Swedish studies on antibiotic use in primary care have been based on one-week registrations of infections. In order to study adherence to guidelines, analyses based on large databases that provide information on diagnosis linked prescriptions, are needed. This study describes trends in management of infections in Swedish primary care particularly with regards to antibiotic prescribing and adherence to national guidelines.A descriptive study of Sweden’s largest database regarding diagnosis linked antibiotic prescription data, the Primary care Record of Infections in Sweden (PRIS), for the years 2008, 2010 and 2013.p = .032). The antibiotic prescribing rate for respiratory tract infections (RTIs) decreased from 40.5% in 2008 to 24.9% in 2013 while those for urinary tract infections and skin and soft tissue infections were unchanged. For most RTI diagnoses there was a decrease in prescription rate from 2008 to 2013, particularly for the age group 0–6 years. Phenoxymethylpenicillin (PcV) was the antibiotic most often prescribed, followed by tetracycline. Tonsillitis and acute otitis media were the two RTI diagnoses with the highest number of prescriptions per 1000 patient years (PY). For these diagnoses an increase in adherence to national guidelines was seen, with regards to treatment frequency, choice of antibiotics and use of rapid antigen detection test. The frequency in antibiotic prescribing varied greatly between different Primary Healthcare Centres (PHCCs).Although the consultation rate for all infections remained around 30% each year, antibiotic prescribing rates decreased significantly over the years from 53.7% in 2008, to 45.5% in 2010, to 38.6% in 2013  contains supplementary material, which is available to authorized users. Antibiotic use is associated with the development of antimicrobial resistance –6. It haThe frequency of antibiotic prescribing varies greatly between countries in Europe –17. In SRespiratory tract infections (RTIs) represent the majority (50–60%) of all infections in Swedish primary care and therefore are at focus for efforts to optimise antibiotic prescribing , 19 MediWithin the last ten years, national guidelines regarding Lower Respiratory Tract infections (LRTIs), acute otitis media (AOM) and tonsillitis have been revised and all recommend a more restrictive use of antibiotics than earlier versions. The guidelines for LRTIs were revised in 2007 and they recommend Phenoxymethylpenicillin (PcV) as first line treatment for pneumonia and to abstain from antibiotics in acute bronchitis. When in diagnostic uncertainty, a C-reactive protein (CRP) test could be used. Guidelines for AOM (revised in 2010) recommend wait and see for three days and no immediate antibiotic treatment in children 1–12 years of age without complicating factors. The guideline for tonsillitis (updated in 2012) require the presence of 3 out of 4 Centor criteria  and a positive rapid antigen detection test for Beta-haemolytic Streptococcus group A (RADT-Strep-A) before antibiotic treatment is initiated.Gathering and analysing data on management of patients with infections in primary care forms an essential knowledge-base in order to identify new strategies for prudent antibiotic use. In Sweden, prudent use of antibiotics is defined as prescribing in adherence with current guidelines.Previous Swedish studies on antibiotic use in primary care have been based on one-week registrations of infectious disease and therefore have included few data , 24. DatIn 2007 the Primary care Record of Infections in Sweden (PRIS) was introduced. PHCCs were invited to participate on a yearly basis and contribute with data on consultations with an infectious diagnosis and all antibiotic prescriptions. In PRIS the majority of antibiotic prescriptions can be linked to a particular diagnosis, as can information on age, gender, results of CRP and RADT-Strep-A, if applicable, for each patient encounter. Preliminary results have shown a very variable prescription rate as well as variable adherence to guidelines , 27.The aim of this study was to describe how antimicrobials were used in Swedish primary care, 2008–2013. We aim to identify trends over time, particularly for respiratory tract infections with regard to antibiotic prescriptions and adherence to national guidelines.This retrospective, descriptive study was based on the information retrieved from the PRIS register, from the years 2008, 2010 and 2013.The number of Primary Healthcare Centres (PHCC) contributing data was 47 in 2008, 58 in 2010 and 88 in 2013. In Sweden there were about 1200 PHCCs in total in 2010. In a typical PHCC in Sweden there are about 3–10 General Practitioners working. Together, the PHCCs represented in PRIS, served 460,529 registered patients in 2008 and 785,070 in 2013, of all ages, in both urban and rural areas of Sweden. Accordingly, the number of registered patients in 2013 represented 8% of Sweden’s total population of 9,644,864 inhabitants in 2013 . Due to For each year all registered contacts were traced either due to the presence of an infectious diagnosis according to the International Classification of Disease and Related Health Problems- Tenth Revision (ICD-10), introduced by the WHO, or because an antibiotic prescription by Anatomical Therapeutic Chemical Classification (ATC)-code had been registered. The ATC coding system is used to classify drugs to facilitate studies of drug use in Sweden. With each registered encounter information on the patient’s age, sex and if applicable laboratory testing and antibiotic prescription was recorded. Common near patient tests in Sweden are CRP and RADT-Strep-A.Some patient encounters had more than one registered diagnosis and sometimes an extra symptom diagnosis had been added, such as cough. The diagnoses in PRIS have therefore been ranked, so that the highest ranked is the diagnosis most likely to cause an antibiotic prescription according to clinical practice. Of the most common RTI diagnoses,, pneumonia has the highest rank, followed by AOM, tonsillitis, sinusitis, acute bronchitis, pharyngitis, common cold and symptom diagnoses like cough. If a contact had generated more than one diagnosis only the highest ranked was retained. The same ranking system was used all years.The different diagnoses were divided into four groups according to type of infection; respiratory tract including ear infections (RTI), urinary tract (UTI), skin and soft tissue (SST) and other infections. The latter group consisted of diagnoses found in Chapter A and B of the ICD-code system, including relatively few patients, and some other diagnoses such as diverticulitis, gynaecological-, and sexual transmitted infections.RTIs were further divided into upper RTIs (URTIs) and lower RTIs (LRTIs). URTIs were organised into groups that share similarities in clinical practice or national guidelines, such as common cold, sore throat, acute otitis media (AOM) and sinusitis. The group of sore throat consisted mainly of tonsillitis but also acute pharyngitis, mononucleosis, quinsy and scarlet fever. AOM was divided into age groups, due to specific Swedish national guidelines regarding children 1–12 years old.The LRTIs were divided into chronic obstructive disease (COPD), cough, acute bronchitis, pneumonia and influenza as these groups differ with regard to recommended treatment. Nonspecific chronic bronchitis was included in the COPD.The consultation and antibiotic prescription rates regarding RTIs in Table In order to calculate the number of antibiotic prescriptions per 1000 patient year (PY) each PHCC provided information on the number of registered patients at their clinic, calculated as a mean for each year. PY refers to the number of registered patients in the participating PHCCs, per year.In Table Data from the 37 PHCCs who participated all three years, with approximately 380,000 registered patients, was compared to that of all PHCCs. Percentage of consultations for infections, prescribed antibiotics and number of prescription per 1000 PY were calculated  and SPSS .http://epitools.ausvet.com.au. P-values ≤ .05 were considered statistically significant.For descriptive statistics, proportions and standard deviations were used. Comparisons between proportions of categorical variables in more than two independent groups were performed with the Chi-square test for trend, using an on-line calculator, p = .032). . Table  The pres). Table .Table 1CRTIs, including AOM, was the most common group of infections, representing more than half of the consultations and resulted in the highest number of prescriptions per 1000 PY. UTIs and SST infections were the second and third most common reason for consultation. The prescribing of antibiotics per 1000 PY decreased significantly for RTIs, but remained unchanged for UTIs and SST infections Table .Table 2CThe frequency of prescriptions without a diagnosis decreased from 22% in 2008 to 13% in 2013. Antibiotic prescriptions with no registered diagnosis could, due to the type of antibiotic prescribed, be allocated to probable RTIs  in 53% (2008), 46% (2010), 41% (2013) of the cases and to UTIs  in 28% (2008), 29% (2010) and 40% (2013). SST type antibiotics  represented about 20% of the prescriptions with no diagnosis in all three years.PcV was the most common antibiotic prescribed for RTIs followed by tetracycline. The proportion of PcV increased and all other antibiotic subclasses decreased from 2008 to 2013 . Although the consultation rate for acute bronchitis remained stable from 2008 to 2013, the antibiotic prescription rate decreased from 59.7% in 2008 to 26.2% in 2013. The most commonly used antibiotic for acute bronchitis was tetracycline (54.1%).The antibiotic prescribing rate per diagnostic group varied between different PHCC. The largest difference between the lowest and the highest prescribing PHCC was found for the diagnoses acute bronchitis and sinusitis. The mean antibiotic prescription rate per diagnoses per PHCC has decreased for all diagnoses between 2008 and 2013, but the standard deviation (SD) as an indicator for variability has increased for all diagnoses except pneumonia Table .Table 4VCRP was analysed in almost one fourth of all consultations throughout the three years. Almost one third (28.5%) of the patients with sore throat diagnosed in 2013 had a CRP taken. Among the lower respiratory tract infections CRP was used most often among patients diagnosed with pneumonia (60.4%) and acute bronchitis (49.4%).The consultation rate decreased for all age groups, except for 65 years and older, from 2008 to 2013.  and the Netherlands. In contrast to Sweden, the antibiotic prescribing for coughs/cold is reported to have increased in the UK over the years 1995–2011 despite implementation of national guidelines . The incIn the Netherlands antibiotic prescribing has been rather stable and not decreased between 2007 and 2010 . Antibio, The reason for the relatively high consultation rate for sore throat in Sweden is believed to be associated with the high availability of the RADT-Strep A at every PHCC, as well as other factors.Sore throat, including tonsillitis and pharyngitis, is noted to have similar treatment frequencies in the UK (62%), the United States (60%) and Sweden (60%) but a little lower in the Netherlands (55%) –34, The The PRIS database contains the largest number of patient registrations with diagnosis linked data on antibiotic prescribing in primary care in Sweden. The PHCCs represented in the database are participating on a voluntary basis, which might cause a selection of more motivated PHCCs and hence, better prescribing rates. On the other hand, the fact that in some of the geographic areas, all of the PHCCs participate contradicts a selection bias. In addition, private and public PHCCs in both rural and urban areas are represented. The number of participating PHCCs increased from 2008 to 2013 and accordingly, the number of consultations, but the percentage related to an infection remained constant (30%), which reduces the likelihood of a very strong selection bias. It can be argued that all calculations should be based only on the PHCCs participating all years, however that would make some of the data less reliable, for example AOM and age groups. We therefore compared the prescription rate and number of prescriptions per 1000 PY for the 37 PHCCs that participated all years with the total and did not find any differences  of these antibiotics were prescribed without a patient visit. Possible explanations are prescriptions for patients at nursing homes, from consultations by telephone or prescriptions being registered later after receiving results from cultures or x-rays.This study supports that primary healthcare in Sweden has succeeded to improve prescribing of antibiotics in accordance to national guidelines. Both consultations and and antibiotic prescribing for RTIs have decreased. The significant decrease in prescribing rate for AOM and tonsillitis indicate that updating and implementing national guidelines is an important tool to reduce antibiotic use.There are still large variations in prescribing frequencies between different PHCCs, indicating that irrational antibiotic prescribing still exists. Continuous evaluation of diagnosis linked prescribing data and feedback to doctors is essential in order to make changes towards more prudent antibiotic use.


Different groups developed wearable robots for walking assistance, but there is still a need for methods to quickly tune actuation parameters for each robot and population or sometimes even for individual users. Protocols where parameters are held constant for multiple minutes have traditionally been used for evaluating responses to parameter changes such as metabolic rate or walking symmetry. However, these discrete protocols are time-consuming. Recently, protocols have been proposed where a parameter is changed in a continuous way. The aim of the present study was to compare effects of continuously varying assistance magnitude with a soft exosuit against discrete step conditions.Continuous-up, peak exosuit ankle moment linearly increased from approximately 0 to 38% of biological moment over 10 min. Continuous-down was the opposite. In Discrete, participants underwent five periods of 5 min with steady peak moment levels distributed over the same range as Continuous-up and Continuous-down. We calculated metabolic rate for the entire Continuous-up and Continuous-down conditions and the last 2 min of each Discrete force level. We compared kinematics, kinetics and metabolic rate between conditions by curve fitting versus peak moment.Seven participants walked on a treadmill wearing a soft exosuit that assists plantarflexion and hip flexion. In Powered-off was smaller in Continuous-up than in Continuous-down at most peak moment levels, due to physiological dynamics causing metabolic measurements in Continuous-up and Continuous-down to lag behind the values expected during steady-state testing. When evaluating the average slope of metabolic reduction over the entire peak moment range there was no significant difference between Continuous-down and Discrete. Attempting to correct the lag in metabolics by taking the average of Continuous-up and Continuous-down removed all significant differences versus Discrete. For kinematic and kinetic parameters, there were no differences between all conditions.Reduction in metabolic rate compared to Continuous directions). The shorter time and higher resolution data of continuous sweep protocols hold promise for the future study of human interaction with wearable robots.The finding that there were no differences in biomechanical parameters between all conditions suggests that biomechanical parameters can be recorded with the shortest protocol condition (i.e. single The online version of this article (doi:10.1186/s12984-017-0278-2) contains supplementary material, which is available to authorized users. In recent years, there have been a growing number of different academic and industry groups developing wearable robots for walking assistance in able-bodied