Following one of the world’s largest nuclear accidents, occured at Fukushima, Japan in 2011, a significant scientific effort has focused on minimizing the potential adverse health effects due to radiation exposure. The use of natural dietary antioxidants to reduce the risk of radiation-induced oxidative DNA damage is a simple strategy for minimizing radiation-related cancer rates and improving overall health. The onion is among the richest sources of dietary flavonoids and is an important food for increasing their overall intake. Therefore, we examined the effect of an onion extract on cyto- and geno-toxicity in human lymphocytes treated with bleomycin (BLM), a radiomimetic agent. In addition, we measured the frequency of micronuclei (MN) and DNA damage following treatment with BLM using a cytokinesis-blocked micronucleus assay and a single cell gel electrophoresis assay. We observed a significant increase in cell viability in lymphocytes treated with onion extract then exposed to BLM compared to cells treated with BLM alone. The frequency of BLM induced MN and DNA damage increased in a dose-dependent manner; however, when lymphocytes were pretreated with onion extract (10 and 20 μL/mL), the frequency of BLM-induced MN was decreased at all doses of BLM and DNA damage was decreased at 3 μg/mL of BLM. These results suggest that onion extract may have protective effects against BLM-induced cyto- and genotoxicity in human lymphocytes. After one of the world’s largest nuclear accidents at the Fukushima Daiichi Nuclear Power Plant, caused by the East Japan Earthquake of 2011, the potential adverse health effects of radiation exposure have become a major concern worldwide .Bleomycin (BLM) is considered a radiomimetic agent because of the similarities between its genotoxicity profile and that of IR . Most noDiets high in fruits and vegetables are known to be protective against a variety of diseases. Particularly, antioxidants are thought to be the principal nutrients responsible for the protective effects of fruits and vegetables . There iAllium cepa L.) are one of the most widely consumed vegetables worldwide. Since onions contain large amounts of flavonoids and constitute one of the major sources of flavonoids in the human diet [Onions , removed from the interface, washed twice, and resuspended in RPMI 1640  that was supplemented with 10% fetal bovine serum (Gibco) and 100 U/mL penicillin/streptomycin . The cultures were stimulated with 1% phytohemagglutinin . The PBMCs were cultured in 5% CO2 at 37 °C.Peripheral blood was obtained from one consenting healthy female donor aged 28. The donor was a non-smoker at the time of blood draw. Separation of peripheral blood mononuclear cells (PBMCs) was carried out under sterile conditions on Ficoll-Paque gradients by the method of Boyum . In brieAllium cepa L., Turbo) were purchased from a local market. The onions were washed with sterile distilled water and allowed to air dry for one hour. The outer covering of the onions were manually removed and the peeled onions were then placed into alcohol (60%) and extracted for 12 h at stable pressure (1–2 kg/cm2) and temperature (85 °C–90 °C). Prior to treatment, the onion extracts were filtered using filter paper .Fresh onions  were added to the culture and incubated for an additional 3 h. The onion extract and BLM treated cells were washed with phosphate-buffered saline (PBS) twice and onion extracts and PHA were reapplied to the cells a second time.6 cells/mL for the trypan blue exclusion assay [The PBMCs were grown at a density of 1 × 10on assay . Cells wper 2000 BN cells.The cytokinesis block micronucleus assay was performed as described by Fenech . The oniet al., [2EDTA, 10 mM Tris-HCl, 1% Triton X-100, and 10% dimethyl sulfoxide at pH 10. The slides were placed on a horizontal gel electrophoresis unit. DNA was allowed to unwind for 20 min in an electrophoretic alkaline buffer  and electrophoresed in the same buffer for 25 min at 0.78 V/cm and 330 mA. Once electrophoresis was complete, the slides were gently washed in a neutralization buffer . Subsequently, the slides were dried and fixed with absolute ethanol for at least 60 s. Immediately before examination, the slides were stained with 100 μL ethidium bromide . For visualization of DNA damage, observations were made using the 40× objective of a fluorescent microscope  that was equipped with an excitation filter of 515–560 nm and a barrier filter of 590 nm. Images of 60 randomly selected cells from each sample were examined using the KOMET5.5 image analysis system  [The alkaline comet assay was performed as described by Singh et al.,  to assesast, UK) ,27.LC-MS/MS analysis was performed by triple quadrupole mass spectrometry coupled with an HPLC system  to determine the concentration of quercetin, the major flavonoid in onion extract. Quercetin was separated on a Zorbax Extend-C18 column . The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) with a flow rate of 0.2 mL/min. The gradient conditions were set as follows: 10%–50% B for 10 min, isocratic 50% B for 2 min and 10% B for 7 min to equilibrate the column. Injection volume was 10 μL. The concentrations used for the standard curve were 0.1, 0.2, and 0.5 μM. Quercetin was monitored in the negative ion mode with electrospray ionization. The optimum MS conditions were as follows: spray voltage 3000, capillary temperature 300 °C, and sheath gas pressure and aux gas pressure of 40 (psi) and 10 (Arb), respectively. Quantitative analysis of quercetin was performed using selective reaction monitoring mode (m/z 301/150.980). Data processing was performed using the Xcalibur software (Thermo Scientific).p < 0.05 was considered statistically significant.Statistical analysis was performed using the SPSS 10.0 for Windows statistical package . All experiments were performed in triplicate, and the inter-group difference among three experiments was tested by analysis of variance (ANOVA). The association of abnormal yields with BLM doses was tested by the Kendall rank correlation coefficient (τ). The variations in cell viability, MN frequency, and cells with DNA damage among different treatment groups were tested with the Mann-Whitney and Wilcoxon tests. Data are expressed as means ± SD or SEM of three different experiments, and p < 0.001; p < 0.05), suggesting the onion extract at a high dose caused cytotoxicity. After treatment of 1 μg/mL of BLM to the PBMCs, there was a significant decrease in the number of trypan blue-negative cells at all time points examined compared to the control cells (p < 0.01). In contrast, supplementing BLM-treated cells with onion extract (20 μL/mL) increased the number of trypan blue-negative cells compared to cells treated with BLM alone (p < 0.05).The viability of PBMCs treated with various concentrations of BLM  was determined using a Trypan blue-exclusion assay. BLM-treated cells exhibited significantly decreased viability compared to the controls and/or DMSO treated cells after 24 hours. BLM treatment decreased the number of viable cells in a dose-dependent manner as indicated by the number of trypan blue-positive cells (p < 0.001). The total MN frequencies in control was 18 ± 2.65, and increased to 36 ± 6.24 and 60 ± 4.36 after 1 and 3 μg/mL of BLM, respectively. The frequency of MN induced by 1 μg/mL of BLM exposure was significantly reduced by pretreating the cells with 20 μL/mL of onion extract, while 3 μg/mL of BLM-induced MN was significantly reduced by both 10 and 20 μL/mL of onion extract (p < 0.05).The CBMN assay was used to determine the effect of onion extract on BLM-induced genotoxicity. The micronucleated cytokinesis-blocked (MNCB) cells and MN frequencies were significantly increased after BLM exposure in a dose-dependent manner as shown in p < 0.001). Pretreatment of cells with both 10 and 20 μL/mL of onion extract significantly decreased the DNA damage induced by 3 μg/mL of BLM (p < 0.05).The comet assay, a conventional measure of DNA damage, was used to determine DNA single-strand breaks induced by BLM with or without onion extract. HPLC analysis confirmed the presence of quercetin in onion extracts with a concentration of 17.1 μM.After the Fukushima tragedy, the potential adverse health effects of radiation exposure became a major worldwide concern. The World Health Organization (WHO) is currently developing a health risk assessment to provide a preliminary evaluation of radiation exposure . In partCurrently, there is a great deal of interest in the use of dietary interventions to assist in protecting the public’s health from chronic radiation exposure. Specifically, dietary inhibitors of mutagenesis and carcinogenesis may be helpful for preventing human cancer and improving overall health . It has et al., recently showed that onion extracts have a protective effect on cadmium-induced oxidative stress [et al., reported that pretreatment with onion extract protects against doxorubicin-induced hepatotoxicity due to its antioxidant properties [et al., reported anti-clastogenic effects of flavonoid derivatives against induction of chromosomal aberrations following BLM exposure [The results of this study demonstrate that pretreatment of normal human PBMCs with an onion extract results in a significant increase in cell survival upon exposure to BLM-suggesting that the onion possesses significant activity as a cyto-protective agent. We determined the protect effects of onion extract on BLM-induced genotoxicity in lymphocytes by using CBMN and single-cell gel electrophoresis assays. Data presented in e stress . Similaroperties . In addioperties ,41. Simiexposure . Furtherexposure ,44.et al., showed that flavonoids can scavenge free radicals and chelate iron, thereby preventing hydroxyl radical-promoting Fenton reactions [et al., [The antioxidant effect of onion extract is mostly due to phenolic acids and flavonoids, which constitute the most predominant functional components . These ceactions . The proeactions . A study[et al.,  reported[et al., . HoweverExtraction of flavonoids using water is known to be inefficient since the solubility of flavonoids in water at ambient conditions is low . TherefoOnions contain various flavonoids such as quercetin, ferulic acid, gallic acid, protocatechuic acid, and kaempferol, which contribute to the radical scavenging and metal ion chelating properties and result in significant anti-genotoxic and protective effects . Howeverin vitro experiment. Therefore, caution is required when extrapolating this data to estimate the daily onion intake required for prevention of BLM-induced toxicity in vivo. Despite these limitations, our study provides useful information. To our knowledge, this is the first study to evaluate the anti-genotoxic and protective effects of onion extract against BLM-induced DNA damage in human lymphocytes in vitro.An epidemiology study measuring cancer risk among a group of atomic bomb survivors showed daily intake of vegetables and fruit reduced the risk of radiation-associated cancer . HoweverThe present study demonstrates protective effects of onion extract against the cytotoxicity and genotoxicity of BLM, a radiomimetic agent. Here for the first time, we provide confirmatory evidence for nutritional protection, particularly by onion extract containing abundant flavonoids, against DNA damage induced by a radiomimetic agent in human lymphocytes. Our results may provide useful information that can be used to set up strategies to minimize radiation-induced health effects.


