 78%, and 8%, respectively.[Apolipoprotein E is a glycoprotein best known as a mediator and regulator of lipid transport and uptake, but also has several additional physiological and pathological roles. The APOEprotein 3kD.[APOE APOE locus impacts survival, and alters risk of hyperlipidaemia, atherosclerosis, cardiovascular disease, and in particular dementia.[APOE-ε4 allele, which is associated with an adverse impact on these risk factors and outcomes, including shortened survival compared with the more common ε3 allele. More recent data suggest that the APOE-ε2 allele might prolong survival,[APOE-ε2 carrier status, above and beyond the absence of the ε4 allele, could lead to novel preventive and treatment options for a wide variety of conditions to promote healthy aging and longevity. Yet, studies of the ε2 allele have been hampered by its low allele frequency, which results in only 1% of the population being homozygous ε2 carriers. Larger studies are therefore warranted, requiring collaborative efforts to design well-powered studies to address these questions.Various studies, dating back as far as 25 years ago, have shown that allelic variation at the dementia. Initial survival,–11 but osurvival,,13 and hsurvival, dysbetalsurvival, and aggrsurvival, A betterAPOE-ε2 allele on survival in the general population. In addition, we studied potential vascular or lipid-mediated mechanisms that might account for this association.We aggregated data from six large cohorts, and aimed to determine the impact of the This study population consisted of participants of European ancestry from six population-based cohort studies: the Framingham Heart Study (FHS), the AGES-Reykjavik Study (AGES), the Rotterdam Study (RS), the Cardiovascular Health Study (CHS), the Long Life Family Studies (LLFS), and the Health, Aging, and Body Composition study (HABC), of which FHS and LLFS are family-based cohorts. Details of the design and characteristics of participating studies have been described previously,–23 and aFramingham Heart Study (FHS) was initiated to study determinants of cardiovascular disease. The original cohort was recruited in 1948 and the offspring of the Original cohort participants and offspring spouses were enrolled in 1971.[st December 2013 were included in the present analyses.The  in 1971.,18 DNA wAge, Gene/Environment Susceptibility -Reykjavik Study (AGES) was initiated to examine potential genetic susceptibility and gene/environment interaction.[st December 2015 via linkage to electronic medical records and vital status registry.The eraction. Between Rotterdam study (RS).[st January 2015.Between 1990 and 1993 all inhabitants of the Ommoord district in Rotterdam, The Netherlands, aged ≥55 years were invited to participate in the udy (RS). The cohoCardiovascular Health Study (CHS) is a prospective population-based cohort study of cardiovascular disease and mortality in >65 year old Medicare-eligible adults living in four United States communities.[The munities. RecruitmLong Life Family Study (LLFS) enrolled families enriched for longevity via 4 field centers  between 2006 and 2009.[>7 consisting of 1493 probands, their siblings and 192 spouses in the older generation, and 2437 offspring and 809 of their spouses. Information collected on onsets of diseases was assessed retrospectively at baseline from self-reports and prospectively during in-person visit (home or clinic), self-administration, or telephone interview through 2015. Death was assessed annually by interview of proxies or from nationwide survival and health register (Denmark) through 2015.The and 2009. The recrHealth, Aging, and Body Composition study (HABC) is a prospective cohort study of 3,075 community-dwelling black and white men and women living in Memphis, TN, or Pittsburgh, PA, and aged 70–79 years at recruitment in 1996–1997.[The 996–1997. ParticipAPOE genotype was determined directly (i.e. not using genetic imputations) in all cohorts. Methods that were used include polymerase chain reaction on coded DNA samples  and bi-allelic Tacqman assays (rs7412 and rs429358) .Fasting serum total cholesterol, high-density lipoprotein (HDL), and triglycerides were measured at baseline. Low-density lipoprotein (LDL) was computed from total cholesterol, HDL and triglycerides, using Friedewald’s formula. Use of lAPOE genotype and death, while adjusting for age, sex, and center of ascertainment (if applicable). Analyses included a comparison of each of the APOE genotypes to ε3:ε3, as well as a comparison of ε2 carriers (ε2:ε2 or ε2:ε3) versus ε3 homozygotes, and ε4 carriers (ε3:ε4 or ε4:ε4) versus ε3 homozygotes. For appropriateness of comparison, heterozygote ε2:ε4 carriers were excluded from the latter comparisons. In additional analyses, we investigated whether results were mediated by dementia, by excluding all participants with dementia at baseline and censoring at time of incident dementia diagnosis (upon reviewer’s request). We also explored potential interaction of APOE with age and sex, by testing for multiplicative interaction in the Cox model. To further assess effect modification by age, we performed a sensitivity analysis among participants aged <80 years, while censoring at age 80 (upon reviewer’s request). Additionally, we adjusted these models for ethnicity, educational attainment, and smoking (upon reviewer’s request). In studies for which information on traumatic injury was available , only a fraction of deaths (328/14848 = 2.2%) was attributable to trauma (upon reviewer’s request).For each cohort separately, we used Cox proportional hazard models (with robust variance for family cohorts) to determine the association between APOE genotypes, using linear regression (mixed effects model for family cohorts), adjusting for age, sex, ascertainment center, and use of lipid-lowering medication. In two cohorts (FHS and RS), we assessed the additional variance explained by APOE genotype. We then repeated the survival analyses with additional adjustment for measured lipid fractions, and prevalent cardiovascular disease, as well as after adjustment for ethnicity, educational attainment, and smoking.Next, triglyceride levels were log-transformed to obtain a roughly normal distribution of data. We then determined per cohort differences in cholesterol, HDL, triglycerides, and LDL across 2 statistic).