and clinical populations. Typically, devices have been evaluated and optimized for specific objectives by means of studies where a device parameter is systematically varied while changes in an objective parameter are measured –12. DiffInitially, most experiments with wearable robots used discrete step protocols , 9, 11. When focusing specifically on metabolic rate, several challenges arise in the use of discrete step protocols. Due to noise in indirect calorimetry measurements, this type of protocol requires averaging a large number of breaths –20 and sIn the field of exercise physiology, continuous sweep protocols have been proposed for identifying cardiopulmonary thresholds and have shown promise as a quicker alternative to discrete step protocols which is especially useful for clinical populations that cannot engage in extended activity , 25, 26.The primary focus of the present study was to evaluate differences in metabolic rate and gait biomechanics between evaluation protocols that use continuous and discrete variations of actuation parameters. In a continuous sweep protocol, it is known that there is an apparent lag or delay between parameter changes and the corresponding changes in metabolic rate. This can be due to the bulk effect of physiological dynamics and biomechanical response delay. The apparent delay in metabolic rate is not an absolute delay but is known to follow an exponential evolution over time. Conversely, we define the time that is required by a trained user to change biomechanics in response to a parameter change as “biomechanical response delay”.Our specific aims and hypotheses are as follows: (1) Measure if there is biomechanical response delay in continuous conditions by comparing biomechanical metrics between continuous and discrete conditions. We hypothesize that potential biomechanical response delay is small or non-existent when the continuous changes in wearable robot parameters are gradual and thus would be easy for participants to adapt to, except if the parameter change is such that the user can never adapt to it, such as if the force is too high to allow walking. (2) Evaluate the physiological dynamics during the continuous conditions by comparing reduction in metabolic rate between continuous and discrete conditions. We hypothesize that measured reductions in metabolic rate in continuous conditions will be lagging behind the values that would be expected during steady-state testing. In turn we expect this will result in higher reductions measured in the continuous condition where the peak force goes from high to low, and lower reductions in the continuous condition where the peak force goes from low to high and the reductions in discrete conditions being halfway between both continuous condition directions. (3) Evaluate two methods to correct for physiological dynamics during testing with a continuous protocol: taking the average of two sweep directions of a continuous condition and using the instantaneous metabolic cost estimation from Selinger et al.  and FeltTo validate the use of continuous and discrete protocols for optimizing wearable robotics we compared both protocols in a parameter study with a soft textile based exoskeleton, called exosuit, developed by our group , 11, 17.We tested seven participants . The study was approved by the Harvard Longwood Medical Area Institutional Review Board and all experiments were conducted in accordance with this approved protocol. All participants provided written informed consent.−1 under two conditions while parameters were varied: A Discrete step condition and two Continuous sweep conditions (Continuous-up and Continuous-down).Participants walked on a treadmill at 1.5 m sDiscrete, participants underwent a series of 5-min periods with steady peak forces at zero (Powered-off), 18.7 (Low), 37.5 (Med.), 56.2 (High), and 75.0% body weight (Max.). This is similar to how the majority of standard parameter studies were conducted in the literature . Th. ThContiContinuous-up, Continuous-down and Continuous-bidirectional versus Discrete is that Discrete is also affected by normal noise and variability in metabolic rate and wearer adaptation. It should also be noted that our findings from comparing different protocols cannot be taken as general guidelines, since the results dependent on the protocol modalities [Discrete as well as in the steady-state periods before Continuous-up and Continuous-down. While the time that we allowed should have been sufficient to approach the steady-state value assuming a time constant of 42 s the fact that we found higher time values when analyzing the individual time constants indicates that this could have affected our results. Results could also have been different if participants were given a shorter or longer training and warm-up time. With shorter training the relative importance of small time savings would be larger but it could also be possible that participants would be less able to quickly adapt to the continuous conditions given less training beforehand. In future studies with discrete and continuous sweeps it could be interesting collect data from the training day to investigate if there is more biomechanical response delay when participants first experience changes in exosuit assistance. While we did not find evidence of biomechanical response delay in kinematics and total body kinetics it could be possible that there would changes in muscle activation (e.g. co-contraction) that could cause and explain changes in metabolic cost. However, in this study we did not measure EMG data and so future studies would be required to investigate if there was a link between changes in muscle activation and metabolic rate. For the Adjusted Continuous-up and Adjusted Continuous-down calculations it could also be interesting to rigorously calculate and use the individual time constant based on extra tests with a large number of small discrete parameter changes. Other limitations are the number of participants that were tested and the fact that we did not use correction for multiple testing. Because of all these limitations the results of this study should be rather interpreted as an explorative analysis of the possible advantages and disadvantages of discrete and continuous protocols rather than a recommendation for either of them over the other. Finally, it should also be noted that aside from continuous protocols other protocols could provide improvements and are being studied such as gradient based methods [A limitation of all comparisons of dalities , 25. Londalities  confound methods , 15.Continuous-up; or decreasing, Continuous-down) with a soft exosuit, we can achieve a shorter time and higher resolution data collection for specific applications compared to a protocol with discrete step conditions (Discrete). As expected, reduction in metabolic rate was smaller in Continuous-up than in Continuous-down at most peak moment levels due to the physiological dynamics associated with metabolic measurements. When evaluating the average slope of metabolic reduction over the entire peak moment range there was no significant difference between Continuous-down and Discrete. Thus, a unidirectional continuous sweep, starting from the steepest side of the landscape of metabolic rate versus parameter change, may replace a discrete step protocol for evaluating the average slope of metabolic rate over peak moment. Attempting to correct for the physiological dynamics by taking the average of Continuous-up and Continuous-down successfully removed all significant differences versus Discrete. However, this result should not be interpreted as an argument against the use of the adjustment methods from Felt et al. [Adjusted Continuous-up and Adjusted Continuous-down correctly estimated Discrete.Different groups successfully developed wearable robots for walking assistance, but there is still a need for methods to quickly tune actuation parameters for each robot and population. Discrete step protocols have traditionally been used for evaluating responses to parameter changes, but these are time-consuming. Recently, protocols have been proposed where a device parameter is changed in a continuous way. In the present study, we show that by continuously varying assistance magnitude  impact the wearer response . However, in this study, we have shown that using continuous sweep protocols for examining changes in kinematic or kinetic objectives has potential for improving over discrete step protocols. This could be useful for certain applications where metabolic rate is not the prime objective or participants have limited walking ability.Additional file 1:Choice of curve fitting order for fitting metabolic rate versus peak exosuit ankle moment. (PDF 97 kb)Additional file 2:Simulation of physiological dynamics. (PDF 71 kb)Additional file 3:Inter-stride variability . Biomechanical delay analysis in Additional file 5:Change in metabolic rate plotted against exosuit ankle peak moment plus minus 95% confidence interval. (PDF 59 kb)