Two types of waste material of corncob  and concrete  were used as the carrier for microorganisms' attachment. During operation, both reactors achieved absolutely high performance of ammonium removal (up to 99%) and total nitrogen removal (up to 95%). The significant advantage of corncob carrier was that the corncob was able to be a source of carbon for biological denitrification, leading to no external carbon requirement for operating the system. However, the corncob caused an increasing turbidity of the effluent. On the other hand, the concrete carrier required the minimal external carbon of 3.5 C/N ratio to reach the good performance. Moreover, a longer period for microorganisms' adaptation was found in the concrete carrier rather than the corncob carrier. Further, the same physiological and biochemical characteristics of active bacteria were found at the two carriers, which were negative gram, cocci shape, and smooth and white-turbid colony. Due to the effluent quality, the concrete was more appropriate carrier than the corncob for wastewater treatment.This research focused on the ammonium-nitrogen (NH Domestic wastewater contains high concentration of nitrogen which is from products through household consumption and human metabolism. When the large volume of domestic wastewater is discharged to the natural water resource, it can cause the environmental problem of eutrophication or algae bloom. The consequences are depletion of dissolved oxygen, water quality degradation, death of aquatic lives, and odor and taste problems. The enormous damage to ecological, health, and economic values from the eutrophication has become serious issues in recent decades such as swimming bans in De Kuil Lake in Netherlands , less ab4-N) is converted to nitrite-nitrogen (NO2-N) and continued to nitrate-nitrogen (NO3-N) by autotroph (named nitrification process), and later NO3-N is reduced to nitrogen gas (N2) by heterotroph (named denitrification process). Since the organic carbon contained in the domestic wastewater was low and insufficient for complete denitrification, the external carbon including acetate and ethanol is necessary for the SND system. However, an overdose of external carbon can cause another contamination of organic carbon which presents in the parameter of biological oxygen demand (BOD), and the BOD contamination requires further treatment. In addition, the traditional SND of suspended sludge has some drawbacks including long lag phase for biomass adaptation, washout of biomass, and sensitive system to pH and loading [Recently, various biological technologies have been proposed for efficient nitrogen removal from wastewater including simultaneous nitrification and denitrification: SND , anammox loading .An attached growth reactor has been proposed to overcome the drawbacks of the traditional SND system. In the attached growth reactor, the carrier is used for biomass attachment and resulted in high active microorganisms in the system. The carrier is broadly categorized into two types: biodegradable and nonbiodegradable materials. The common biodegradable carriers are organic polymers such as polycaprolactone and polyacetic acid. The small diameter of 4 mm polycaprolactone carrier was packed in the reactor, which was operated under the nitrogen concentration of 40–50 mg/L and the COD/TN of 3.5–8.0. The nitrogen removal efficiency was around 57–60% . The devIn this research, two waste materials of corncob and concrete were used as carrier in the attached growth reactor. The corncob which is biowaste represented the biodegradable material and the concrete which is construction waste represented the nonbiodegradable material. The performance of using both carriers on simultaneous nitrification and denitrification was compared. Further, the preliminary study on the attached biomass was also discussed.The corncob waste was collected from a local market in Phitsanulok, Thailand. The corncob was washed several times to remove the dirt and minerals and then cut into a 2 × 2 × 2 cm size. After that, the corncob carrier was dried at 105°C for two days in a furnace to remove moisture and fungus. Similarly, the concrete waste was collected from a constructed site in the same area. The concrete was washed, cut in a 2 × 2 × 2 cm size, and dried at 105°C for two days. The corncob and concrete carriers were added to the dense activated sludge tanks under continuous air supply for two days. The biomass carriers were transferred to the reactors; the reactor containing corncob carrier was named the corncob reactor and the reactor containing concrete carrier was named the concrete reactor.The schematic of attached growth reactor is shown in The corncob reactor was continuously operated for 25 days under the condition of no external carbon  in the influent. On the other hand, the concrete reactor was operated under three phases: no external carbon for 18 days, low carbon (C/N = 2.5) for 30 days, and high carbon (C/N = 3.5) for 30 days : NHt 0.5 mL . The NH44-N, NO2-N, and NO3-N analysis in accordance with the standard method [4-N removal efficiency and total nitrogen removal efficiency were calculated as present in  [4-N concentration was approximately 20 mg/L in the effluent. The low NO3-N was observed, while the NO2-N concentration was high of 15 mg/L  , 18. Und 15 mg/L . The res4-N and total nitrogen removal efficiencies increased to 85% and 60%, respectively. This is because the amount of active biomass was increasing by operation time. The low NH4-N and NO2-N concentrations were detected, while NO3-N was suddenly increased to the maximum value of 12 mg/L. The low concentration of carbon in this phase verified that the low C/N ratio of 2.5 was insufficient for complete denitrification. At the high external carbon of the C/N of 3.5, the excellent NH4-N and total nitrogen removal efficiencies of 95% were achieved, and all the concentrations of NH4-N, NO2-N, NO3-N, and organic carbon met the effluent standard. In addition, the water quality of effluent was relatively good; the pH was around 7.8 and turbidity was <1 NTU. The concrete was more appropriate carrier than the corncob for biological nitrogen removal at 40 mg/L of NH4-N concentration. It has to be noted that the result might be different at other NH4-N concentrations because at high NH4-N concentration the effluent carbon and the turbidity may be dropped.Under the low external carbon of the C/N ratio of 2.5, the NH4-N of 40 mg/L, the NH4-N concentration was immediately dropped to 8 mg/L, due to the dilution of water in the reactor. In 4-N was sharply reduced and changed to NO3-N. This phenomenon refers to the fact that the nitrification completely occurred. However, the NO3-N concentration kept increasing and reached the highest concentration of 7.3 mg/L at hour 4. This is because the nitrification still occurred using the remaining oxygen. At hours 4 to 8 (nonaeration period), when the DO was nearly zero, the NO3-N concentration was decreasing by denitrification and the NH4-N was kept to increase. The increase in NH4-N concentration was due to the continuous influent feeding and ineffective nitrification occurred. It can be concluded that there were two mechanisms of nitrification and denitrification which occurred in the attached growth reactor; the nitrification mainly occurred in aeration and the denitrification mainly occurred in nonaeration.Since the concrete reactor obtained better performance in terms of removal efficiency and water quality, its mechanism of nitrogen removal was identified by collecting the in situ water for eight hours of a cycle period. During continuously feeding the influent NHIn this research, the physiological and biochemical characteristics including gram stain, shape, and colony were used to broadly categorize the group of bacteria on the carriers. In the corncob reactor, the bacteria on the corncob surface consisted of eight isolates which were categorized into two different groups see . Both grOn the other hand, the characteristics of abundant bacteria on the concrete carrier were the same as found in the corncob carrier. However, the other minor group of bacteria was found at the surface and core. Since no initial bacteria were detected in the unused concrete carrier, the attached bacteria were from the initial biomass. It can be concluded that the bacteria responsible for nitrogen removal in the corncob and concrete reactors were negative gram, cocci shape, positive catalyst test, and smooth and white-turbid colony. However, further microbial analysis is required to identify the bacteria species.4-N and total nitrogen removal. The significant advantage of using corncob carrier was no external carbon. This is because the corncob carrier can release the soluble carbon which was used for denitrification. However, the effluent of corncob reactor contained high turbidity and carbon from corncob degradation. On the other hand, the concrete reactor required the external carbon to achieve high efficiency, in which the minimum value was the C/N ratio of 3.5. In comparison, the concrete was more appropriate carrier than the corncob for nitrogen removal. In addition, the characteristics of abundant bacteria for both carriers were similar which were negative gram, cocci shape, and smooth and white-turbid colony.To utilize the waste material for water treatment, the simple two attached growth reactors were developed to use corncob and concrete as the biomass carrier. The results show that both carriers were good carrier for biomass attachment and the performances were greater than 95% for NH


In 2006, a study investigating knowledge and attitudes regarding antibiotic use and resistance in Sweden, indicated high level of knowledge but also areas in need of improvement.(i) To provide an update on the knowledge and attitudes to antibiotic use and resistance of the Swedish population, and (ii) to identify which groups within the population are in particular need of improved knowledge or attitudes.A questionnaire was sent by post in 2013 to 2,500 randomly-selected individuals aged 18–74, living in Sweden. Latent class analyses were conducted to group respondents based on their responses. The association between socio-demographic characteristics and the probability of belonging to each latent class was assessed.The response rate was 57%. Ninety-four per cent of the responders knew that bacteria could become resistant to antibiotics and the majority answered correctly to the questions regarding antibiotic resistance development. The respondents expressed confidence in doctors who decided not to prescribe antibiotics. Three latent classes related to ‘knowledge regarding antibiotic use and resistance’, two regarding ‘attitudes towards antibiotic accessibility and infection prevention’ and three regarding ‘attitudes towards antibiotic use and effects’ were revealed. Men, younger and more educated people were more knowledgeable but males had a less restrictive attitude. Respondents with high levels of knowledge on antibiotics were more likely to have appropriate restrictive attitudes to antibiotics.Knowledge on antibiotic use and resistance is maintained high and has improved in Sweden compared to 2006. People with lower education and elderly are especially in need of improved knowledge about antibiotic use and resistance. Antibiotic treatment is a pre-requisite for modern healthcare. Despite the fact that antibiotic resistance is becoming an increasing global health challenge, very few new antibiotics have reached the market in the last 30 years.,2 One imIn 2006, we conducted a study on knowledge and attitudes regarding antibiotic use and resistance in a random sample of the Swedish population. The results indicated a relatively high level of knowledge. However, some areas needed improvement; in particular, respondents showed uncertainty as to whether antibiotics are indeed effective against viruses.To further improve antibiotic use and contain resistance through actions such as information campaigns, it is important to have a concrete understanding of the knowledge and attitudes towards antibiotics within different groups of a population, such as age groups, family structure (with or without children), etc. Although antibiotics are prescription-only medicines in Sweden, and this is strictly followed, it is vital that the population is empowered with knowledge on how antibiotics should be used, as well as the risk of resistance. There is a risk that leftover antibiotics may be taken on a later occasion, or given to someone else; alternatively people may consider buying antibiotics abroad or online. Furthermore, understanding attitudes towards antibiotics among the general population is also important; research shows that in cases where patients or patient guardians expect an antibiotic prescription, the likelihood that a doctor will provide a prescription is higher.