[We used inverse variance weighted fixed and random effects models to pool hazard ratios and mean differences from separate cohorts. We formally assessed for heterogeneity between studies, determining the share of variation across studies that was due to heterogeneity rather than chance (Higgins’ Iatistic). In case Analyses were done using IBM SPSS Statistics version 23.0  or R statistical software version 3.1.1 . Alpha level (type 1 error) was set at 0.05.None of the funders were involved in study design, data collection and analysis, preparation of the manuscript, or the decision to submit for publication.APOE-ε2, ε3, and ε4 alleles was 7.9%, 78.6%, and 13.5%, respectively. Observations lay within Hardy-Weinberg equilibrium.A total of 38,537 participants were included from the 6 cohort studies. Baseline characteristics of the entire sample as well as per cohort are presented in P = 1.1*10−2; APOE-ε4 carriers were at increased risk of death , 17,021 participants died. Carrying one or two copies of the ε2 allele was significantly associated with reduced mortality risk (hazard ratio (HR), 95% confidence interval: 0.94, 0.90–0.99, carriers . Risk foAPOE-ε2 , but attenuated for APOE-ε4   or sex , also witnessed by similar effect estimates when restricting analyses to participants under the age of 80  were attributed to cardiovascular causes, and 3922 (23.0%) to cancer. After exclusion of patients with dementia at baseline, and censoring in the main analysis at time of incident dementia diagnosis, effect estimates for mortality remained similar for 01–1.12) . All ass01–1.12) , and thege of 80 . These age of 80 .APOE genotype was associated with all measured lipid fractions, generally in a dose-dependent manner . Similarly, adjustment for prevalent cardiovascular disease did not materially change risk estimates of mortality .Effect estimates of E2-CHARGE is the largest collaboration of cohort studies to date to determine the impact of APOE and, in particular, the APOE-ε2 allele, and aggregates data of 38,537 individuals from 6 population-based cohort studies. In this first analysis of the data, we found that the APOE-ε2 allele is associated with prolonged survival, whereas APOE-ε4 is associated with increased mortality. Adjustment for the prevalence of cardiovascular disease or measured lipid fractions had trivial effects on these estimates.APOE in longevity,[APOE in survival. While most of these studies indeed show that APOE allele frequencies shift with age,[APOE is associated with longevity as study endpoint, they did not reach genome wide significance,[APOE locus from that at the TOMM40 or APOC1 loci.[APOE genotype with survival and longevity, but with contrasting findings. Two Scandinavian studies reported hazardous effects of the ε4 allele,[APOE genotype with survival, which were consistent across participating cohorts. Our pooled effect estimates suggest prior studies were likely underpowered to detect these differences, in particular for the ε2 allele. Survival and selection bias at older ages, whereby ε4 carriers die prior to entry or are less likely to be enrolled due to poor health, may have led to underestimation of hazardous effects of the ε4 allele in others.Since the first implication of ongevity, several with age, or confiificance,–30 or weOC1 loci.,32 Sever4 allele,,12 while4 allele, These fi4 allele, as well 4 allele,,11 Never4 allele, We foundAPOE-ε4, but were virtually unaltered for APOE-ε2. This suggests that a vast part of excess risk for APOE-ε4 is via its effect on dementia pathology, but that other mechanisms also play a role in carriers of the APOE-ε2 and to a lesser extent APOE-ε4 allele. Mounting evidence indeed suggests pleiotropic effects of APOE on various organ systems. In addition to the well-established link with dementia, more recent clinical and population studies have linked APOE gene variation to atherosclerosis,[APOE locus by DNA methylation.[APOE genotype with lipid fractions align well with those in a prior European study.[APOE-ε2 carriers, but found no evidence of mediation. In addition, estimates changed only minimally after taking into account clinically manifest vascular disease at baseline. Although this may in part reflect limitations of single measurements of lipid fractions, it suggests other  mechanisms could be involved.[After accounting for dementia in our study, associations with mortality attenuated for clerosis, cerebralclerosis, stroke,[clerosis, lung disclerosis, multipleclerosis, and neopclerosis, Precliniclerosis,,39 neuroclerosis, inflammaclerosis, type IIIclerosis,,42 prostclerosis, and epighylation. The assoan study. We invesinvolved.APOE genotype directly rather than by imputation, we did not investigate other genetic variants that might modify the effect of APOE through epistatic interactions. Second, (impact of) serum lipid levels on health and disease may differ over time, which is not captured by one-time measurement at study baseline, and may cause underestimation of any mediation. Third, Friedewald’s formula for computation of LDL levels assumes that all triglycerides are carried on VLDL, and that the triglyceride-to-cholesterol-ratio of VLDL is constant at 5:1, which may not apply in all individuals. Fourth, although the mean age of participants at study entry was only 65 years, we cannot rule out attenuation of effect estimates, in particular for ε4 carriers, due to selection bias at older ages. Fifth, albeit the largest study of APOE-ε2 in relation to mortality to date, precision may still be lacking with respect to separate genotypes to fully reveal a dose-effect response. Finally, the study population was entirely of European ancestry, and findings may not be applicable to other ethnicities.Certain limitations should be taken into account. First, although we only determined E2-CHARGE brings together data from several population-based studies worldwide. In this paper, we describe the details of each population and the first analysis of the data. We find that APOE-ε2 prolongs survival in the general population of European descent, which appears only in part explained by commonly determined lipid fractions, or prevalent vascular disease. Further studies are needed to determine the role of APOE-ε2 in vascular, as well as other types of disease, above and beyond the absence of the APOE-ε4 allele. Various other population studies have collected or are collecting data on APOE genotype and disease outcomes, and inclusion of these data–in particular from ethnically diverse populations–may aid in elucidating the role of APOE-ε2 in health and disease.In conclusion, S1 Supporting Information(DOCX)Click here for additional data file.S2 Supporting Information(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


In recent years, surgical outcomes have improved, and positive reports on surgery for type A aortic dissection (AAD) in the elderly are increasing. However, the difference between surgical and conservative treatments in the elderly remains unclear. Therefore, we conducted this study to determine whether surgery should be performed for Stanford (AAD) in elderly patients.Data of patients aged 80 years or older who were hospitalized for AAD from April 2014 to March 2016 were extracted from the Japanese national inpatient database. Outcome measures were all-cause in-hospital death, stroke, acute kidney injury and tracheotomy, and composite adverse events , and we compared them between surgical and conservative treatments using propensity score matching.p < 0.001; 16.7% vs. 31.6%, p < 0.001, respectively); however, there was no significant difference in composite adverse events after matching , and adjusted odds ratio was 1.06 and 95% confidence interval was 0.86–1.29 (p = 0.61) with reference to conservative treatment.The study cohort included 3258 patients, with 845 matched pairs (1690 patients) in the propensity score matching. All-cause in-hospital death was significantly lower in the surgical treatment group than in the conservative treatment group before and after matching  contains supplementary material, which is available to authorized users. The ageing of populations is presenting new challenges