The determination of compressive strength is affected by many variables such as the water cement (WC) ratio, the superplasticizer (SP), the aggregate combination, and the binder combination. In this dataset article, 7, 28, and 90-day compressive strength models are derived using statistical analysis. The response surface methodology is used toinvestigate the effect of the parameters: Varying percentages of ash, cement, WC, and SP on hardened properties-compressive strengthat 7,28 and 90 days. Thelevels of independent parameters are determinedbased on preliminary experiments. The experimental values for compressive strengthat 7, 28 and 90 days and modulus of elasticity underdifferent treatment conditions are also discussed and presented.These dataset can effectively be used for modelling and prediction in concrete production settings. Specifications TableValue of the data•The present data can be used to predict the strength of auto-compacting concrete at varying days.•The dataset can be used to determine the trend of strength associate with concrete.•The dataset can be used to detect the effect of SP.•The dataset can be used to determine the nature of concrete, and the corresponding degree of hydration.•The dataset can serve as an experimental framework for the analysis of other basic properties of concrete.•The dataset can help in developing experimental programme for the evaluation of model accuracy and precision.1Strength data presented here are from seventy-two (72) different POFA concrete samples fabricated to compare with normal concrete without ash. We make reference to 1.1In this investigation, concrete samples were prepared using Palm Oil Fuel Ash (POFA) at varying percentages, with ordinary Portland cement. The POFA was replaced at 5%, 10%, 15%, 20%, 25%, and 30% with cement and superplasticer at 2%.22.1Besides the statistical software used in the data analysis, is a predictive quadratic model defined as follows:2.2For It is noted from 2, 80.5 N/mm2 respectively at 90 days.The plots of the experimental values of the compressive strength at varying days vs the predicted strength using Matlab are shown via 2.3Matlab statistical software was used to analyse and investigate the effect of the parameters  ratio, POFA and superplasticiser (SP) on the hardened properties (compressive strength at 7, 28 and 90 days. Determination of the independent parameters with respect to their percentage replacement was made on initial experiments as shown in


Human dental pulp cells have been known to have the stem cell features such as self-renewal and multipotency. These cells are differentiated into hard tissue by addition of proper cytokines and biomaterials. Hydroxyapatite-tricalcium phosphates (HA-TCPs) are essential components of hard tissue and generally used as a biocompatible material in tissue engineering of bone. Demineralized dentin matrix (DDM) has been reported to increase efficiency of bone induction. We compared the efficiencies of osteogenic differentiation and in vivo bone formation of HA-TCP and DDM on human dental pulp stem cells (hDPSCs). DDM contains inorganic components as with HA-TCP, and organic components such as collagen type-1. Due to these components, osteoinduction potential of DDM on hDPSCs was remarkably higher than that of HA-TCP. However, the efficiencies of in vivo bone formation are similar in HA-TCP and DDM. Although osteogenic gene expression and bone formation in immunocompromised nude mice were similar levels in both cases, dentinogenic gene expression level was slightly higher in DDM transplantation than in HA-TCP. All these results suggested that in vivo osteogenic potentials in hDPSCs are induced with both HA-TCP and DDM by osteoconduction and osteoinduction, respectively. In addition, transplantation of hDPSCs/DDM might be more effective for differentiation into dentin. Periodontal disease and dental caries cause periodontal bone defects, which will lead to tooth loss. The best way to treat periodontal bone defects is the induction of osteogenesis through reconstructive surgery. Recently, bone tissue engineering technology is known to be a promising clinical application for replacing wounded dental tissues –5. Bone α-MEM (HyClone) containing 20% fetal bovine serum  and 1% antibiotics (Lonza) at 37°C in humidified atmosphere supplemented with 5% CO2. To observe osteoinduction efficiency in vitro, 40,000 cells per well were cultured with 0.8 g of HA-TCP  or DDM  in a hanging insert cell culture dish (SPL Life Sciences). For transplantation, 1 × 106 cells were resuspended in 100 μL of thrombin, and 40 mg of HA-TCP or DDM granules was added. Then, 100 μL of fibrinogen  was added and mixed immediately to allow polymerization at room temperature. Fibrin blocks of hDPSCs/HA-TCP or DDM were incubated in culture at 37°C in humidified atmosphere supplemented with 5% CO2 till transplantation.For primary culture of human dental pulp stem cells (hDPSCs), human third molars were collected from patients who were 15 to 27 years old under guidelines approved by the Institutional Review Board (IRB) of Dankook Dental Hospital (DKUDH IRB 2016-12-005). Human dental pulp tissue was obtained from the internal part of the tooth, chopped into small fragments, and enzymatically digested with 3 mg/mL collagenase type I (Millipore) and 4 mg/mL dispase (Sigma) at 37°C for 1 h. Cell suspension was incubated in α1 radiation (λ = 1.5405 Å). The patterns of XRD were measured for step-scan of 0.02° and rate of 3° per minute over a 2θ range from 20 to 80°. Surface morphologies of HA-TCP and DDM were examined by scanning electronic microscopy . The HA-TCP and DDM were sputter-coated  with platinum and sliced to a thickness of 5 nm prior to SEM measurement. The pore size of DDM in SEM images was evaluated to use ImageJ software .The crystallographic properties of HA-TCP and DDM were investigated to use X-ray diffraction  measurement. The XRD was activated at 40 kV and 40 mA with Cu Kα-MEM (HyClone) without FBS at 37°C for 3 days to extract their components. Extracts were then concentrated. To detect cytokines and growth factors released from each inorganic granule, proteins in extracts were separated by SDS-PAGE, transferred to PVDF membrane, and probed with anti-collagen type-1 antibody (Santa Cruz Biotechnology) followed by incubation with horseradish peroxidase- (HRP-) conjugated secondary antibody. Protein signals were visualized by using ECL™ reagent .Each HA-TCP (0.7 g) and DDM (0.7 g) granule was incubated with μM EthD-1 and 2 μM calcein-AM in PBS at room temperature for 45 min in the dark. The fluorescent signals were detected by using confocal microscope .Cell viability was determined using calcein-AM staining solution (Thermo Scientific). The fibrin blocks of hDPSCs/HA-TCP and hDPSCs/DDM were incubated in culture media for 4 days, washed with PBS, and incubated with 4 Twenty immunocompromised nude mice  were used in this study. Animal study protocol was approved by the Institutional Animal Care and Use Committee of Dankook University. Two experimental groups were used in this study. Each group had 10 animals. Group 1 was used for fibrin gel with HA-TCP granule with or without hDPSCs. Group 2 was used for fibrin gel with DDM granule with or without hDPSCs. For transplantation, mice were anesthetized by intraperitoneal injection of a mixture of tiletamine/zolazepam  and xylazine . Two subcutaneous 1 cm incisions were made in each flank pocket from the dorsal midline of mice. Prepared fibrin gels with granule with or without hDPSCs were placed into each pocket. After 1 week and 8 weeks, animals were sacrificed and implants were retrieved for further studies.