–13 SinceIn previous studies analysing knowledge and attitudes, typically either individual items or arbitrary scores have been used. However, we decided to use Latent Class Analysis (LCA) in order to provide additional data on differences in the results from different groups of respondent. LCA is a method which strives to place people into classes based on their responses to items in the questionnaire and not arbitrarily by the researchers.In this population-based cross-sectional study, a questionnaire was sent to 2,500 people between 18 and 74 years of age, selected randomly from the Swedish population using SPAR (statens personadressregister), a governmental register which includes all people residing in Sweden, both Swedish and foreign citizens. SPAR provided a list with selected participants’ names and addresses; data on sex and age were not included.The questionnaire in English and Swedish (supplementary data), which was developed in Swedish, was based on the questionnaire used in our earlier study. Some queData were entered manually and checked using EpiData 3.1 . Analyses were carried out using SPSS Software Version 21  and the poLCA package for the open-source software R Version 3.0.2 .Descriptive statistics were used to summarise the numerical (mean and standard deviation) and categorical (frequencies and percentages) variables. Cross-tabulation with chi-square tests and independent t-tests were performed to compare sex and mean age respectively, between respondents and non-respondents. The significance level was set to 0.05.LCA was used to discover underlying response patterns, thus allowing the identification of respondent groups with similar knowledge and/or attitudes.Three fields of interest were defined using selected items from the questionnaire for LCA: (i) knowledge regarding antibiotic use and resistance (10 items from the section on side effects and resistance), (ii) attitudes towards antibiotic accessibility and infection prevention (8 items from the sections on antibiotic accessibility and infection prevention), and (iii) attitudes towards antibiotic use and its effects (9 items from the sections on antibiotic use and its effects). The responses to these items (i.e. manifest variables) were used to categorise respondents into groups with similar response profiles (i.e. latent classes). Consequently, the items were selected assuming that more variation in the answers could be observed, rendering them more suitable for a LCA analysis.Selection of the final LCA models was based on two main criteria: to minimise the values for Akaike Information Criteria (AIC) and Bayesian Information Criteria (BIC), and to obtain a meaningful and interpretable class structure. AIC and BIC are recognised tools for comparing different models in terms of the balance between fit and parsimony. Cases wiMultinomial and binary logistic regression analyses were conducted to investigate which explanatory variables influenced latent class membership. Odds ratios and 95% confidence intervals were estimated. Groups representing the highest level of knowledge or the most appropriate attitudes were used as reference groups. Respondents who had one or more missing values in the manifest variables were excluded from further analyses.I am confident in a doctor’s decision if s/he does not prescribe antibiotics”  was used to create the outcome variable in the ordinal logistic regression model. The new three-level ordinal categorical variable ‘level of confidence’  excluded the ‘do not know’ responses.In addition, the specific item “Six explanatory variables were used for all the regression analyses. These were: (i) sex, (ii) age , (iii) education , upper secondary school , university ), (iv) monthly income ; 1 EURO ≈ 9 SEK), (v) children in the household , and (vi) any medical or healthcare-related education  (yes/no). The ordinal categorical variable ‘knowledge level’ was derived from the LCA model and used as an explanatory variable in the regression models regarding attitudes.Explanatory variables were tested separately with the significance level set to 0.20 for crude associations, while for the final model the significance level was set to 0.05. The parallel lines test was performed to assess possible violations of the proportional odds assumption. In all the models created, the explanatory variables were tested for collinearity and there was none.This study was approved by the Regional Ethics Review Board in Stockholm (Registration number: 2012/1993-31).P<0.001). The mean age of respondents (48 years) was significantly higher than that of non-respondents (40 years), P<0.001. Nine percent (n = 101) of the non-respondents contacted the researchers providing explanations for non-participation. The reasons stated were: did not want to (73), presently abroad (13), unable to answer (10), did not use antibiotics (2), never answered questionnaires (1), perceived the questions as too difficult (1), or having a medical education (1).The overall response rate was 57% (n = 1426) . Women hPeople can become resistant to antibiotics”.Of the respondents, 94% (n = 1329) knew that bacteria can become resistant to antibiotics . A largeDue to missing values on the manifest variables 41 cases were removed (list wise deleted) before estimating the model, therefore the LCA was based on 1385 responders. A three-class model was chosen to describe patterns on knowledge according to our LCA criteria . The maiAccurate knowledge’. Class 2  included respondents who had a higher number of incorrect answers compared to class 1 and 3 and was labelled, ‘Some misconceptions’. Older people (>65 years) were more likely to have ‘Some misconceptions’ than younger people  was the class with the highest number of correct answers compared to the other classes. This class was labelled ‘r people . Class 3Accurate knowledge’, compared to those with the lowest educational level. Respondents with any medical or healthcare-related education, as well as men, were less likely to belong to any category other then ‘Accurate knowledge’ than women and those without any medical or healthcare-related education. The results do not show any evidence that income, or having at least one child in the household, influences one’s level of knowledge.Respondents with higher education were less likely to belong to this group than to ‘Of the responders, 6.1% stated that leftover antibiotics could be saved for personal future use or to give to someone else (data not shown).Appropriate restrictive attitude’, comprised of respondents who consistently reported a high degree of Appropriate restrictive attitudes regarding how antibiotics should be accessed and how to prevent infections.Due to missing values on the manifest variables 155 cases were removed (list wise deleted) before estimating the model, therefore the LCA was based on 1271 responders. A two-class structure  was chos‘Fairly appropriate attitude’. Men and those with ‘Some misconceptions’ or ‘Limited knowledge’ about antibiotics, were more likely to have a ‘Fairly appropriate attitude’ compared to those with ‘Accurate knowledge’  and 26,000–40,900 SEK (borderline significant) were less likely to have a ‘Fairly appropriate attitude’ compared to people within the lowest income level.Class 2  was characterised by slightly less consistent appropriate restrictive responses than the first group and was labelled, owledge’ . People Of the respondents, 13.4% and 29.5% stated incorrectly that antibiotics make people recover faster when having a cold and that ear infections in a 3–6 year old child always need to be treated with antibiotics, respectively (data not shown).Appropriate restrictive attitude’ and ‘Fairly appropriate attitude’, respectively. These groups had almost equivalent appropriate responses in relation to antibiotic use for symptoms indicating common cold or flu. Furthermore, both classes had responses which were not consistent with evidence-based recommendations for antibiotic use for specific infections, e.g. tonsillitis, ear infections in children and urinary tract infections in women.[‘Appropriate restrictive attitude’ reported a more restrictive approach in antibiotic use for sore throat and cough. Class 3  consisted of respondents with the highest number of ‘do not know’ answers. This class was labelled ‘Uncommitted’.Due to missing values on the manifest variables 164 cases were removed (list wise deleted) before estimating the model, therefore the LCA was based on 1262 responders. The most appropriate LCA model describing attitudes towards antibiotic use and antibiotics effects consisted of three classes . Classesin women. However,‘Uncommitted’ than to demonstrate an ‘Appropriate restrictive attitude’ towards antibiotic use and its effects than female respondents  in doctor’s decision to not prescribe antibiotics, while 88.7% expressed confidence in doctor’s decision to prescribe antibiotics (data not shown).P = 0.389).After initial cross-tabulation, the explanatory variable ‘monthly income’ was grouped into three categories  due to the low frequencies observed in the four original categories. The analysis showed that younger respondents (significant) and women (borderline significant) were less likely to have high confidence in a doctor’s decision not to prescribe antibiotics than older respondents . The parOur results show that the Swedish population’s level of knowledge about antibiotic use and resistance represented by the participants has not only remained high but has also increased since 2006. A decrease was observed in the percentage of people who agreed to the wrong statement that antibiotics make one recover faster when having a cold (19.1% in 2006 and 13.4% in 2013), that ear infections in a 3–6 year old child always need to be treated with antibiotics (49.5% in 2006 and 29.5% in 2013) and that people become resistant to antibiotics (84.7% in 2006 and 70.8% in 2013). Regarding whether bacteria can become resistant to antibiotics the percentage of respondents who agreed increased from 80.7% in 2006 to 93.7% in 2013.Similarly, a recent European survey showed that Swedish respondents had the highest knowledge compared to other European participants about antibiotic ineffectiveness against viruses (77% vs EU27 average of 40%) and common colds (77% vs EU27 average of 52%). PreviousPeople can become resistant to antibiotics”. In our current study, this has improved, however it still requires attention. This misinterpretation of antibiotic resistance has been confirmed and explored further in qualitative studies elsewhere,[Although respondents in the present study understood the relationship between unnecessary antibiotic use and antibiotic resistance development, some misconceptions still exist. In the 2006 survey, confusiolsewhere,,24 highlOverall, respondents had a comprehensive knowledge-base, in line with the main messages promoted by Strama and otheFairly appropriate’ attitude towards antibiotic accessibility and infection prevention Click here for additional data file.S2 Appendix(DOC)Click here for additional data file.S3 Appendix(DOC)Click here for additional data file.S4 Appendix(PDF)Click here for additional data file.S5 Appendix(PDF)Click here for additional data file.S1 File(R)Click here for additional data file.S2 File(R)Click here for additional data file.S3 File(XLS)Click here for additional data file.S4 File(R)Click here for additional data file.S5 File(R)Click here for additional data file.