to developed countries worldwide. In 2015, 26.7% of the Japanese population was over 65 years of age, and 8.0% of the population was over 80. The fraction of the Japanese population aged 80 or older is estimated to reach 10% by 2025 , surgeryAlthough retrospective cohort studies have compared conservative and surgical management of AAD, the patient cohorts of these studies were not adjusted for background. Thus, many inoperable patients with severe conditions may have been included in the non-surgical treatment group, making comparison of the two groups by randomization difficult. Therefore, this study compares the conservative and surgical management of patients with AAD aged > 80 years after adjusting for severity by propensity scores using data from the national inpatient database in Japan.We performed a retrospective cohort study using the Japanese Diagnosis Procedure Combination (DPC) database. The DPC system was introduced in Japan in 2003 as a comprehensive daily evaluation system for acute-phase hospital medical care. In 2016, DPC-participating hospitals accounted for approximately 80% of acute-phase beds in Japan. Clinical information and other data for patients were registered in the DPC database and classified by a 14-digit DPC code. The system holds the following clinical information: patient demographics, diagnoses, comorbidities and complications , drugs and devices used, procedures performed, healthcare costs and status at admission and discharge.Data were extracted from the DPC database between April 2014 and March 2016. First, we identified patients aged 80 years or older with acute aortic dissection  recorded as either the “main diagnosis,” “admission-precipitating diagnosis,” or “most resource-consuming diagnosis” and identified patients with dissection in the ascending aorta with “Stanford type A,” “DeBakey I,” or “DeBakey II” as the disease name. We excluded patients who had planned admission and unknown admission pathway. The final cohort was divided into the conservative and surgical treatment groups.We matched propensity scores between the surgical and conservative treatment groups. Propensity scores were calculated using a logistic regression model with the following variables: age, sex, body mass index , admission pathway , ambulance use, hypertension, diabetes mellitus, chronic obstructive pulmonary disease (COPD), chronic kidney disease, ischemic heart disease, chronic liver disease, cancer, dementia, cardiopulmonary arrest, cardiac tamponade, shock, coronary malperfusion, limb ischemia, aortic insufficiency, hospital volume  and consciousness level upon admission . Outcome measures were all-cause in-hospital death, stroke, and acute kidney injury (AKI) requiring renal replacement therapy (RRT), tracheotomy, composite adverse events , length of hospital stay, length of intensive care unit (ICU) stay, inpatient costs, home discharge rate, and activities of daily living (ADL) of surviving patients at discharge. ADL was evaluated using the Barthel index (BI), which assesses the ADL using a 100-point scale comprising the following 10 items: “Bowels,” “Bladder,” “Grooming,” “Toilet use,” “Feeding,” “Transfer,” “Mobility,” “Dressing,” “Stairs,” and “Bathing,” and we set the BI cut-off at 60 points, which indicates having functional dependency. , 13.P < 0.05 was considered significant, and hypothesis tests were two sided. This study was performed with the approval of the Ethics Committee, Kyoto University Graduate School and Faculty of Medicine .Summary statistics are presented as frequencies and percentages for categorical variables and median and interquartile ranges for continuous variables . Data were compared using the Mann–Whitney U test for continuous variables and the chi-squared test for categorical variables before matching; after matching, the Wilcoxon signed rank and McNemar tests were performed. We performed one-to-one pair matching using nearest neighbor matching without replacement within 0.2 standard deviations of the logit of the propensity score as caliper width . Covarian = 1241; conservative treatment group, n = 2017) who were hospitalized in a total of 634 hospitals. The study had 845 matched pairs (1690 patients) in the propensity score matching. As shown in Fig. The process used to select eligible patients is shown in Fig. p < 0.001), but complications and tracheotomy during hospitalization were high in the surgical treatment group both before and after matching: stroke (3.5% vs. 17.2%)/(4.5% vs. 17.5%), acute kidney injury (1.0% vs. 8.8%)/(1.7% vs. 8.6%), and tracheostomy (0.8% vs. 8.7%)/(1.1% vs. 9.2%). Composite adverse events occurred in 54.0 and 36.6% in conservative and surgical treatment groups, respectively, before matching but occurred in 36.0 and 37.2%, respectively, after matching, which was not significant.Outcomes are shown in Tables p < 0.001]/.The hospital length of stay and ICU length of stay were significantly longer in the surgical treatment group before/after matching, although the difference was lower after matching. The medical expenses were also significantly higher in the surgical treatment group than in the conservative treatment group before/after matching [$4100  vs. $36,600 ; The results of functional status at discharge of surviving patients are shown in Table The AOR for postoperative outcomes by each type of surgeries is shown in Table Multiple sensitivity analyses about missing data showed similar point estimates and 95% CIs , the risk difference was 14.9% even after matching. These results certainly indicate that surgery is a good treatment option; however, on the other hand, the percentage of patients who could be discharged to home after surgery was approximately 40, and 40% of the patients in the surgical treatment group had BI < 60 and required a lot of assistance. These results support the observation by Hata et al. that AAD surgery poses issues including the risk of being bedridden and/or depressed and a burden on the family . In addiExtremely positive outcomes have been reported for elderly patients undergoing hemiarch replacement and a short-time/less-invasive ascending aortic replacement for entry closure , 21, andFew reports have compared conservative and surgical treatment of the elderly, and the results of this study may be useful for choosing treatment plans in patients with AAD. The strengths of this study are that we examined an unprecedented number of AAD cases in the elderly and are the first to compare surgical and conservative treatments using propensity scores and that we compared short-term mortality as well as event-free survival rate and functional status at discharge, which is particularly important in the elderly. However, this study also has some limitations. Because the study cohort includes a very specific population over 80 years of age, the results cannot be extrapolated to different populations. The retrospective design of this study has inherent limitations, especially selection bias and dependence on historical records. Other limitations include a lack of clinical information, including preoperative vital signs, laboratory data/diagnostic imaging, specific anaesthesia method/surgical technique, and postoperative management. Other underlying