μm pixel with a 0.5 mm aluminum filter and a rotation step of 0.4. Three readings were obtained for each sample. Bone volume was determined using a CT analyzer program . 3D images were obtained using a 3D-visualization program. Statistical analyses for three readings were carried out using Student's t-test. Statistical significance was considered when p value was less than 0.05. Data are expressed as means with error bars representing standard error of the mean (SEM). Student's t-test was performed using GraphPad Prism 6 program.Mineralization and bone formation were evaluated by micro-CT. Micro-CT images were obtained using a micro-CT scanner  at a resolution of 15 Total RNAs of hDPSCs and frozen tissue were extracted by using Easy-spin™ Total RNA Extraction kit  and RNA Easy Mini Extraction kit (Qiagen), respectively. cDNA was synthesized from total RNA by using the ReverTra Ace™ qPCR RT kit (Toyobo Corporation), and the qRT-PCR was performed by using iTaq™ Universal SYBR™ Green Supermix (Bio-Rad) system. Used primers are listed in t-test and p values of less than 0.05 were considered statistically significant. Data are expressed as means (n = 3) with error bars representing standard error of the mean (SEM). Student's t-test was performed using GraphPad Prism 6 program.GAPDH was used as an internal control to normalize the variability in target gene expression. Statistical analyses on three readings were carried out using Student's μm by a microtome . Sectioned samples were deparaffinized in xylene, hydrated with a series of graded ethanol, and stained with hematoxylin/eosin and Golder's trichrome staining. An optical microscope  was used to obtain images of the stained samples for confirming newly the deposition of mineralization by HA-TCP and DDM.After 8 weeks, the retrieved samples from dorsal midline of athymic nude mice were fixed in 4% paraformaldehyde (PFA) overnight. The fixed samples were rinsed with PBS for removing residual PFA and dehydrated using 70% ethyl alcohol. And then, the dehydrated samples were decalcified using decalcifying solution-Lite  for 16 hrs and embedded in paraffin wax. The embedded samples were transversely cut to obtain different cross-sectional images. The samples were sectioned with thickness of 6 n = 6 per group) and immersed in xylene and preceded a preconditioning process for double immunofluorescence staining of osteogenic and dentinogenic markers with human nuclear antigen (HNA). The samples were incubated in blocking buffer for 1 hr at room temperature. After blocking, samples were stained for 16 hrs with primary human specific antibodies; a 1 : 100 dilution of antibone sialoprotein antibody (Abcam), 1 : 200 dilutions of antihuman nuclear antigen (Abcam) and antiosteocalcin antibody (Abcam), a 1 : 500 dilution of antiosteopontin antibody (Abcam), a 1 : 1000 dilution of antiosteonectin antibody (Millipore), and 1 : 100 dilution of antidentin sialophosphoprotein antibody (Abcam). Fluorescein-isothiocyanate conjugated secondary antibodies  and 4′,6′-diamidino-2-phenylindole  were used to visualize the signals of the primary antibodies and nuclei, respectively. Samples were measured by using confocal microscope .Sectioned samples were selected randomly  used in this study was a congeneric biomaterial produced from human dentin for this research. It was prepared by grinding, degreasing, and decalcification . Two different specimens were randomly selected to determine their pore size and surface morphologies by SEM analysis. Results of SEM analysis are shown in μm , lanes 4μm , lanes 3μm , lanes 2To determine the osteoinduction potential of HA-TCP or DDM, hDPSCs were cultured under the inserts with 800 mg of HA-TCP or DDM. Actively growing hDPSCs are generally detected as well-spreading and flat shapes , panel 1For transplantation, hDPSCs were mixed with HA-TCP or DDM granules. To keep cells gathering in transplantation area and promote cell survival, it is necessary to encapsulate cells and biomaterial granules within fibrin gel. Culturing cells in a three-dimensional matrix is an important technique for tissue engineering as well as for studying cellular responses under culture conditions with biomaterials in vitro. hDPSCs were encapsulated with HA-TCP and DDM-fibrin gel, which had a similar size of 1 cm in width and 0.4 cm in height Figures . Regardl3 and 2.91 mm3, respectively, at 8 weeks compared to those at 1 week (∗p < 0.001), but the expression of other genes was not significantly different in between HA-TCP and DDM. Although DDM had higher osteoinduction potential than HA-TCP, osteoconduction ability of HA-TCP could also be induced after in vivo transplantation experiment for 8 weeks. Goldner's trichrome staining results showed nearly no bone formation in implantation of HA-TCP or DDM without hDPSCs after 8 weeks of subcutaneous transplantation in mice. Implantation of HA-TCP or DDM resulted in regeneration of fibrous-like tissue  DDM might also be useful as a promising scaffold in bone substitution. Furthermore, microsized pores are expected to be able to efficiently supply nutrients and oxygen in vivo  D in vivo . hDPSCs  in vivo . Express in vivo . Reverse in vivo , demonst in vivo , and lac in vivo . Finally in vivo , indicatHuman dental pulp stem cells have been known to be able to form hard tissues through osteo/dentinogenesis. In current studies, HA-TCP and DDM have been used for regeneration of dentin or bone in tissue engineering. Here, we investigated the effect of osteo/dentinogenic potential of hDPSCs on DDM compared to that on HA-TCP in vitro and in vivo. Osteoinduction effect of DDM was clearly observed in vitro, but osteogenic potential was similar in both cases in in vivo transplantation. Interestingly, dentinogenic potential was detected in a higher efficacy in transplantation of DDM/hDPSCs, suggesting that DDM might be more effective than HA-TCP on dentin regeneration of hDPSCs.


Met treatment significantly improved left ventricular (LV) function, reduced infarct size and CK-MB release in comparison with IR group. Decreased TUNEL staining positive cells were also observed in IR+Met group ex vivo. Met treatment markedly inhibited IR inducing cell death and significantly decreased apoptosis with few generations of reactive oxygen species (ROS) in H9c2 cells in comparison with IR group. Up-regulated expressions of phosphorylated LKB1/AMPK/ACC, as well as down-regulated expressions of apoptotic proteins (Bax and cleaved caspase 3) were found in IR+Met group when compared to the IR group. Importantly, Met significantly up-regulated the expression of antioxidant enzymes  during IR procedure either ex vivo or in vitro. Compound C, a conventional inhibitor of AMPK, abolished the promoting effect of Met on antioxidant enzymes, and then attenuated the protective effect of Met on IR injury in vitro. In conclusion, Met exerted protective effect on myocardial IR injury, and this effect was AMPK/ antioxidant enzymes dependent.The objective was to examine the protective effect of metformin (Met) on myocardial ischemia-reperfusion (IR) injury and whether the mechanism was related to the AMPK/ antioxidant enzymes signaling pathway. Rat Langendorff test and H Myocardial infarction (MI) is one of the most common cardiovascular diseases with high mortality. Early reperfusion by primary percutaneous coronary intervention (PCI) remains the most effective treatment for MI . HoweverIt is well known that the excessive generation of reactive oxygen species (ROS) resulting in redox imbalance during reperfusion period plays key roles in the pathogenesis of IR injury . ExcessiMetformin (Met), an orally administered biguanide drug, is now widely used to lower blood glucose concentration in patients with type 2 diabetes mellitus (T2D). The cardioprotective effect of Met has been documented as well –12. InteTherefore, the present study investigated the cardioprotective effect of Met on myocardial IR injury and determined whether this effect was involved in AMPK/ antioxidant enzymes signaling pathway.Male Sprague-Dawley rats (10 weeks old) were purchased from the Institute of Laboratory Animal of Chinese Medical Science Academy, Beijing, China. Rats were maintained in a temperature-controlled room (25°C) with a natural day/night cycle and fed with a standard rodent diet and water. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of Nankai University. The protocol was approved by the Animal Care Committee of School of Medicine, Nankai University, (Permit Number: 10017). The rats were anesthetized with i.p. injection of 10% chloral hydrate, followed by cervical dislocation prior to removal of the hearts. All efforts were made to minimize suffering.Thirty-two rats were randomly divided into three groups as follows: (1) Control (Con) group ; (2) IR group (n = 8/11); (3) IR +Met group (n = 8/12). Rat hearts were quickly removed from heart cavity and connected to Langendorff apparatus by inserting a perfusion cannula into the aorta. Isolated hearts were stabilized for 30 min with Krebs-Henseleit (K–H) solution, and then subjected to global, no-flow ischemia for 30 min and 60 min reperfusion with a flow speed of 4 ml/min in IR group. No ischemia was subjected to isolated heart in Con group. Met (100μM) was added into K-H solution in IR+Met group 15min prior to the ischemia period. Left ventricular developed pressure (LVDP), left ventricular end diastolic pressure (LVEDP), maximal rate of rise of left ventricular pressure (+dp/dp max) and maximal rate of decrease of left ventricular pressure (-dp/dt max) were recorded by a data acquisition device . Perfusion solutions  were collected for further assessments. When the Langendorff test was finished, the heart samples were collected to examine the area of myocardial infarction, apoptosis of myocardial cells and expression levels of related mRNA and proteins.Rat hearts were removed at the end of reperfusion and immediately frozen in -20°C refrigerator for 1h. Then, hearts were incubated in 1% triphenyl tetrazolium chloride (TTC) solution  at 37°C for 15min, followed by immersion in 4% paraformaldehyde solution for 30min. Five cross-sectional slices were taken and imaged on a digital camera, and the percentage of infarct area (white area) to total ventricular area was calculated using Image J software.Perfusion solution samples were collected at 10, 70, 90 and 120 min. The CK-MB levels were measured using a rat CK-MB ELISA kit  with a microplate reader  according to the manufacturer’s instruction.After Langendorff test, the heart tissues were fixed in paraformaldehyde solution for 24h, embedded in paraffin, and 5 μm sections were obtained. Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining in heart tissues was performed according to the protocol of TUNEL Staining Kit  to determine the apoptosis of cardiomyocytes. The apoptotic rate was determined by Image J software.2. Once cells reached 60–70% confluence, subsequent experiments were performed. Briefly, H9c2 cells were cultured with 500 µmol/L H2O2 in DMEM containing 2% FBS for 18 h to mimic IR procedure. Cells were pretreated with Met (50μM), Compound C , AICAR  for 24h to confirm the effect on AMPK signaling pathway.Rat embryonic cardioblasts H9c2 cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. Cells were cultured in the DMEM medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100U/mL penicillin, and 100μg/mL streptomycin and incubated at 37°C with 95% air and 5% COCalcein-AM/PI double staining was performed as described previously  to deterApoptosis was also assessed by flow cytometry. Cells were stained with Annexin V and PI by a FITC Annexin V apoptosis detection kit  as described previously . ApoptotIntracellular ROS level was measured using dichlorofluorescein diacetate (DCFH-DA) staining kit . In brief, DCFH-DA was diluted by PBS to the final concentration of 10μmol/L and incubated at 37°C for 30min. Then the cells were washed twice with PBS. The images of cells were captured by a fluorescence microscope  and fluorescent intensities were analyzed using Image J software.5′-AGGTCGCTTACAGATTGCC-3′ and 5′-CATTCTCCCAGTTGATTACATT-3′. catalase: 5′-CCTCGTTCAAGATGTGGT-3′ and 5′-CACCTTTGCCTTGGAGTA-3′. GAPDH: 5′-TATCGGACGCCTGGTTAC-3′ and 5′-CTGTGCCGTTGAACTTGC-3′. The qPCR was performed using SYBR Green Mix  in a Bio-Rad IQ5 detection system. The amount of each gene expression was normalized to GAPDH mRNA expression.Total RNA in heart tissues and H9c2 cells were isolated with Trizol reagent  as described previously . In brieWestern blot analysis was performed as described previously . In brieAll experiments were repeated three times, and all results were expressed as the mean ± S.E.M. Significant differences between three or more groups were tested by one-way ANOVA followed by multiple comparisons performed with LSD test (SPSS ver. 17). Statistical significance was defined as p < 0.05.To evaluate the myocardial IR injury, parameters for LV function were recorded and calculated in Langendorff apparatus. No significant differences in the LV function parameters were found between three groups in 10 min of perfusion (stabilization period). IR resulted in decreased ±dp/dtmax, LVDP, and increased LVEDP in comparison with Con group in 70, 90, 120min (reperfusion period), respectively. However, Met treatment significantly improved LV functions in reperfusion period .As shown in ex vivo study. Met increased the p-LKB1, p-AMPK, p-ACC protein levels in comparison with IR group, indicating that Met treatment activated AMPK signaling pathway  and phosphorylated ACC (down-stream of AMPK) in  pathway . To dete pathway . In orde pathway . Additio pathway .AICAR (an activator of AMPK) and Compound C (an inhibitor of AMPK) were further used in H9c2 cells IR model to determine whether Met exerted cardioprotective effect via AMPK signaling pathway. We found that 50μM Met treatment markedly inhibited cell death during IR procedure; therefore, the concentration of 50μM of Met was selected to serve as the working concentration in the subsequent studies . AICAR tTo assess whether Met exerted anti-apoptotic effect via AMPK signaling pathway, flow cytometry test was performed in H9c2 cells. Met and AICAR treatment markedly attenuated IR-induced apoptosis of cardiomyocytes, while Compound C treatment abolished this attenuation by inhibition of AMPK signaling pathway .ex vivo study, Met increased phosphorylated Akt, decreased Bax and cleaved caspase-3 protein levels in comparison with IR group. Above mentioned effects of Met were abolished in the presence of Compound C  and increased apoptosis were found in IR group in comparison with Con group. Met treatment increased ΔΨm and reduced apoptosis when compared to the IR group, whereas, Compound C significantly blunted anti-apoptotic effect of Met . Meanwhimpound C .2O2 treatment, while Met obviously lessened the generation of ROS. Combination of Met and Compound C increased the ROS generation when compared to Met alone treatment , then inhibited the generation of ROS, reduced apoptosis, finally protected myocardiocytes against IR injury. Noticeably, this cardioprotective effect of Met was AMPK dependent .Metformin, a first line oral anti-diabetic drug for T2D, has been documented for its cardioprotective effect not only in animal studies but also in clinical trials. However, the underlying mechanism for this effect remains controversial. Zhou et al.  first reex vivo Click here for additional data file.