As the HIV/AIDS pandemic has evolved over recent decades, Africa has been the most affected region, even though a large proportion of HIV/AIDS deaths have not been documented at the individual level. Systematic application of verbal autopsy (VA) methods in defined populations provides an opportunity to assess the mortality burden of the pandemic from individual data.To present standardised comparisons of HIV/AIDS-related mortality at sites across Africa and Asia, including closely related causes of death such as pulmonary tuberculosis (PTB) and pneumonia.Deaths related to HIV/AIDS were extracted from individual demographic and VA data from 22 INDEPTH sites across Africa and Asia. VA data were standardised to WHO 2012 standard causes of death assigned using the InterVA-4 model. Between-site comparisons of mortality rates were standardised using the INDEPTH 2013 standard population.The dataset covered a total of 10,773 deaths attributed to HIV/AIDS, observed over 12,204,043 person-years. HIV/AIDS-related mortality fractions and mortality rates varied widely across Africa and Asia, with highest burdens in eastern and southern Africa, and lowest burdens in Asia. There was evidence of rapidly declining rates at the sites with the heaviest burdens. HIV/AIDS mortality was also strongly related to PTB mortality. On a country basis, there were strong similarities between HIV/AIDS mortality rates at INDEPTH sites and those derived from modelled estimates.Measuring HIV/AIDS-related mortality continues to be a challenging issue, all the more so as anti-retroviral treatment programmes alleviate mortality risks. The congruence between these results and other estimates adds plausibility to both approaches. These data, covering some of the highest mortality observed during the pandemic, will be an important baseline for understanding the future decline of HIV/AIDS. The human immunodeficiency virus (HIV) and the consequent acquired immune deficiency syndrome (AIDS) caused a globally devastating pandemic starting in the late twentieth century. This pandemic caused mortality of such a magnitude as to distort population age–sex distributions in the worst affected areas . Now witIn the absence of laboratory testing and physician diagnosis, one way of determining the magnitude of HIV/AIDS-related mortality is by using verbal autopsy (VA), involving a structured interview with family or friends after a death . The intIn this paper, we present HIV/AIDS-specific mortality rates as determined by computer-interpreted VA from 22 INDEPTH Network Health and Demographic Surveillance Sites (HDSS) across Africa and Asia . These fThe overall INDEPTH dataset  from whiBox 1. Summary of methodology based on the detailed description in the introductory paper  probabilistic model was used for all the cause of death assignments in the overall dataset . InterVAOverall datasetThe overall public-domain dataset  thus conThe InterVA-4 ‘high’ HIV/AIDS setting was used for sites in Kenya, Malawi, and South Africa. All other sites used the ‘low’ setting; the ‘very low’ setting was not used. The InterVA-4 guideline is that the ‘high’ setting is appropriate for an expected HIV/AIDS cause-specific mortality fraction (CSMF) higher than about 1%, though it does not result in any great dichotomisation of outputs; the clinical equivalent is a physician’s knowledge that his/her current case comes from a setting where HIV/AIDS is more or less likely, irrespective of that current case’s particular symptoms. The validity of the InterVA-4 model in assigning HIV/AIDS as a cause of death in relation to HIV sero-status has been extensively explored in conjunction with the ALPHA Network , and fouDeaths assigned to HIV/AIDS, and the closely related causes of PTB and pneumonia, were extracted from the overall data set together with data on person-time exposed by site, year, age, and sex. As each HDSS covers a total population, rather than a sample, uncertainty intervals are not shown.For the sake of comparison with other estimates of HIV/AIDS-related mortality, unadjusted data were extracted for all sites for the period 2008–2012 . These data were grouped into three age bands  and aggregated by country, to facilitate comparison with contemporaneous national point estimates for 2010.In this context, all of these data are secondary datasets derived from primary data collected separately by each participating site. In all cases, the primary data collection was covered by site-level ethical approvals relating to on-going health and demographic surveillance in those specific locations. No individual identity or household location data were included in the secondary data and no specific ethical approvals were required for these pooled analyses.In the overall dataset, there were 10,455.4 deaths attributed to HIV/AIDS , with a further 10,363.4 deaths attributed to acute respiratory infections (including pneumonia), and 12,874.8 attributed to PTB.The age–sex–time standardised CSMFs for HIV/AIDS at each site are shown, together with the population-based HIV/AIDS-specific mortality rate per 1,000 person-years, in Against the background of extensive modelling approaches that have been applied to HIV/AIDS mortality, this dataset presents results from individually documented deaths at a range of sites across Africa and Asia. The expected huge differences in HIV/AIDS mortality rates between Africa and Asia were evident from these results, and, to a lesser extent, the substantial differences that occurred within the African continent. The good news is that HIV/AIDS deaths declined in recent years in all the sites with high mortality rates , as the The validity of VA cause of death assignment for HIV/AIDS is not straightforward. In these results, the similar and marked changes over time in the high mortality sites  added veThe WHO 2012 VA standard  includesThere are other major pieces of work describing HIV/AIDS mortality patterns across Africa and Asia, but these largely relied on modelling estimates from whatever specific sources of data were available, and therefore carried large degrees of uncertainty given the sparse nature of the data from many settings. The two major sources of contemporaneous estimates for HIV/AIDS mortality come from the UNAIDS Spectrum model  and the Similarly there were relatively few appreciable differences between GBD 2010 and InterVA-4 estimates. The differences may reflect local disparities in rates between sites and national populations, given that the relationships between symptoms and causes would not be expected to vary substantially between countries. It also has to be remembered that, although all these VAs have been processed in a standardised way using the WHO 2012 protocol, they were collected in the field in slightly different ways before 2012, and some observed differences may also reflect that. Overall, however, there was appreciable congruence in mortality rates between these various sources.Measuring HIV/AIDS mortality continues to be a highly challenging area, particularly in Africa, where rates are high and data are often unavailable. This is the largest single systematic study that has applied common methodologies to HIV/AIDS mortality at the individual level across Africa and Asia, and it largely confirms the corresponding findings coming from modelled estimates. This mutually adds plausibility to both existing estimates and to these population-based findings. The challenges involved in measuring HIV/AIDS mortality will grow as ART coverage and individual duration on treatment increase; in many ways, these results represent an important baseline for future studies of the treated pandemic.


Although often co-expressed, the distinct functional roles of c-Met and RON are not fully understood. c-Met and RON form both activated homodimers and heterodimers with themselves and other families of phosphotyrosine kinase receptors. Inhibitors for c-Met and RON including small molecular weigh kinase inhibitors and neutralizing antibodies are in pre-clinical investigation and clinical trials. Several of the tyrosine kinase inhibitors have activity against both c-Met and RON kinases whereas the antibodies generally are target specific. As with many targeted agents used to treat solid tumors, it is likely that c-Met/RON inhibitors will have greater benefit when used in combination with chemotherapy or other targeted agents. A careful analysis of c-Met/RON expression or activity and a better elucidation of how they influence cell signaling will be useful in predicting which tumors respond best to these inhibitors as well as determining which agents can be used with these inhibitors for combined therapy. The semThe mature forms of c-Met and RON are approximately 180 kD heterodimeric proteins composed of an extracellular 35 kD α-chain and a 145 kD transmembrane β-chain linked by disulfide bonds. RON shares 25% homology with c-Met in its extracellular domain and 63% homology within the tyrosine kinase domain . c-Met aHepatocyte growth factor (HGF) and macrophage stimulating protein (MSP) are ligands that activate c-Met and RON, respectively , 32, 33.MSP shares a high level of sequence and structural homology with HGF . MSP is c-Met gene but not the RON gene was amplified. Experimentally, c-Met is shown to have stronger kinase activity than RON [RON and c-Met are reported to be co-expressed in many tumor types -42 and cthan RON  and thusthan RON .As is the case with heterodimers from the EGFR family of receptors, signaling from heterodimers creates signaling diversity. Thus, depending on the relative abundance of each receptor type RON expression may in part modulate c-Met activity and vice versa. In this context, we recently showed that knockdown of RON enhanced the level and duration of HGF mediated activation of MAPK and AKT . The funin vivo is yet to be firmly established. A separate study showed that activated EGFR is able to phosphorylate c-Met indirectly through Src [Studies also indicate that c-Met and RON may interact with other phosphotyrosine kinases. Lowy and his colleagues recently showed that MSP stimulated RON was unable to activate IGF1-R but that IGF1 or EGF treatment caused phosphorylation of RON , 48. Thuough Src . RegardlThe recruitment and binding of substrates/adaptor proteins to the phosphorylated carboxy-terminal docking sites of activated c-Met and RON provides the platform to activate signaling cascades. As described above, the docking sites are Tyr-1349 and Tyr-1356 for c-Met and Tyr-1353 and Tyr-1360 for RON. Potential signaling cascades are illustrated in Figure Numerous cellular responses are attributed to c-Met and RON signaling and these induce but are not limited to cytoskeletal changes, EMT, migration and invasion, stemness, resistance to apoptosis, angiogenesis and proliferation. It is likely that activation of down stream molecular targets and subsequent biologic responses are cell context dependent and require critical levels of adaptor and singling molecules and are highly dependent on cross talk with other signaling molecules. c-Met can directly interact with E-cadherin, disrupting adherens junctions and leading to nuclear accumulation of β-catenin which potentially drives EMT in epithelial derived tumors , 51. SimThere is increasing evidence that c-Met is involved in expansion and maintenance of cancer stem cells. However, evidence of RON signaling in stemness is not clear. The function of c-Met in maintenance of cancer stem cell phenotype is consistent with its role in embryogenesis and tissue development. The suggestion that c-Met was involved in stem cells has been around for a number of years including a study by Kmiecik et al  who showc-Met and RON are over expressed in many cancer types leading to aberrant signaling that contributes to cancer development and progression , 62. METThe major successes of biologic targeted therapies along with preclinical findings showing that c-Met and RON are aberrantly expressed in cancer cell lines and tissues led to efforts to develop agents that inhibit their function or activities. As with other tyrosine kinase receptors, various approaches are being investigated for inhibiting c-Met and RON. These approaches include natural inhibitors of ligand binding, ribozymes and siRNAs, decoy receptors that capture ligand, neutralizing antibodies to ligands or antibodies to the receptors that block signaling and small molecular weight tyrosine kinase inhibitors (TKIs). These approaches and preclinical testing are detailed in numerous reviews including , 70, 71.Tivantinib  a c-Met TKI has gone through phase II trials as a single agent or in combination with other targeted agents or chemotherapy . Based oA number of recent studies, as described below, have linked aberrant activity of c-Met or RON with resistance of tumor cells to cancer therapies. These studies imply that up regulated signaling through c-Met and RON may be induced in response to chemotherapy or biologically targeted therapy. For example, amplification of MET has been associated with EGFR TKI resistance. Patients who harbor EGFR mutations get treated with erlotinib or gefinitib, and will invariably develop TKI resistance, and ~20% of which can be associated with MET amplification . It has Targeting VEGF or VEGFR represents the first major success in the clinic for inhibiting angiogenesis. Bevacizumab, a humanized anti-VEGF antibody, is approved for metastatic colorectal and renal cell carcinomas and for glioblastomas and non-small cell lung cancers. Tyrosine kinase inhibitors (TKIs) that target VEGFR with various levels of specificity including sunitinib, sorafenib, pazopanib, tivozanib, axitinib, cabozantinib and dovitinib are approved or in clinical trials as reviewed , 87. HowC-met and RON are structurally related tyrosine kinase receptors that contribute to tumor progression and promote resistance to chemotherapy. Further understanding of the pathogenesis and signaling pathways of these receptors may provide valuable insight into their role in cancer development and progression of disease. C-Met and RON are activated by separate ligands and following ligand activation they can homo- or heterodimerize with each other. Moreover, they may interact with and modulate signaling of other receptor tyrosine kinases. Expression of these receptors are associated with worse prognosis in various tumor types, and increasing expression of them have been seen in the progression of aggressive tumors. Moreover, c-Met and possibly RON are associated with maintenance of cancer stem cells. As a result, there continues to be advances in the study of these receptors and their signaling pathways with respect to cancer drug development. Numerous clinical trials are in progress using tyrosine kinase inhibitors directed at these receptors as potential molecular targets, with promising results. More studies remain to be seen if these drugs are effective and safe in improving patient survival and tumor response rates, as well as preventing development of tumor resistance to chemotherapy.