diseases may not have been identified because of the extraction of comorbidity or findings on admission from registered disease names. Propensity scores may have been influenced by the presence of unmeasured factors. Because our study was short term, the results do not reflect subsequent death after discharge or the later need for surgery. Thus, our results do not address the mid- or long-term outcomes after discharge.Our results indicate that AAD surgery is a feasible treatment option for the elderly and has a significantly lower hospital mortality rate than does conservative treatment, even in patients over 80 years of age. Despite these findings, many elderly patients with AAD are not treated surgically. This may be due to postoperative complications and poor condition at discharge. With the growth of older populations in Japan and other countries worldwide, postoperative outcomes of the elderly will be an increasingly important issue, and increase in event-free survival rate will support the choice of AAD surgery for the elderly patients.Additional file 1:Additional table. Sensitivity analysis for BMI missing data (DOCX 19 kb)


The objective of this study is to evaluate the effect of simvastatin on the jaw bone microstructural defect and autophagy in rats with high cholesterol diet (HCD).n = 10/group). The static histomorphometric changes in the jaw bone tissues in individual rats were evaluated. The relative levels of OPG, RANKL, NF-κB, LC3, and p62 in the jaw bone tissues were determined by quantitative RT-PCR and/or immunohistochemistry. Male Sprague-Dawley rats were fed a standard rodent chow (NC group) or a high cholesterol diet for 32 weeks and the HCD-fed rats were treated with vehicle (HC group) or simvastatin (5 mg/kg orally daily for 8 weeks, HC + SIM group, and κB activation and autophagy. Simvastatin treatment inhabited these changes, including the decreased levels of serum proinflammatory cytokines and increased autophagy. Compared with the NC group, the HC groups had lower trabecular bone volume, trabecular thickness and trabecular number, and increased ratios of RANKL/OPG in the jaw bone, accompanied by enhanced NF- Simvastatin treatment could inhibit the hyperlipidemia-induced jaw bone microstructural defect in rats by increasing autophagic flux. High fat or cholesterol diet can cause hyperlipidemia, which is associated with oxidative stress and inflammation, leading to osteoporosis, a high prevalent metabolic disorder in developed and developing countries . PreviouAutophagy is an intracellular process of degradation of cytoplasmic components in lysosomes to maintain cellular homeostasis. During the process of autophagy, many stimuli can induce the formation of autophagosomes, in which the microtubule-associated protein 1A light chain 3 (LC3-I) on the autophagosomal inner membrane converts to LC3-II by conjugating to phosphatidylethanolamine. The LC3-II can recruit p62/SQSTM1 promotes the degradation of cargo proteins . Recent Statins are common drugs for the treatment of hyperlipidemia and can modulate lipid and bone metabolism, improving osteoporosis –14. PrevIn this study, we tested the hypothesis that treatment with simvastatin could mitigate hyperlipidemia-induced jaw bone microstructural defect by upregulating autophagy flux in rats.n = 10) with 100% standard rodent chow; or (2) high cholesterol diet  with 2% cholesterol + rodent chow  normal control diet , total cholesterol (TC), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) were measured using enzymatic methods in an AutoBiochemical Analyzer .μm) were stained with hematoxylin and eosin (H&E). Three to five areas selected randomly from each sample were photoimaged under an Olympus microscope  using the Image Pro Plus software, version 6.0 . The trabecular bone volume relative to the tissue volume , trabecular number , trabecular thickness , and trabecular separation  in each image were analyzed in a blinded manner.After being sacrificed, the jaw bone tissue samples from individual rats were dissected out and fixed in 4% paraformaldehyde for 48 hours. After fixation, the jaw bone and tooth samples were decalcified in 10% EDTA solution (pH 7.2) for 2 weeks at room temperature. The decalcifying solution was changed with fresh one every 5 day. The samples were paraffin-embedded, and the longitudinal tissue sections (5 2O2 in methanol to inactivate endogenous peroxidase and were incubated with anti-NF-κB (1 : 200), anti-LC3 (1 : 100), and anti-p62  at 4 °C overnight. After being washed, the sections were incubated; biotinylated secondary antibodies and the bound antibodies were detected with peroxidase-labeled streptavidin at 37 °C for 30 min. The stained signals in the sections were visualized using Diaminobenzidine  and the sections were counterstained with haematoxylin. The vehicle PBS was used as the negative control in every step.The bone tissue sections were deparaffinized using xylene and rehydrated by serial concentrations of ethanol. After antigen retrieval using 0.125% pancreatin at 37°C for 30 min, the sections were treated with 3% HκB to determine the relative expression levels. The images were analyzed by two specialized pathologists in a blinded manner.The intensity of the IHC staining was evaluated semiquantitatively using Image Pro Plus software, as described previously , 21. SixκB, LC3, p62, and GAPDH are shown in μl of 1 × SYBR Premix Ex TaqII, 0.2 μmol/l each of primers, and 20 ng cDNA template. The amplifications were performed in duplicate at 95°C for 30 s and subjected to 40 cycles of 95° C for 5 s and 60°C for 30 s. The relative levels of each target gene mRNA transcripts to the GAPDH were analyzed using 2-△△Ct method [The dissected jaw bone tissues were frozen in liquid nitrogen and powdered. Total RNA was extracted from each jaw bone sample using Trizol reagent (Ambion), according to the manufacturer's instruction. The RNA samples were reversely transcribed into cDNA using the PrimeScript™ RT reagent kit with gDNA Eraser , according to the manufacturer's instruction. The relative levels of target gene mRNA transcripts to the control GAPDH were analyzed by quantitative RT-PCR using the FastStart DNA Master SYBR Green I kit  and specific primers in a LightCycler system 480 (Roche). The sequences of primers for OPG, RANKL, NF-t method  and exprP value of < 0.05 was considered statistically significant.Data are expressed as mean ± standard deviation (SD). The difference among groups was assessed by one-way analysis of variance (ANOVA) and post hoc Fisher's Least Significant Difference (LSD) test using the SPSS, version 17.0 . A P > 0.05, To determine the effect of simvastatin on HCD-induced hyperlipidemia, SD rats were fed with HCD for 24 weeks, randomized, and treated orally with vehicle or simvastatin daily for 8 weeks. A group of rats received vehicle PBS as the NC controls. Their body weights were measured weekly in P < 0.001). However, the levels of serum TC and LDL in the HC + SIM group were significantly lower than that in the HC group (P < 0.01) but remained significantly higher than that in the NC group , while the Tb.Sp values were significantly lower than that of the HC group of rats (P < 0.05). More importantly, there was no significant difference