In vivo experiments were performed to test the effects of c-MET inhibitor on tumor growth in orthotopic mouse xenografts of OCCC cell line RMG1 and a patient-derived tumor xenograft (PDX) model of OCCC. c-MET expression was significantly greater in OCCCs compared with serous carcinomas and normal ovarian tissues (p < 0.001). In in vitro study, inhibition of c-MET using c-MET inhibitors (SU11274 or crizotinib) significantly decreased the proliferation, and increased the apoptosis of OCCC cells. SU11274 decreased expression of the p-c-MET proteins and blocked the phosphorylation of down-stream proteins Akt and Erk. Furthermore, SU11274 treatment significantly decreased the in vivo tumor weight in xenograft models of RMG1 cell and a PDX model for OCCC compared to control .In this study, we investigated the therapeutic effects of c-MET inhibition in ovarian clear cell carcinoma (OCCC). Expression levels of c-MET in the epithelial ovarian cancers (EOCs) and normal ovarian tissues were evaluated using real-time PCR. To test the effects of c-MET inhibitors in OCCC cell lines, we performed MTT and apoptosis assays. We used Western blots to evaluate the expression of c-MET and its down-stream pathway.  Epithelial ovarian cancer (EOC) is one of the leading causes of cancer-related deaths in women, and the most lethal gynecologic malignancy45c-MET is a receptor tyrosine kinase with a high-affinity ligand, hepatocyte growth factor/scatter factor (HGF/SF). In tumor, deregulation of c-MET activity can trigger important cellular processes including to cell proliferation, invasion, survival, and angiogenesis78101112in vitro and in vivo studies reported that inhibition of c-MET using small interfering RNA and small molecule inhibitors reduced EOC growth and metastasis161720in vivo as well as in vitro experiments including an orthotopic mouse model using an established cell line (RMG1) and a patient-derived tumor xenograft (PDX) model.In EOC, overexpression of c-MET is found in about 7% to 27%15161717c-MET expression was estimated in human ovarian tissues, including 16 normal ovarian, 47 serous carcinoma and 16 OCCC tissues. The expression level of c-MET was significantly higher in OCCC tissue compared with normal ovarian tissue or serous carcinoma tissue .in vitro and in vivo experiments with SU11274.We used two kinds of c-MET inhibitors to block the endogenous activity of c-MET in OCCC cells. SU11274 is a selective small molecule c-MET inhibitor, and crizotinib  is a multikinase inhibitor with known action against c-MET, anaplastic lymphoma kinase (ALK) and c-ros oncogene 1 (ROS1). In the MTT assay, SU11274 and crizotinib significantly reduced cell viability in a dose- dependent manner in both OCCC cell lines, including ES2 and RMG1 . The SU1To ascertain whether the expression of downstream proteins in the c-MET signaling pathway would be affected by a c-MET inhibitor, OCCC cells were stimulated with 25 ng/mL HGF for 20 mins. We examined the effects of SU11274 on phosphorylation of c-MET and the downstream signaling effectors, Akt and Erk. ES2 cells were pretreated with the indicated dose of SU11274 before stimulation with 25 ng/mL HGF for 20 mins. Treatment with SU11274 effectively decreased expression of the p-c-MET proteins and blocked phosphorylation of Akt and Erk, with inhibition of the c-MET downstream proteins in a dose-dependent manner . Phosphon = 10)An orthotopic model was established in nude mice by intraperitoneal injection (IP) of RMG1 cells  resulted in a decline of cell viability in MET-amplified OCCC cell lines due to increased apoptosis and senescence, suggesting that the MET signaling pathway plays an important role in OCCC carcinogenesiset al. reported that c-MET overexpression was associated with chemoresistance and worse prognosis in 86 OCCC patients. Collectively, these data indicate that inhibition of c-MET is a novel strategy for the clinical management of OCCC patientsIn OCCC, previous studies found that MET amplification was observed in >20% of cases and that OCCC with MET amplification exhibited a poor prognosis19in vivo orthotopic mouse model of OCCC (RMG1), treatment with SU11274 significantly decreased total tumor weight compared with that of control. These results were corroborated in a PDX model derived from a patient with OCCC, in which treatment with SU11274 significantly reduced tumor growth. These findings indicate that targeting c-MET with SU11274 represents a potential strategy for novel therapeutics for treatment of OCCC. To our knowledge, this study, for the first time, demonstrates the direct effect of c-MET inhibition on tumor growth of OCCC in vivo as well as in vitro.In this study, we found that c-MET expression was significantly higher in OCCC, compared with serous carcinoma or normal ovarian tissue. In addition, c-MET inhibitors (SU11274 or crizotinib) significantly reduced cell viability and enhanced apoptosis in OCCC cells. Moreover, in an et al. have found MET gene amplification in 37% of OCCC caseset al. have found c-MET overexpression in 41% of OCCC casesc-MET overexpression has been noted in various cancers. In a recent large study of 1115 patients with advanced solid cancers, MET amplification was detected in 2.6%. The highest prevalence was observed in renal cell (14%) and adrenocortical tumors (15%) and MET amplification was observed in 4% of ovarian cancers, all of which were serous carcinomaset al. reported that MET signaling is involved in tumorigenesis, such as tumor proliferation, protection from apoptosis, angiogenesis, and motility1516171917202118313218A systematic review from Furlan et al. recently reported that c-MET inhibitors with multikinase activity like crizotinib may exhibit less activity in ovarian cancer than c-MET specific drugs35et al. reported that SU11274 reversed the increased migration and invasion ability of sorafenib-resistant hepatocellular carcinoma cellsin vitro study, demonstrated that SU11274 reduced cell growth, motility, and invasive activity of EOC cellsTang in vitro experiments. Second, the biological effects of HGF and c-MET interactions were not investigated. According to recent study, Kwon et al. reported that anti-proliferative activity of DCC-2701, c-MET inhibitor, was dependent on c-MET activation which is induced by stromal derived HGF, and lesser extent exogenous HGF. They suggested that post-translational modifications of growth factors, such as HGF, might be one of the mechanisms that promotes tumor growth via factors produced by the microenvironmentsWe can consider several limitations in this study. First, this study was performed with relatively small number of OCCC cell lines for in vitro and in vivo experiments. This work suggests that c-MET targeting therapy may be promising for OCCC treatment. Further clinical studies focused on c-MET inhibitors in OCCC are warranted.In conclusion, we found that unique activation of c-MET in OCCC tissues. In addition, inhibition of c-MET with the small molecule c-MET inhibitor (SU11274) showed potent anti-tumor activity in OCCC using This study was reviewed and approved by the Institutional Review Board’s approval at Samsung Medical Center, Seoul, Korea (IRB # 2012-09-045). All experiments were performed in accordance with relevant guidelines. Written informed consent was obtained from each patient who participated in the investigation. Study subjects were enrolled from the Department of Obstetrics and Gynecology at Samsung Medical Center from July 2003 to November 2010. A total of 79 tissue specimens were collected including 16 normal ovarian tissues, 47 serous cancer tissues, and 16 clear-cell cancer tissues. Normal ovarian tissues were obtained from patients who underwent hysterectomy for benign disease, and tumor tissues were obtained from patients with ovarian cancer during surgery. All specimens were immediately snap frozen at −80 °C. The specimens were stained with hematoxylin and eosin (H&E) and evaluated by a gynecologic pathologist (ME Hong). Specimens with more than 90% tumor cells were used in the analysis. Histological typing of each tumor was conducted in accordance with the World Health Organization guidelines.