Processing of homologous recombination intermediates is tightly coordinated to ensure that chromosomal integrity is maintained and tumorigenesis avoided. Decatenation of double Holliday junctions, for example, is catalysed by two enzymes that work in tight coordination and belong to the same ‘dissolvasome’ complex. Within the dissolvasome, the RecQ-like BLM helicase provides the translocase function for Holliday junction migration, while the topoisomerase III alpha-RMI1 subcomplex works as a proficient DNA decatenase, together resulting in double-Holliday-junction unlinking. Here, we review the available architectural and biochemical knowledge on the dissolvasome machinery, with a focus on the structural interplay between its components. RecQ helicases play a key role in genome stability maintenance. In humans, five distinct proteins containing a RecQ-like module are involved in a set of diverse nucleic acid transactions, including DNA replication, recombination and repair. Three of these proteins  are found mutated in rare genetic disorders, characterised by chromosomal aberrations that are in turn associated with cancer predisposition and premature aging . MutatioDNA double-strand breaks can be repaired by homologous recombination, whose key intermediate is the four-way Holliday junction. During meiosis, Holliday junctions are cut by endonucleases to generate crossovers that are key to the proper segregation of chromosomes ,5. In soRecQ C-terminal (RQC) domain contains a zinc finger motif and a duplex-DNA binding Winged-Helix subdomain [in vitro[Helicase and RNaseD C-terminal (HRDC) domain is a separate, globular entity that confers substrate specificity to BLM [BLM Sgs1 in yeast) provides the ATP-dependent motor function to convergently migrate a dHJ ,16 and c in yeastubdomain , which pubdomain ). Unexpe[in vitro. Lastly,y to BLM ,26, beiny to BLM .Various reports indicate that BLM can be found in distinct oligomeric states that co-exist in solution, ranging from monomers and dimers ,29 to tei) relaxases (such as the E. coli TopoI), which efficiently remove negative supercoils from a covalently closed plasmid [ii) decatenases (e.g. E. coli TopoIII), which can unlink catenated DNA molecules [E. coli TopoI and TopoIII overall share the same fold, TopoIII contains small additional elements, as for example a short domain IV insertion (‘decatenation loop’), which lines the topoisomerase central cavity and is important for catenane unlinking  olecules . AlthougE. coli TopoIII decatenase (Figure trans, which could be sufficient to turn a relaxase into a decatenase. Although the crystal structure of TopoIIIα-RMI1 was solved in a closed configuration, the insertion loop appears poised to push the gate open, locking the topoisomerase in a covalently-attached, open, DNA-bound form that would promote DNA decatenation. Consistent with this model, a scrambled insertion-loop mutant, which does not interfere with complex formation, fails to stimulate dHJ dissolution, reverting yeast Top3 back to a modest decatenase [RMI1 stimulates the double-Holliday junction dissolution reaction and directly interacts with both the BLM helicase and TopoIIIα ,14,40. Icatenase .Drosophila, which appears to lack RMI1, a fly-specific C-terminal insertion in TopoIIIα that contacts BLM at an unknown site, is essential for dissolution and might functionally substitute RMI1 [How BLM structurally and functionally interacts with the TopoIIIα–RMI1 sub-complex is still poorly understood. It is known that BLM contacts NTD RMI1 ,14,45 whute RMI1 . It remaDrosophila, for example, suggests that BLM and TopoIIIα could coordinate their activities to achieve branch migration [In vitro studies on this system indicate that, while the isolated BLM is incapable of migrating a topologically constrained dHJ, vigorous migration is observed when Drosophila Top1 is added to the reaction mixture, although branches pause before merging into a hemicatenane. When BLM is instead assayed with TopoIIIα, the reaction goes to completion without any pausing, indicating that dissolution is indeed a highly processive reaction, and hinting at a possible catalytic interplay between the two enzymes [Similarly unclear is the nature of the BLM-TopoIIIα catalytic interplay . Work inigration . In vitr enzymes .Work in yeast, however, indicates that Sgs1/BLM has a role in modulating strand passage by TopoIIIα and RMI1, which does not depend on the catalytic activity of the helicase module . In a caRecent advances shed light on the role of isolated dissolvasome components in the convergent migration of dHJ junctions and hemi-catenane unlinking. Whether BLM and TopoIIIα-RMI1 allosterically influence their reciprocal catalytic activities still remains unclear. A comprehensive view of the molecular basis of dHJ dissolution will be likely achieved once the high-resolution structure of the full dissolvasome assembly is determined.The authors declare that they have no competing interests.PS and AC wrote the manuscript. Both authors read and approved the final manuscript.