in the values of these measures between the HC + SIM and NC groups of rats.Hyperlipidemia can induce oxidative stress, leading to the bone microstructural defect. Next, we examined the effect of simvastatin on the jaw bone microstructure in the HCD-fed rats by histology. The static histomorphometry revealed that the HCD-fed rats displayed significantly lower trabecular bone volume (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N), with the trabecular separation (Tb.Sp) higher than the NC rats (P < 0.05). Treatment with simvastatin significantly decreased the ratios of RANKL to OPG mRNA transcripts in the HC + SIM group . Further immunohistochemistry revealed that the NF-κB was expressed in osteoblasts, inflammatory mononuclear infiltrates, bone-lining cells, and osteoclasts. The mean density of anti-NF-κB immunoreactivity in the bone tissues of the HC group was significantly higher than in the NC (P < 0.01) and HC + SIM groups . Immunohistochemistry revealed similar patterns of the levels of LC3 and p62 protein expression in the jaw bone tissues of the different groups of rats (Figures Hypercholesterolemia can inhibit autophagy in the heart of rats . Finally Figures , Table 4The present study employed a HCD-fed rat model to test the effect of simvastatin treatment on the jaw bone microstructural defect. We found that treatment with simvastatin not only improved the HCD-induced hyperlipidemia but also mitigated the HCD-induced jaw bone microstructural defects by reducing inflammation and increasing autophagic flux in the jaw bone of rats. To the best of our knowledge, this was the first study to show that autophagy regulated the jaw bone metabolism in rats fed with HCD.Feeding with HCD or HFD can induce hyperlipidemia in animals. We found that rats fed with HCD for 32 weeks had significantly higher levels of serum TC and LDL to develop hyperlipidemia, consistent with a previous observation . TreatmeκB signaling to induce c-Fos expression and NFATc1-triggered osteoclastogenic gene transcription, leading to osteoclastogenesis and bone resorption [κB activation to inhibit osteoclast differentiation but promote osteoblast differentiation in the bone. While hyperlipidemia can inhibit the proliferation and differentiation of osteoblasts and statins, such as simvastatin, it can stimulate osteoblast differentiation [κB expression in the jaw bone of rats, which was demolished by simvastatin treatment. Therefore, simvastatin treatment suppressed osteoclast differentiation and promoted osteoblast differentiation by down-regulating the RANKL/OPG ratio and NF-κB activation in rats.It is well known that the balance of RANKL and OPG is crucial for regulating osteoclastogenesis and osteoblastogenesis, contributing to the bone metabolism and remodeling . High-lesorption –33. OPG,ntiation –36. Indentiation . In addintiation –40. In tκB signaling, which promotes osteoclastogenesis and osteoclast formation [A previous study has shown that chronic HFD feeding can decrease the levels of autophagy in mouse hypothalamus . Inhibitormation . In thisormation –51. SabeκB signaling, which modulated autophagy in the jaw bone of rats. Simvastatin treatment improved the hyperlipidemia-induced jaw bone microstructural defects by increasing autophagy flux. Potentially, our findings may provide a significant contribution to further experimental and clinical studies by directly comparing the effects of simvastatin on the jaw bone with other bone tissues.In conclusion, the present study revealed that HCD feeding induced hyperlipidemia and decreased trabecular volume by increasing the RANKL/OPG ratios and activating the NF-


This work provides a new facile strategy for the design and synthesis of sustainable and effective molybdenum-based electrocatalysts as alternatives to non-Pt catalysts for HER.Efficient electrocatalyst plays a significant role on the development of hydrogen energy. In this work, an N,P-codoped carbon layer coupled with MoP nanoparticles (MoP/NPCs) was prepared through a facile high-temperature pyrolysis treatment. The obtained MoP/NPCs presented efficient activity for hydrogen evolution reaction (HER), with low onset potential of 90 mV, and a small Tafel slope (71 mV dec As is known to all, fossil energy  suffers from excessive consumption with rapidly growing economies, which results in global environmental destruction and energy crisis ,2,3,4. RMolybdenum phosphide (MoP) has extensively been investigated for HER, due to its similar electronic structure and excellent catalytic performance for hydrodesulfurization, which have similar reaction processes to HER ,26,27,28−1), as well as extraordinary stability in acidic electrolyte, which demonstrates a new route to prepare sustainable and effective electrocatalysts as alternatives to non-Pt catalysts for HER.Herein, we develop an efficient strategy to form MoP/NPC which exhibit excellent HER performance. The prepared MoP/NPC catalyst exhibits high efficient hydrogen evolution reaction (HER) activity with a low onset potential of 90 mV, and a small Tafel slope .The surface morphologies of different catalyst samples were tested using an S-4800 SEM instrument  and transmission electron microscopy (TEM). X-ray diffraction  was investigated through radiation of Cu-Kα. X-ray photoelectron spectroscopy (XPS) was measured using Escalab 250 xi , which provided a base pressure of 5 × 102SO4 solution. A carbon rod and a reversible hydrogen electrode were used as a counter electrode and reference electrode, respectively. The work electrode was modified with a catalyst layer by dropping 5 µL of catalyst ink on the glassy carbon electrode (GCE) with a diameter of 3 mm. All the linear sweep voltammetry (LSV) curves were obtained at scanning rate of 5 mV s−1. All potentials were calibrated without iR compensation. The frequency of electrochemical impedance spectroscopy (EIS) measurements ranged from 100 to 0.1 Hz at the potential of −0.25 V.Hydrogen evolution reaction activity of the as-prepared catalysts were performed in a standard three-electrode electrochemical cell in argon 0.5 M HThe microstructure and morphology of the as-prepared catalysts were investigated using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). As shown in 4+ (228.2 and 231.6 eV) and Mo6+ (233.2 and 236.3 eV) in MoP/NPCs may be caused by the surface oxidation suffering from air contact [X-ray photoelectron spectroscopy (XPS) tests were conducted to investigate the composition and chemical valence of the prepared catalysts. In line with the elemental mappings in  contact . For the contact . The pea contact . X-ray d contact .j + a  [−1), the linear portions were consistent with the Tafel equation, revealing the HER proceeds through a Volmer–Heyrovsky mechanism. The smaller Tafel slope for MoP/NPC-400 mg, MoP/NPC-800 °C indicates the better catalytic activity for HER. Overall, the optimal dosage of ammonium molybdate is 400 mg, and pyrolysis temperature is 800 °C.Hydrogen evolution reaction electrocatalytic properties of different contents were investigated by a standard three-electrode cell in which the carbon rod was used as counter electrode suppressing interruptions of platinum . Figure e slope) . Tafel s−2). Overpotentials of 205 mV is needed to