Various helicases and single stranded DNA (ssDNA) binding proteins unfold G-quadruplex (GQ) structures. However, the underlying mechanisms of this activity have only recently come to focus. We report kinetic studies on Bloom (BLM) helicase and human telomeric GQ interactions using single-molecule Förster resonance energy transfer (smFRET). Using partial duplex DNA (pdDNA) constructs with different 5′ ssDNA overhangs, we show that BLM localizes in the vicinity of ssDNA/double-stranded DNA (dsDNA) junction and reels in the ssDNA overhang in an ATP-dependent manner. A comparison of DNA constructs with or without GQ in the overhang shows that GQ unfolding is achieved in 50–70% of reeling attempts under physiological salt and pH conditions. The unsuccessful attempts often result in dissociation of BLM from DNA which slows down the overall BLM activity. BLM-mediated GQ unfolding is typically followed by refolding of the GQ, a pattern that is repeated several times before BLM dissociates from DNA. BLM is significantly less processive compared to the highly efficient GQ destabilizer Pif1 that can repeat GQ unfolding activity hundreds of times before dissociating from DNA. Despite the variations in processivity, our studies point to possible common patterns used by different helicases in minimizing the duration of stable GQ formation. Human Bloom helicase (BLM) is classified as a superfamily 2 helicase ,2 . MoreEscherichia coli RecQ are significantly less efficient in GQ unfolding under similar conditions  sequence or the human telomeric GQ-forming sequence and a poly-T spacer between the duplex region and GQ, to accommodate BLM binding in the vicinity of the ssDNA/dsDNA junction. Under these conditions, we observed that BLM localizes in the vicinity of the junction and reels in the ssDNA tail before encountering the GQ. Upon encountering the GQ, BLM unfolds the GQ in 50–70% of the cases, and dissociates from DNA without unfolding the GQ in the other cases. The reeling activity is also observed for poly-T ssDNA overhangs which do not contain a GQ. A comparison of these results with those on Pif1 helicase  suggests642–1290 on partial duplex DNA substrates under physiologically relevant salt  and pH (7.5) conditions. BLM642–1290 comprises the RecQ core of BLM containing the helicase, RecQ C-terminal (RQC) domain and helicase and RNase D C-terminal (HRDC) domain, and it maintains both dsDNA unwinding and ssDNA translocation activities of wild-type protein (We utilized smFRET assay to examine the activity of BLM protein . Two typ−1). Traces that did not show any reeling events were also included in the statistics in order to provide a better representation of the concentration dependence.Prism-type total internal reflection microscopy was used to perform the measurements. Details of the assay conditions are given in Supplementary Data. The total number of observed events in several hundred traces was counted and this number was divided by the total time, which yields the Event Rate . Figure t = 0 s). Figure Unwinding of the 12 bp duplex results in release of donor fluorophore, thus providing a means to detect GQ unfolding . However, a stable GQ and a short spacer (2 nt) are still maintained in this construct and BLM should still be able to make contact with these regions. These measurements did not show any duplex unwinding under these circumstances. In addition, as the GQ overhang was only 4 nt long, BLM could not initiate GQ unfolding by binding to this region, which resulted in all GQ molecules remaining stably folded at even the highest BLM and ATP concentrations we studied. In conclusion, these measurements show that BLM does not bypass a folded GQ and proceed to unwind the duplex by either making contact with the GQ itself without unfolding it, or with the short spacer between the GQ and the duplex.EFRET = 0.23 ± 0.05  take place.Having established that BLM can unfold GQ under these conditions, we proceeded with measurements on pd-30ThGQ construct which has a 30T spacer to enable BLM binding and translocation and a GQ-forming sequence in the overhang  to be reached during the FRET rise to ensure that ssDNA reeling has completed. This threshold was based on separate observations in which FRET rises to EFRET < 0.60 followed by a return to the initial FRET level and is consistent with our data on pd-35T, which will be discussed below. Such events were classified as unsuccessful attempts at unfolding GQ, i.e. BLM reels in some or all of the 30T spacer DNA but either dissociates or releases the DNA before unfolding the GQ. Figure Having established that BLM can unfold GQ after reeling in the ssDNA between the junction and the GQ, we sought to quantitatively characterize these events as a function of BLM and ATP concentrations. We used the following criteria to identify a FRET burst as an event that includes GQ unfolding. The first criteria is the pattern described earlier, a FRET rise followed by at least one significant dip and rise in FRET. We also required a minimum FRET level  for different BLM or ATP concentrations, performed based on these criteria, are shown in Figure +, pd-35T showed a peak at EFRET = 0.35 ± 0.06  and the time it waits at the highest FRET level (will be referred to as waiting time). The reeling time is expected to decrease with increasing ATP concentration. The waiting time is a measure of the time BLM holds on to the reeled-in DNA before either dissociating from it or just releasing it, while still remaining bound to the overhang. There were variations in the shape of the FRET traces among different events. While some had a well-defined high-FRET state, others showed variations in the high-FRET state as shown in the bottom panel of Figure BLM titration measurements were performed at a constant 20 μM ATP concentration on pd-35T construct. The event rates are reported in Figure We further studied the ATP dependence of the event rate Figure , reelingEFRET = 0.19 ± 0.04 at 150 mM K+, as shown in Figure Similar results were obtained with the pd-50T construct, which has a 50T overhang , which represents the BLM-bound unfolded GQ state  from the top FRET level to the bottom FRET level, which is a measure of GQ unfolding time, and we did not observe any ATP dependence. Our measurements resulted in transition times in the Δtuf = 0.17–0.19 s range for 4, 10 and 20 μM ATP. These Δtuf were not significantly different from each other given the uncertainty of our measurements (±0.07 s). The uncertainty in these unfolding times was estimated using the two data point uncertainty that was employed for the reeling and waiting times, which is a more conservative estimate than the standard error of the Gaussian fits to histograms of these transition times. We believe the similarity of the unfolding times at different ATP concentrations is reasonable as unfolding of GQ happens in the form of a collapse of the structure, i.e. removing a few nucleotides from the structure leads to unfolding of GQ, and the time for such a collapse to take place should not depend on ATP concentration. We do not observe a step-wise unfolding of GQ, one strand at a time, as proposed for other proteins . The sa−1 based on a Langmuir binding isotherm fit. The corresponding rate under similar conditions for the pd-30ThGQ substrate is 0.12 ± 0.02 s−1. Given the similar overall spacer lengths for these substrates, we conclude that about 70% of all reeling events result in unfolding of the GQ. A similar comparison with the BLM titration at 20 μM ATP shows saturation rate of 0.35 ± 0.02 s−1 for pd-35T while the corresponding rate for pd-30ThGQ is 0.18 ± 0.07 s−1, suggesting about 50% of reeling events result in GQ unfolding. This reduction in the event rate would result in an overall slowdown of BLM activity as it would have to restart the reeling activity after failed attempts at unfolding GQ, which are often accompanied by dissociation of BLM from the DNA. A recent study showed a related effect on Pif1 helicase in which non-productive ATP hydrolysis events which failed to result in GQ unfolding were observed  with a short spacer demonstrate. BLM-mediated GQ unfolding and GQ refolding repeat several times before BLM dissociates from DNA. GQ unfolding was observed in 50–70% of the reeling attempts. In the other cases, BLM either released the reeled-in DNA, while remaining bound in the vicinity of junction, or dissociated from DNA completely before it could unfold GQ. Such unsuccessful attempts would naturally slow down BLM activity as each unsuccessful attempt would require restarting the process. These observations are similar in nature to those observed with Pif1 helicase, with BLM being significantly less processive in its repetitive patrolling and GQ unfolding activities. Despite the variations in processivity, the common features observed for the two helicases support the view that different helicases might use similar mechanisms for unfolding GQ structures.Supplementary Data are available at NAR Online.


Statistical-sequential learning (SL) is the ability to process patterns of environmental stimuli, such as spoken language, music, or one’s motor actions, that unfold in time. The underlying neurocognitive mechanisms of SL and the associated cognitive representations are still not well understood as reflected by the heterogeneity of the reviewed cognitive models. The purpose of this review is: (1) to provide a general overview of the primary models and theories of SL, (2) to describe the empirical research – with a focus on the event-related potential (ERP) literature – in support of these models while also highlighting the current limitations of this research, and (3) to present a set of new lines of ERP research to overcome these limitations. The review is articulated around three descriptive dimensions in relation to SL: the level of abstractness of the representations learned through SL, the effect of the level of attention and consciousness on SL, and the developmental trajectory of SL across the life-span. We conclude with a new tentative model that takes into account these three dimensions and also point to several promising new lines of SL research. From an ecological point of view, learning about temporal patterns in our environment, and using this information to make predictions about upcoming events and actions, is arguably of primary importance to humans and other higher-order organisms . In the In the current review, we focus in particular on the learning of temporal or sequential patterns of stimuli and therefore use the term “statistical-sequential learning” or simply “sequential learning” (SL) for short. Because it is still an open question as to whether these learning abilities are also governed at least in part by explicit processes e.g., , we avoiTo date, the underlying cognitive and neural mechanisms of SL and the associated cognitive representations are still not well understood. SL has been explored though a combination of cognitive modeling and empirical studies using behavioral and neurophysiological measurements. The current outcome of these heterogeneous approaches is that the proposed theories of SL still need to be confirmed by empirical evidence. The purpose of this review is to provide an initial assessment of the current theories of SL and to identify the areas of empirical research that need further development. Due to the extensive behavioral and neural SL literature, the scope of this review will focus on the exploration of SL with a specific neural approach, the event-related potential (ERP) technique  dimensions: (1) the extent to which SL encodes and manipulates concrete versus abstract representations, (2) whether SL depends on the level of conscious awareness or attention, and (3) how SL changes across the life-span. We consider each of these issues in turn.Sequential learning could in principal encode either: (1) concrete features of the sequence, such as the frequencies of individual items (or exemplars) of the sequence, (2) or abstract features, e.g., abstract rule(s) that organize the to-be-learned sequence . This sePerhaps the best-known empirical demonstration of SL comes from Figure 1). In the same vein, several models have been proposed to account for surface learning based on the to-be-learned type of concrete information. Some models focused on conditional statistics between items of the sequence  or amodal (more abstract) representations. For Reber, SL was a mainly amodal process ; howeverSome models of SL in fact explicitly incorporate a multilayer structure. Related to the issue of modality-specificty, it should be noted that the more concrete-based aspects of SL appear to show similarities to perceptual learning (PL), which allows for the development of spatio-temporal representations of the environment through learning along various levels of cortical processing . InteresOne final way that, together with abstractness and modality, SL representations might be differentiated is by the types of input structures . Three tIt appears likely then that SL involves multiple processes, some that could be characterized as being more domain-general and that manipulate rather abstract representations, and others that are more input-specific and that encode more concrete features. This perspective is similar to the “more-than-one-mechanism” (MOM) hypothesis of language acquisition, stating that language is acquired via the manipulation of both rule-based and statistical representations . SeveralThe issue of the abstractness of the representations manipulated during SL is complex. Perhaps the most promising accounts of SL involve the processing of both concrete and abstract information e.g., . The exaIn addition to dissociating the mechanisms of SL by the level of abstractness of the learned features, the level of attention (and consciousness) has also been recognized