conduct a current density of 10 mA cm–2 for MoPON/C, demonstrating the relatively inferior HER activities of this catalyst compared with that of MoP/NPC (180 mV). In addition, the current density of MoP/NPC is higher than that of MoPON/C at the range of potential. To investigate the HER reaction mechanisms of these catalysts, Tafel plots derived from the polarization curves are shown in −1 for MoP/NPC, slightly larger than that of Pt/C (37 mV dec−1), confirming the highly efficient kinetics of MoP/NPC for HER, which follows a Volmer−Heyrovsky mechanism [echanism . dl) for MoP/NPC was 81 mF cm−2, which was much higher than MoPON/C (12 mF cm−2), indicating a larger ECSA for the MoP/NPC. This phenomenon may be the result of the lattice of MoP, which can expose more electrochemically active sites. Further, electrochemical impedance spectroscopy (EIS), which reflects the charge transfer kinetics [The electrochemical active surface area (ECSA) is regarded as a quantitative index for surface roughness. For that, the cyclic voltammograms (CVs) of MoP/NPC and MoPON/C were measured in the region of 0.1–0.2 V . As demokinetics , were al−1), as well as extraordinary stability in acidic electrolyte. Therefore, this work may open up a new strategy for the design and synthesis of sustainable and effective electrocatalysts as alternatives to non−Pt catalysts for HER.In summary, MoP decorated on NPCs as an efficient electrocatalyst for HER was successfully synthesized via annealing a mixture of ammonium molybdate and phytic acid. The MoP/NPC catalyst delivers highly efficient HER activity with a low onset potential of 90 mV, and a small Tafel slope (71 mV dec


Escherichia coli strains. The aim of this work was to determine whether two isothiocyanates, sulforaphane (SFN) and phenethyl isothiocyanate (PEITC), similarly as in E. coli induce stringent response in Bacillus subtilis, model Gram(+) bacterium, and test their potency against a panel of clinical isolates belonging to Gram(+) or Gram(−) groups. Minimal inhibitory concentrations were determined as well as effect of ITCs on membranes integrity, synthesis of DNA, RNA and stringent response alarmones was assessed. SFN and PEITC are effective against B. subtilis and bacterial isolates, namely E. coli, K. pneumonia, S. aureus, S. epidermidis and E. faecalis. Interestingly, in B. subtilis and E. faecalis the inhibition of growth and nucleic acids synthesis is independent of ppGpp accumulation. In bacteria, which do not induce the stringent response in the presence of ITCs, membrane integrity disruption is observed. Thus, ITCs are effective against different pathogenic bacteria and act by at least two mechanisms depending on bacteria species.Isothiocyanates (ITCs) derived from cruciferous plants reveal antibacterial activity, although detailed mechanism is not fully elucidated. Recently it has been reported that ITCs induce the stringent response in  At the same time, efforts to develop new antibacterial drugs by pharmaceutical industries have declined substantially. Thus, an increasing number of patients suffer from diseases caused by bacteria which are not sensitive to currently available drugs. It results in increased morbidity and mortality, longer hospitalization and health care costs3. They are generated from glucosinolates, secondary metabolites found abundantly in plants from Brassicaceae family, which are hydrolyzed by a β-thioglucosidase (myrosinase) released after plant tissue damage, or myrosinase derived from mammalian gastrointestinal tract microflora4.Plants pose a valuable source of new biologically active compounds with antibacterial activity. Among them, isothiocyanates (ITCs) have received increasing attention due to their chemopreventive, anticancer as well as antimicrobial activities coupled with moderate toxicity towards normal human cells5. Its use was approved in Japan for food preservation6. Another aliphatic ITC, sulforaphane (SFN), has shown activity against Helicobacter pylori both in vitro and in vivo7. It also effectively inhibited a panel of Gram(−) bacteria, including Escherichia coli and Pseudomonas aeruginosa, and Gram(+) bacteria, such as Enterococcus faecalis, Staphylococcus aureus or S. saprophyticus isolated from human gastrointestinal tract8. Antimicrobial activity of aromatic ITC, 2-phenylethyl isothiocyanate (PEITC), has been described for harmful intestinal bacteria  while it had no effect on growth of beneficial bifidobacteria and lactobacilli, as assessed using disc-diffusion assay9. In another study, agar disc diffusion assay showed that PEITC was more effective against Gram(+) bacteria  than Gram(−) bacteria 10. Recently, ten ITCs naturally found in cruciferous vegetables, including SFN, AITC, BITC and PEITC, have been tested for 14 bacterial strains growing in BHI medium . Results indicated that in most cases bacteria were sensitive to the ITC tested, however the extent of antimicrobial activity depended on the ITC and the strain considered11.One of the first investigated ITCs was allyl isothiocyanate (AITC) which has been shown to effectively inhibit different pathogenic microorganisms11. The mechanism of antimicrobial action of ITCs is not well understood. ITC group is highly electrophilic and reacts with amine, thiol or hydroxyl groups, thus it may bind to cellular targets influencing their activity and function. For instance, AITC inhibited activity of thioredoxin reductase and acetate kinase, which are responsible for important metabolic reactions in bacteria12, however, existence of ITC-enzyme conjugates has not been proven. It has been also reported that phenyl ITC (PITC), AITC and PEITC had strong antimicrobial activity through the disruption of the bacterial cell membrane function which led to a loss of its integrity and subsequent cell death14. Recently, it has been shown that PEITC, SFN, AITC, benzyl isothiocyanate (BITC), PITC and isopropyl isothiocyanate (IPRITC) efficiently inhibited growth of Shiga toxin harboring E. coli strains which was accompanied by an inhibition of the lytic development of Shiga toxin-converting bacteriophage and stx gene expression. Moreover, these effects were mediated by alarmones of the stringent response16.Most reports are limited to determination of minimal inhibitory concentration (MIC), which may vary depending on a method used. There is no general rule concerning the efficacy of ITCs toward various bacteria but aromatic ITCs seem to be more active that aliphatic ones18. The stringent response is aimed to reprogram cellular metabolism in order to downregulate the active growth and energy consuming processes and to activate those necessary for survival and adaptation to the challenging environmental conditions. For instance, during amino acid starvation, transcription from promoters of genes coding for stable RNA (rRNA or tRNA) or ribosomal proteins is inhibited and transcription from promoters for amino acids transport and biosynthesis is activated19. One of crucial processes which are affected under conditions of the stringent response is DNA replication. Its downregulation, however, may be achieved at initiation or elongation stage, thus it might not be detectable shortly after stringent response induction but after longer times20.The stringent response is one of the most far-reaching global regulatory mechanisms in bacteria. The unusual nucleotides, ppGpp and pppGpp (referred together to as (p)ppGpp) are synthesized promptly after the onset of starvation and various physical and chemical stresses. In addition to the stress response, (p)ppGpp regulates also other processes such as biofilm formation, quorum sensing, and bacterial virulenceE. coli and some of other gamma- and beta-proteobacteria harbor two enzymes, synthetase RelA  and bifunctional SpoT, responsible for both synthesis and hydrolysis of (p)ppGpp. While most of other bacterial species, including Gram(+) bacteria, have single bifunctional enzyme, named generally RSH (Rel/Spo homolog)21. Nevertheless, in both genetic arrangements, dysfunction of these enzymes is not lethal for bacteria, but leads to the distinct phenotype, named ppGpp-null, characterized by impaired stress response, decreased survival in nutrient limiting conditions and auxotrophy to several amino acids22.(p)ppGpp metabolism in the cell is under the control of specific enzymes able to synthesize and hydrolyze these nucleotides. E. coli the (p)ppGpp-mediated regulation takes place mainly at the level of gene expression, with RNA polymerase as a main target of the alarmone direct and indirect effects. On quite a contrary, in many Gram(+) bacteria, including model Bacillus subtilis, (p)ppGpp acts by different strategy; it does not interact directly with RNA polymerase, but its main effect is exerted by controlling the cellular level of GTP23. Depleting the cells of GTP, achieved both by lowering the pool of available GTP through (p)ppGpp synthesis and inhibiting the enzymes from GTP synthesis pathway by (p)ppGpp, leads to decreasing of transcription from GTP-starting promoters (including rRNA promoters)25. This strategy of (p)ppGpp-mediated regulation has been reported for many Gram(+) bacterial species, including E. faecalis, S. aureus, L. monocytogenes and others28.Despite the presence of (p)ppGpp in majority of free living bacteria, the mechanism of both synthesis and action of this alarmone can differ between bacterial species. In E. coli and B. subtilis, both wild type and defective in stringent response induction (ppGpp-null variants). Next, we tested activity of ITC toward bacteria belonging to opportunistic human pathogens   and aromatic (PEITC) ITC towards different Gram(−) and Gram(+) bacteria and to correlate it with ppGpp production. First, we compared response to ITC of model strains of s) Table . MajoritE. coli16. To determine whether tested ITCs have similar mechanism of action in Gram(+) bacteria, we used B. subtilis as a model representative of this group. E. coli cells were more sensitive to tested ITC than B. subtilis: MIC for SFN was 88.6 and 177.3 mg/L, and for PEITC – 40.8 and 163.3 mg/L, respectively. Interestingly, E. coli strain unable to produce stringent response alarmones (ppGpp-null) was more sensitive to ITCs than wt strain while sensitivities of wt and ppGpp-null B. subtilis strains were similar   Fig. . Inhibitone Fig. . ITC inhone Fig. . It is wlls Fig. .Figure 2E. coli and B. subtilis and their ppGpp-null derivatives were treated with ITC at different concentrations  and their viability was evaluated at different time points. For E. coli lack of ppGpp resulted in an increased sensitivity to high concentrations (2 and 4x MIC) of both tested ITCs  and as high as 555 mg/L  and for PEITC they are as low as 41 mg/L  and as high as 327 mg/L . Since the composition of growth medium may influence the susceptibility to tested compounds, MIC values were determined for bacteria growing on Mueller-Hinton (MH) or brain heart infusion (BHI) media which are both rich media recommended by the National Committee for Clinical Laboratory Standards. Effect of different concentrations of ITCs on growth kinetics of selected bacteria is shown in Fig. We compared activity of SFN and PEITC on clinical isolates of a broader panel of pathogenic bacteria. Table E. coli, K. pneumonia, S. aureus and S. epidermidis. We did not notice the presence of these alarmones in SFN- or PEITC-treated E. faecalis, although PEITC quite efficiently inhibited growth of this bacterium. SHX, which is a positive control for the stringent response induction, elevated (p)ppGpp in each tested bacterial strain which indicates that stringent response in these isolates functions properly. Interestingly, SFN and PEITC inhibited stable RNA synthesis in all tested bacteria, even in E. faecalis ppGpp have been evaluated for selected strains. Results presented in Fig. lis Fig. . DNA synlis Fig. .Figure 513. Our own results indicated that ITCs, including SFN and PEITC, had no significant effect on membrane integrity in E. coli16. However, our experiments were performed in wt strain of E. coli which was able to induce the stringent response upon ITCs treatment, and the induction of the stringent response may, as an indirect effect, lead to the changes in membrane properties29. Thus, we assessed the membrane integrity using propidium iodide test in ppGpp-null E. coli as well as B. subtilis and selected clinically isolated bacteria. As evidenced in Fig. E. coli which are impermeable to propidium iodide, the ppGpp-null derivatives stain positively when treated with SFN or PEITC. Interestingly, in B. subtilis the fluorescence signal indicating the disruption of membrane integrity is visible in both, wt and ppGpp-null strains treated with ITCs. It again supports the notion that ITCs do not induce the stringent response in B. subtilis, which would otherwise protect against membrane stress. Similarly, E. faecalis sensitivity to ITCs relies on the loss of membrane integrity probably due to lack of stringent response induction ppGpp, alarmones responsible for, among others, growth retardation and inhibition of stable RNA synthesis or Shiga toxin-converting phage development16. Here we show that SFN and PEITC inhibit growth of other bacteria, including clinical isolates of E. coli, K. pneumoniae, S. aureus, S. epidermidis, E. faecalis. Their sensitivity to ITC varies and the more susceptible are reference than clinically isolated strains: MIC values for E. coli, S. aureus or S. epidermidis ATCC strains are in 44–177 mg/L range for SFN and 41–82 mg/L for PEITC depending on medium, while for their clinical isolates are 82–89 mg/L for S. aureus 6285 or higher than 555 mg/L for K. pneumoniae 1077 treated with SFN. Similar MIC values have been reported by others14, although Ko et al. showed that E. faecalis is quite resistant to SFN and PEITC (MIC = 1 g/L or higher)30. Such discrepancy between MIC values might result from different strains and media used in our study compared with previously published work.Recently we reported that the antibacterial activity of ITCs against enterohaemorrhagic B. subtilis which does not elevate (p)ppGpp levels after ITC treatment. Interestingly, ITCs in both wild type and ppGpp-null strains of B. subtilis inhibit nucleic acids synthesis and cell viability. The explanation for this phenomenon might be a drop in GTP level which we observed in ITCs-treated B. subtilis cells. The regulation of the GTP levels underlies the effects of the stress response of Gram(+) bacteria, e.g. rRNA