as a critical dimension of SL. The SL literature often refers to this issue in terms of “implicit” and “explicit” processing. Traditionally, SL is generally thought to involve the activation of incidental/implicit, automatic, and even unconscious processes e.g., . SeveralAlternatively, other theories have argued for a more direct role of explicit processing in SL. For instance, Between these two extreme views there exist proposals that acknowledge the development of both conscious and unconscious representations resulting from SL as well as the contribution of explicit and implicit mechanisms to SL. For instance, Clearly, there is far from a consensus on the question of whether SL is subserved by implicit or explicit mechanisms, or a combination of both. Nevertheless, perhaps the most influential view to date is that both types of mechanisms contribute to SL e.g., . ImportaFrom a methodological point of view, one way to explore the extent of explicit and implicit learning in SL paradigms is to use rapid serial visual presentations (RSVP). Even though these new methods have improved our ability to assess the contribution of the level of consciousness to SL mechanisms, the issue is far from settled. Some researchers still believe that the assessment of consciousness needs further improvements . ImportaIn summary, the literature remains highly heterogeneous in terms of the impact of the level of consciousness on SL performance. However, perhaps the most conservative view, similar to that discussed earlier, is that SL might not be governed by a single cognitive mechanism and might not store representations in a single – e.g., unconscious – format. Instead, SL is likely subserved by at least two mechanisms, one that is rather independent of the level of consciousness/attention and results in unconscious representations and one that depends more on attentional resources and leads to more conscious representations. We will see that ERPs can be helpful in testing this assumption.Whether described in terms of the abstractness of the representations or on the consciousness/attentional dimension, SL can hardly be fully investigated without taking into account its developmental trajectory. Although most SL experiments have been performed with young adults, several studies have focused on SL in children  and infaDespite the growing body of research that focuses on SL across the life-span, the developmental progression of SL is still largely unknown. The early literature on implicit learning assumed that this cognitive ability was rather independent of age , while eIn terms of how SL abilities develop later in life, the literature from the elderly population points either to no change in old age in the case of deterministic sequences  but age-The model of Thus, similarly to the dissociation of mechanisms of SL into explicit and implicit components, and into mechanisms encoding concrete and abstract representations, the Figure 2). In order to provide extra empirical validation of this model, we now turn to how ERPs have contributed to a better understanding of the mechanisms of SL.In summary, SL may consist of at least two different systems. The first relies upon bottom-up implicit/perceptual mechanisms that result in unconscious representations, develop early in life, and are likely to exploit surface structure of input and hence can explain some of the impressive language-related abilities present in infants and children. The second system develops later in life, consisting of expert SL mechanisms that rely more on top-down information, are more dependent on the level of attention, and result in explicit knowledge of abstract rules that further improves language processing abilities . We will then focus on how ERPs have been used to explore the three above-mentioned dimensions of SL mechanisms: the abstractness of the manipulated representation, the level of attention/consciousness of the mechanisms and the level of consciousness of the representations, and the development of SL across the life-span. After considering these three dimensions of SL, we then consider new avenues of research and then conclude with a re-evaluation of the two-system model of SL described in Figure 2.We will first summarize the main ERP paradigms that have been used to date in SL research . This paradigm elicits a P300 ERP component, one of the most studied components of ERP research  target stimulus presented along with more frequent (or “standard”) non-target stimuli in a serial input stream , which typically is thought to reflect an automatic discrimination or echoic memory updating between the standard and the deviant stimulus . Response buttons correspond spatially to each location. SL is behaviorally demonstrated by a reduced response time to repeating/familiar sequences compared to novel or random sequences. The SRT has been subsequently adopted and modified by many others for various purposes . The participant is asked to respond to target stimuli without being told that certain predictor stimuli predict the occurrence of the target with fixed contingent probabilities. That is, the occurrence of the predictor allows the participant to predict the target with varying probabilities. The assumption is that this design requires a kind of basic statistical learning of the contingent probabilities that links the predictors to the targets. One final variation of the oddball design comes from Unlike these oddball paradigms where the sequences embody rather simple contingent statistics, other ERP paradigms have been used to explore SL using more complex sequences, such as the “artificial grammar” paradigm.Figure 7), have been designed to mimic the complex structure of natural language while simultaneously removing other potentially confounding parameters such as semantic information. Converging evidence has suggested that this experimental design is a good model for testing the grammatical and structural processing of natural language  paradigms, which incorporate a set of rules that govern the structure of sequences , and the developmental trajectory.As previously discussed, SL is thought to stem from at least two different types of mechanisms, one that acts on rather concrete information and the other that acts on more abstract information. With concrete feature-learning mechanisms, SL is explained by the encoding of distributional properties of the sequence of items, such as item co-occurrences or the transitional probability between items. The alternative (or complementary) mechanism assumes that the perceiver encodes abstract rules .One of the crucial results from ERP studies of SL is provided by Conversely, other studies have found ERP correlates – specifically, the MMN – related to concrete feature encoding . For insIn summary, some of the few ERP studies that explored the level of abstractness of the encoded information during SL have been interpreted as evidence that SL is governed only by abstract rule-learning mechanisms. On the other end, other ERP research were taken as evidence that concrete-rule encoding can also be indiced by ERPs. Overall, it appears that the ERP research supports the assumption that both concrete and abstract feature encoding occurs in SL. The apparent inconsistency between these studies may be due simply to variation in experimental designs and a lack of sensitivity of ERP to adequately index particular mechanisms of SL.Across various paradigms, not just those specifically looking at SL, almost all ERP components have been reported to be modulated by the level of attention . Thus, oper se. However, even if this top-down account is true, the ERP components still reflect an outcome of the SL process, that is, a learning-related change of attention to stimuli based on whether or not the stimuli are consistent with the previously learned patterns.The literature contains several other ERP studies that attribute to ERP components the property of indexing SL while often ignoring the alternative top-down attentional explanation . For insWhether SL requires conscious awareness is a hotly debated topic. The relation between implicit SL, explicit SL, and ERPs has been mostly explored through two approaches: by dissociating implicit from explicit learning according to whether participants acquired explicit knowledge of the patterns e.g.,  or by diIn line with early behavioral studies of SL , 1997, sStill using the oddball paradigm and measuring the P300, but under rapid stimulus presentation - the so-called RSVP paradigm – other studies tested the perception of a deviant within an attentional blink . These sImportantly, none of the above-mentioned ERP studies rule out the possibility that explicit mechanisms of SL also contribute to the reported ERP effects. Indeed, the ERP research on SL mechanisms has abundantly explored the explicit component(s) of SL . For insFigure 8). In addition, these authors reported P300 effects to sequence violations that were, when explicit SL occurs, more than two times larger than those observed when only implicit SL was permitted . A similar “effect size doubling” on behavioral performance was reported by Eimer et al.  using 10-letter sequences with standard and deviant sequences. The effect size increase was even larger when measuring the amplitude of the N200. In the same vein, In addition, larger effects of learning (as measured by behavior and ERP) appear to be found in explicit compared to implicit conditions. For instance, However, robust effects of explicit SL are not systematically reported. For instance, In summary, the ERP literature seems to support the existence of both implicit and explicit mechanisms of SL. Furthermore, the effect size of the SL measured behaviorally or neurophysiologically appears to increase with the intention to learn the rules and with the explicit knowledge of these rules. Therefore, when attempting to understand the mechanisms of SL, a very critical aspect appears to be the attentional/consciousness dimension. Importantly, since the level of attention can affect almost all ERP components, the interpretations of ERP correlates of SL must be cautious as in some instances there may be an alternative top-down explanation.In general, there is a paucity of ERP research examining SL in young children. However, neural signatures of infant and children’s early language learning mechanisms – presumably dependent in part on SL – have been documented using ERPs. Indeed, ERP studies have provided some evidence that the ability to extract statistical dependencies between adjacent elements in the speech stream appears to be present from birth, and infants can learn non-adjacent dependencies in a natural, non-native language by 4 months of age . From abAlthough SL is assumed to be important for language acquisition, few studies have directly examined the relationship between SL and language outcomes. Recently, the link between SL and children’s language performance has received new support. As concerns older populations, the literature about ERP correlates of SL is scarce and mostly involves oddball paradigms that elicit, for example, the MMN and the P300 . In lineClearly, the developmental trajectory of SL still has many unexplored fundamental questions. We believe the ERP technique has not been used to explore SL across the lifespan to its fullest potential. This research gap in the developmental dimension as well as opened questions left by the previously discussed models of SL models lead us now to consider several new lines of ERP research that we believe could offer new insights into SL, some of which are amenable to developmental approaches.As mentioned earlier, SL mechanisms can be explored on the dimensions of the abstractness of the manipulated representations  and attention . For these two approaches, ERPs, allowing the assessment of “online” cognition, could make a nice contribution if new paradigms are applied to control for the amount of concrete information available in the input and the level of attention (or consciousness) brought to bear. In this regard, the control of concrete information could be performed using the so-called “balanced chunk strength design” e.g., . This prper se and not indirect effects of increased attention to newly learned materials.We mentioned earlier that almost all ERP effects observed in SL paradigms can be interpreted either as indices of the SL process itself or as a consequence of SL, which modulates the level of attention to the learned material . Future approaches could control the level of attention using for instance subliminal stimulation . FindingAnother area where ERPs can be fruitfully used is to explore the nature of multisensory SL and the ways in which different subsystems of SL interact and integrate information across domains. For instance, In addition, future ERP research could focus more on examining the developmental time-course of SL. This issue can be assessed either on a short time scale, with for instance the analysis of the development of SL across trials within a single experiment; or on a longer time scale, with groups of participants of various ages. Both approaches have been followed for instance by Importantly, developmental approaches should not be restricted to comparisons between age groups. What is also needed to better explore SL at a longer time-scale are longitudinal studies . As indicated earlier, ERPs are particularly well-suited to explore each of these dimensions and could also be used to explore combinatory modulations of each of these dimensions.Finally, it is essential that future research also recognizes the need for exploring several dimensions of SL together, because by only assessing one dimension alone, we may suffer from an overly simplistic and perhaps inaccurate view of the underlying mechanisms of SL. For instance, it might be that different aspects of SL such as the level of abstractness of the encoded representations and the level of consciousness of the learned patterns may develop along different developmental trajectories .SL mechanisms can be described along several partially-overlapping dimensions: the level of abstractness of the encoded sequential information, the level of attention/consciousness of these representations and the mechanisms that manipulate them, as well as the developmental trajectory. Based on these descriptors, several cognitive and computational models have been proposed. Although many disagreements and unanswered questions remain about these views, a general picture emerges. As an integrative model, we propose that SL is most likely governed by at least two types of systems whose respective contributions vary across the life-span  is correct, then this helps frame our understanding of the nature of many related aspects of cognition including motor skill development, perceptual processing, and language acquisition. One potential outcome of an improved understanding of the mechanisms of SL is the ability to design novel language rehabilitation interventions, capitalizing on the assumption that improving performance on SL could have a transfer effect and thereby improve the performance of other cognitive processes, such as language, that stem from it.In sum, this review has explored to what extent ERP findings can be used to better understand the neurocognitive mechanisms of SL. Rather than continuing to argue over a simple dichotomy of abstract versus concrete feature encoding or implicit versus explicit mechanisms, future research must be more aware of the potential complex relationships among multiple neurocognitive mechanisms that may differ along one or more of these dimensions based on the task at hand. Furthermore, ERPs can be used to shed light on the developmental progression of these various mechanisms. If the two-system view of SL (The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