promoter transcription. The decrease in GTP level under ITC treatment may be responsible for observed RNA synthesis down-regulation. In our case we did not observe (p)ppGpp production in B. subtilis treated with ITCs, thus it seems that GTP drop is mediated by some other mechanisms. Similar effect, i.e. lack of stringent response induction but inhibition of growth and nucleic acids synthesis by ITCs accompanied by decrease in GTP level, we observed in another Gram(+) bacterium, namely E. faecalis.What is interesting, growth inhibition by ITC is independent of (p)ppGpp production in some of tested bacteria. The example is E. coli, L. monocytogenes, S. aureus and P. aeruginosa, and when used at high concentrations , they modified physicochemical properties of bacterial surface which led to cytoplasmic membrane permeabilization14. Here we confirm that SFN and PEITC compromise the integrity of cytoplasmic membrane, however this is evident only in these strains which do not produce (p)ppGpp upon ITCs treatment, i.e. B. subtilis, E. faecalis and E. coli ppGpp-null variant, and might be the main mechanism underlying antimicrobial activity of ITCs. Interestingly, strains able to induce stringent response, in addition to the observed inhibition of RNA and DNA synthesis, do not react to ITCs treatment with increased membrane permeability, even treated with very high concentrations of ITC . This is in the agreement with reports showing that ppGpp changes the rate of phospholipid synthesis and increases proportion of saturated to unsaturated fatty acids which results in reduction of membrane permeability in amino acid starved E. coli32 and the generalized response to the envelope stress, e.g. in S. aureus27, and in E. coli33. Such response provides a survival advantage in challenging conditions with the example presented by our results  where wild type E. coli exposed to high concentrations of ITCs (2 or 4x MIC) reveals better survival than its ppGpp-null derivative in prolonged tests. The difference in the response to ITCs treatment dependent on the presence of (p)ppGpp indicates that in the wild type cells induction of the stringent response prevents the membrane disruption by ppGpp-mediated gene expression changes leading to the higher tolerance to the envelope stress.It has been shown that AITC and PEITC were effective against E. coli in the presence of PEITC15; and glycine, cysteine, arginine, tryptophan and aspartic acid decreased sensitivity to SFN16. These results suggest that ITCs might bind and titrate these amino acids or block the aminoacylation of relevant tRNA (e.g. by inhibiting a specific aminoacyltransferase), thus elevating their level in cells would prevent the stringent response induction. It is also possible that in some bacteria titration of specific amino acids by ITCs is not a rate limiting step for stringent response induction, however, such hypothesis needs further experimental validation.The question arises about the mechanism of species-specific induction of the stringent response by ITCs. Our previous results indicate that supplementation with high concentrations of specific amino acids reversed inhibitory potential of ITCs. For example, supplementation of glycine, arginine, or to lesser extent lysine, methionine, phenylalanine, serine or threonine restored growth of 34 and modulate numerous cell signalling pathways, some – specific for particular cell line2. Here we show that ITCs act in at least two ways in bacteria, but – interestingly – the outcome is similar, namely bacterial growth inhibition. This is of great importance for the potential use of ITCs as the antimicrobial agents in the pathogenic bacteria infections.Concluding, our results indicate that ITCs may have more than one target in the bacterial cells, and the mechanism of their antibacterial activity is species-specific. Noteworthy, SFN was reported to have various targets in eukaryotic cellsBacterial strains used in this study are listed in Table Antimicrobial activity of SFN and PEITC  was assessed by determination of the MIC in accordance with CLSI guideline M07-A9. MICs were assessed in two-fold broth microdilution assay and concentrations range from 12.5 to 800 mg/L were used. Zone inhibition tests were assessed by Kirby Bauer disc diffusion method using 6 mm membrane discs . Cell concentration of tested microorganisms was adjusted at 0.5 McFarland turbidity standards and inoculated on MHA plates and diameters of growth inhibition were measured after 20-h incubation in 37 °C.15. Briefly, bacteria were grown in MH in 37 °C to A600 of 0.1 and the radioactive precursor of DNA or RNA synthesis, [3H]thymidine and [3H]uridine, respectively, was added at 5 µCi. This time was set as time zero and SFN and PEITC in range of concentrations were added to the cultures. Samples (50 µl) in indicated time were collected on Whatmann 3MM filter papers and transferred to ice-cold 10% trichloroacetic acid (TCA) for 10 min, washed in 5% TCA and in 96% ethanol. After drying of filters, the radioactivity was measured in a scintillation counter MicroBeta . Results (expressed in cpm) from three independent experiments were normalized to bacterial culture density (A600) and presented as mean values with SD.The assessment of nucleic acids synthesis was performed as described600) in the presence of relevant concentrations of ITCs. Bacterial cultures were grown in 37 °C to early exponential phase A600 of 0.1 and treated with indicated compounds. Independent experiments were assessed at least in triplicate and results were expressed as mean ± standard deviation (SD).Growth inhibition was measured spectrophotometrically  minimal medium with the addition of [32P]orthophosphoric acid (150 μCi/ml) and grown for at least two generations in 37 °C. Then, culture was divided into new flasks and ITCs (1/4xMIC) or SHX, RHX (1.5 mg/ml both) were added at time zero. The untreated culture was used as a negative control. Bacterial samples, collected at time points, were lysed with formic acid (13 M) in three cycles of freeze-thaw procedure. After centrifugation, nucleotides extracts were separated by thin-layer chromatography, using PEI cellulose plates in 1.5 M potassium phosphate buffer. The chromatograms were analysed in phosphorimager .pppGpp and ppGpp levels were measured basically as described600 of 0.1 in the same conditions as for growth inhibition experiment. Then, 1 ml of cultures were transferred to Eppendorf tubes 1.5 ml and treated with ITCs in 4xMIC concentration by 1 hour in 37 °C. To determine membrane integrity bacterial cells were washed and resuspended in 1 ml PBS and stained with propidium iodide (IP) 10 µM for 10 min. Bacteria were collected by centrifugation  and washed twice in PBS to remove remaining dye from supernatant. Fluorescence signal from permeabilized cells was analysed using Leica DMI4000B microscope with dedicated filter No. N2.1. Images were collected and processed using LAS AF 3.1 software (Leica). Experiments were repeated at least three times. Results were expressed on photographs as merge of transmitted light- Differential Interference Contrast (DIC) and fluorescence channel.Bacteria were grown with shaking in 37 °C to early exponential phase ASupplementary data