As antibiotic consumption rates between hospitals can vary depending on the characteristics of the patients treated, risk-adjustment that compensates for the patient-based variation is required to assess the impact of any stewardship measures. The aim of this study was to investigate the usefulness of patient-based administrative data variables for adjusting aggregate hospital antibiotic consumption rates. Data on total inpatient antibiotics and six broad subclasses were sourced from 34 acute hospitals from 2006 to 2014. Aggregate annual patient administration data were divided into explanatory variables, including major diagnostic categories, for each hospital. Multivariable regression models were used to identify factors affecting antibiotic consumption. Coefficient of variation of the root mean squared errors (CV-RMSE) for the total antibiotic usage model was very good (11%), however, the value for two of the models was poor (> 30%). The overall inpatient antibiotic consumption increased from 82.5 defined daily doses (DDD)/100 bed-days used in 2006 to 89.2 DDD/100 bed-days used in 2014; the increase was not significant after risk-adjustment. During the same period, consumption of carbapenems increased significantly, while usage of fluoroquinolones decreased. In conclusion, patient-based administrative data variables are useful for adjusting hospital antibiotic consumption rates, although additional variables should also be employed. Antibiotic consumption can vary between hospitals depending on a number of factors including implementation and adherence to antibiotic policies, antibiotic resistance rates, and hospital function which depends on the patient characteristics -3.A few reports have focused on risk adjustment models that account for differences in specific health risks that patients bring to their healthcare facilities, thus ‘levelling the playing field’ when comparing rates of antibiotic consumption between hospitals with varied case mix -6. TheseVariables relating to a variety of patient characteristics from public acute hospitals, based on administrative data, are readily available in Ireland . Unlike The rates of antibiotic use in hospitals are dynamic and have been shown to change over time not only in Ireland but in many countries . HospitaThe study was an observational, retrospective analysis of aggregate data on antibiotic use and patient administration from 34 public acute hospitals in Ireland. The participating hospitals in this study represented all tertiary/referral hospitals in Ireland and all general hospitals bar two facilities that were unable to provide consistent antibiotic consumption data. Single-speciality hospitals  were excluded.Clinical antimicrobial dispensary data from hospital pharmacy systems were extracted and converted into defined daily doses (DDD) using the World Health Organization (WHO) Anatomical Therapeutic Chemical (ATC) classification method  via the 1. Carbapenems, which included agents such as meropenem,2. Fluoroquinolones such as ciprofloxacin,3. Glycopeptides such as vancomycin and teicoplanin ,4. Macrolides such as erythromycin,5. Penicillins with enzyme inhibitors such as amoxicillin/clavulanic acid,6. Third-generation cephalosporins such as cefotaxime,7. Total antibiotic use, all systemic anti-bacterial agents.Hospital In-patient Enquiry (HIPE) data following patients’ discharge or death in the hospital were used to obtain aggregate annual patient administration variables from 2006 to 2014 for all participating hospitals. These were accessed through the Health Intelligence Ireland secure online healthcare data analytical system . Data onR software was used for all statistical analyses . We consResults for these models are reported using incidence rate ratios (coefficient estimate) and 95% confidence intervals for each outcome variable. Coefficients of variation of the root mean squared errors (CV-RMSE) are reported for each model.Satisfactory models were used to generate expected values of antibiotic consumption for all facilities for each year in the study period, given the patient administration parameters for the facilities during the relevant time points. The difference between the observed antibiotic use and the estimated use is the residual, and the standardised residual is a ratio of the residual divided by the standard deviation of the residuals. Data points for any facility–year combination that had standardised residual values of less than − 2 or greater than + 2 were considered as having lower or higher than expected consumption, as estimated by the statistical model, respectively.The total antibiotic usage rate for the 34 participating public acute hospitals decreased from 82.5 in 2006 to 80.0 DDD/100 BDU in 2009, and then increased to 89.2 DDD/100 BDU in 2014. Rates for carbapenems, glycopeptides and penicillins with enzyme inhibitors increased, those for macrolides and third-generation cephalosporins stayed level and those for fluoroquinolones decreased between 2006 and 2014 .A heat map of the 29 explanatory variables grouped into eight sections is shown in The proportion of patients aged 30 to 49 years ranged from 14% to 31% between hospitals and accounted for the highest proportions of all discharged patients, i.e. 22% of the all patients. The age group five to 14 years represented the lowest proportion of patients. The proportion of female patients ranged from 45% to 66% between hospitals and overall, 54% of all patients were female.The most common type of admission was emergency, representing 70% of all discharges. Note that other admission types includes newborn and maternity admission. Among the admission sources, home was the most common type (88–97%), while ‘other source type’  was the least common at 0–2%. Among discharge locations, home was again the most common type (73–92%), while ‘other location type’  was the least common at 0–2%. Just over half of the patients (58%) did not have private health insurance and the proportion of patients under a public payment scheme ranged from 36% to 79% per hospital.Length of stay of one day (overnight) ranged from 14% to 31%, while a larger proportion stayed for between two to four days (28–45%); the remainder stayed five days or longer (28–55%). Overall, 7% of the patients had a stay in intensive care. Diseases and disorders of the circulatory system (MDC-5) and diseases and disorders of the digestive system (MDC-6) were the most common MDC overall at 15% each. Diseases and disorders of the respiratory system (MDC-4) were also frequent at 13%. Of interest for antibiotic consumption are infectious and parasitic diseases (MDC-18) which was uncommon at just over 1%.The final seven regression models for the antimicrobial groups are shown in Different age groups were associated with increased risk of consumption of the different antibiotic groups. In particular, there was a high degree of association between decreased use of carbapenem and hospitals with a higher proportion of patients in the age group of five to 14 year-olds. Female sex was not significantly associated with any of the indicators of consumption, and neither was admission type .Hospitals that had a higher proportion of patients admitted from ‘other source type’ had much reduced consumption of carbapenems, fluoroquinolones, glycopeptides, macrolides and total antibiotics. Similarly, hospitals that had a higher proportion of patients discharged to ‘other location type’ had much reduced consumption of glycopeptides, macrolides, third-generation cephalosporins and total antibiotics.While only 0.1% of patients overall were classed under the MDC for burns (MDC 22), the category was associated with increased use of carbapenems and glycopeptides and with reduced use of fluoroquinolones and third-generation cephalosporins. The group of infectious and parasitic diseases (MDC 18) was associated with increased use of fluoroquinolones, penicillins with enzyme inhibitors, third-generation cephalosporins and total antibiotics.Year as a categorical variable was significant for consumption of two antibiotic groups: carbapenems, which increased, and fluoroquinolones, which decreased over the study period. Two individual year values for macrolides (2009 and 2010) and one for total use (2009) were significant decreases.Our analysis identified three aspects of surveillance of hospital antimicrobial consumption: it identified factors that are important in driving antimicrobial use; it identified antibiotic groups for which the changes in consumption rate occur faster than could be explained by changes in patient profiles at the individual hospital alone; and it identified outliers so that stewardship strategies can be followed at those facilities to improve patient care.On the first aspect, the main patient profile factors including MDC, and their variation both over time and across different facilities, may explain the dynamics that are evident in hospital antimicrobial consumption in Ireland. The range of factors that were significant for the different antimicrobial groups shows that using only a single factor such as the cost-based case mix index would not have been adequate . AdditioEnterobacteriaceae are becoming more frequent across Europe [On the second aspect, fluoroquinolones were the only antimicrobials in this study for which consumption decreased. There has been a concerted effort since 2008 by pharmacists in Ireland to reduce fluoroquinolone use as a whole and to switch to oral preparations as fluoroquinolones have a good bioavailability . The incs Europe . All hosOn the last aspect of outliers, our analysis showed that there were hospitals that had consistently higher antibiotic consumption than would be expected given the characteristics of patients cared for in those hospitals. It is likely that these few hospitals have services that were not included in the parameters of our models. It is important to address the presence of and adherence to antibiotic prescribing policies in these institutions.Our analysis has limitations. Firstly, there is a debate about how to appropriately measure antibiotics usage. We selected the WHO ATC/DDD system as it is the one chosen by the European Surveillance of Antimicrobial Consumption Network (ESAC-Net). Furthermore, direct measures of antibiotic usage such as days of therapy could not be used as the pharmacy computer systems used in Ireland do not yet support it, unlike hospitals elsewhere . The secBased on the findings of this study we recommend that the national guidance documents for antimicrobial stewardship should be updated to strengthen prescribing practice for carbapenems in particular and to incorporate a mechanism to ensure good adhere to antibiotic prescribing. We also recommend that performance-linked measures are put in place to ensure that when hospitals demonstrate reduction in the use of one antibiotic group, this does not lead to increases in another group of antibiotics. However, high antibiotic use among outliers may not imply poor performance and the hospitals not found to be outliers may still have substantial inappropriate use. Therefore, the findings of this study should be used in conjunction with other information and as part of a broader stewardship strategy. Finally, we suggest that a Europe-wide hospital antimicrobial study based on a unified methodology of risk adjustment is undertaken that takes into account the limitations of this and other similar studies. Risk adjustment may even be required to compare the wide variation in hospital antibiotic consumption as driven by diverse healthcare delivered to the populations in different jurisdictions.In conclusion, patient-based administrative data variables are useful for adjusting hospital antibiotic consumption rates, although additional variables relating to clinical services should also be employed.