−ΔΔCT method.Total RNA, including mRNA, was extracted from the tissues using the mirVana miRNA isolation kit . cDNA was synthesized from total RNA using oligo dT primer  in accordance with the manufacturer’s TaqMan RNA assay protocol . Real-time PCR was performed using an Applied Biosystems 7900HT Sequence Detection system (Applied Biosystems) in accordance with the manufacturer’s protocol. Data normalization was performed using GAPDH. To avoid amplification of genomic DNA, the primers and probes for amplifying c-MET (Met) and GAPDH were chosen to hybridize at the junction between the 2 exons as follows: c-MET (Met)  and GAPDH . Relative quantification of c-MET expression was calculated using the 22 at 37 °C. The c-MET kinase inhibitors, cirzotinib (3-[(1 R)-1-ethoxy]-5-(1-piperidin-4-ylpyrazol-4-yl)pyridine-2-amine)  and SU11274 ((3Z)-N-(3-Chlorophenyl)-3-methylidene]-N-methyl-2-oxo-1H-indole-5-sulfonamide) , were resuspended in dimethyl sulfoxide (DMSO) at a concentration of 100 μg/ml. Cells were seeded at 3 × 103 cells/well in 96-well microplates in culture medium with 10% fetal bovine serum (FBS). Cells were treated with inhibitor based on previous reportsES2 was obtained from the American Type Culture Collection  and RMG1 and RMG2 were purchased from Japan Health Science Research Resources Bank . A2780 was purchased from the European Collection of Cell Cultures . SKOV3ip1, HeyA8, A2780-CP20, HeyA8-MDR and SKOV3-TR were a gift from Dr. Anil K. Sood, Department of Cancer Biology, University of Texas M.D. Anderson Cancer Center, TX, USA. ES2, RMG1 and RMG2 cells were originally derived from ovarian clear cell carcinomas (OCCCs); the other cell lines are non-OCCC cellsCells or tissues were lysed in PRO-PRE Protein Extraction Solution . Whole cell lysates were prepared from tumor cell lines or tumor tissues and cells were lysed with lysis buffer containing 1 mM EDTA, 1% TX-100, 100 mM NaCl, 50 mM NaF, TrisHCl pH 7. 5, phosphatase inhibitor cocktail . Protein concentration was determined by Bradford assay kit . Cell or tissue lysates  were separated in 8% acrylamide gels by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Hybond-ECL nitrocellulose filter paper . Membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 for 1 hour at room temperature. Protein bands were probed with antibodies against c-MET and phospho-c-MET (p-c-MET) at a 1:200 dilution (Abcam ab74217 & ab5662), or total-Akt, phospho-Akt (p-Akt), total-ERK, phospho-ERK (p-ERK) , and β-actin at 1:3000 , and then labeled with horseradish peroxidase-conjugated anti-rabbit antibody . Bands were visualized by enhanced chemiluminescence using an ECL kit  in accordance with the manufacturer’s protocol.The MTT assay is based on the conversion of MTT to insoluble MTT-formazan by cleavage of the tetrazolium ring by mitochondrial dehydrogenase enzymes of living cells. Cells were treated with 1, 2, 3 and 5 μM of SU11274 and 0.1, 0.5, 1 and 2 μM of crizotinib for 48 h and 72 h, respectively. MTT solution  was added to each well. After 4 hours of incubation, the medium was discarded, and 100 μl of acidic isopropanol (0.1 N HCl in absolute isopropanol) was added and the plate gently shaken. Absorbance was measured on an enzyme-linked immunosorbant assay (ELISA) reader at a test wavelength of 540 nm. Each sample was assayed in triplicate and the experiment was repeated three times.6 cells/mL. Annexin V FITC and propidium iodide (PI) were added (5 μL per 105 cells). Samples were gently mixed and incubated for 15 mins at room temperature in the dark before fluorescence activated cell sorter (FACS) analysis. Cell apoptosis was analyzed by staining Annexin V with using flow cytometry after 48 h of 5 μM of SU11274 or 0.5 μM of crizotinib incubation. Both early apoptotic  and late apoptotic  cells were defined as apoptosis.The relative percentage of apoptotic cells was assessed at 48 hours using the Annexin V-FITC Apoptosis Detection Kit- 1  in accordance with the manufacturer’s protocol. Briefly, cells were washed twice in PBS and treated with SU11274 or crizotinib. The pellet was re-suspended in annexin V binding buffer at a concentration of 10The primary antibodies used were against p-c-MET (Abcam ab5662). Tissue sections were deparaffinized three times in xylene for a total of 15 mins and subsequently rehydrated. Immunostaining for p-c-MET was performed using a Bond-maxTM automated immunostainer  and the BondTM Polymer Refines Detection kit . Briefly, antigen retrieval was carried out at 97 °C for 20 mins in ER1 buffer. After blocking the endogenous peroxidase activity with 3% hydrogen peroxidase for 10 mins, primary antibody incubation was carried out for 15 mins at room temperature at an antibody dilution of 1:200. Negative controls (substitution of primary antibody for TBS) were run simultaneously. Immunohistochemical staining for Ki67 was done as described previouslyFor the analysis of caspase-3, tumor tissues were lysed with lysis buffer containing 1 mM EDTA, 1% TX-100, 100 mM NaCl, 50 mM NaF, Tris-HCl ph 7.5, phosphatase inhibitor cocktail (sigma-Aldrich). Lysates were clarified at 13000 rpm for 20 min at 4 °C. Total lysates samples were assayed for concentrations of caspase-3 by using a ELISA kits. ELISA kits were used as described by the manufacturer to measure concentrations of human caspase-3  The samples were measured in triplicate.6 cells/0.2 mL HBSS) cells were injected into the peritoneal cavity of micen = 10 per group) were monitored daily for tumor development and sacrificed either on day 70–90 after the injection of cancer cells (orthotopic RMG1 model), or on day 60–70 (PDX model) or when any of the mice seemed moribund. We recorded total body weight, tumor weight and the number of tumor nodules. Tumors were fixed in formalin and embedded in paraffin or snap frozen in OCT compound  in liquid nitrogen.Female BALB/c nude mice were purchased from Orient Bio, Seongnam, Korea. This study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Samsung Biomedical Research Institute (protocol No. H-A9-003) which is a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International  that performs in accordance with the Institute of Laboratory Animal Resources (ILAR) guide. All methods were carried out in accordance with the relevant guidelines. To establish orthotopic models, RMG1  was used for all statistical analyses. All statistical tests were two-sided and p values less than 0.05 were considered to be statistically significant.Each experiment was carried out at least three times in triplicate. The results are presented as the means ± standard deviation (SD) of three independent experiments. The Mann–Whitney How to cite this article: Kim, H.-J. et al. c-MET as a Potential Therapeutic Target in Ovarian Clear Cell Carcinoma. Sci. Rep.6, 38502; doi: 10.1038/srep38502 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


However, clinicopathological and prognostic value of c-Met in breast cancer remains elusive.PubMed and EMBASE databases were searched for eligible studies. Correlation of c-Met overexpression with survival data and clinicopathological features was analyzed by using hazard ratio (HR) or odds ratio (OR) and fixed-effect or random-effect model according to heterogeneity. All statistical tests were two-sided.32 studies with 8281 patients were analyzed in total. The c-Met overexpression was related to poor OS  ) of 18 studies with 4751 patients and poor RFS/DFS  ) of 12 studies with 3598 patients. Subgroup analysis according to data source/methods/ethnicity showed c-Met overexpression was related to worse OS and RFS/DFS in Given by author group, all methods group and non-Asian group respectively. Besides, c-Met overexpression was associated with large tumor size, high histologic grade and metastasis.Our results showed that c-Met overexpression was connected with poor survival rates and malignant activities of cancer, including proliferation, migration and invasion, which highlighted the potential of c-Met as significant candidate biomarker to identify patients with breast cancer at high risk of tumor death. Breast cancer is the most common cancer type and the second leading cause of cancer death in women worldwide and is expected to account for 29% all new cancer diagnoses for female . BesidesThe tyrosine kinase c-Met, also called MET and hepatocyte growth factor receptor (HGFR), is a key regulator of organ development and cancer progression and has been studied in many cancer types such as lung cancer, gastric cancer, prostate cancer and so on –7. c-Metin vitro experiment and reviews. Among the remaining studies, three studies were performed in the same institution and only the most recent study was included. Finally, 32 studies were included and the detailed literature search and study selection could be seen in Figure 507 records were identified in total and then 70 candidate studies were selected. Through further screening, 33 studies were excluded because of There were 32 studies with 8281 patients in total involved in our meta-analysis. Thereinto, 18 studies with 4751 patients were available for OS survival data and 12 studies with 3598 patients were available for RFS/DFS survival data. There were 24 (75%) articles using immunohistochemistry method to determine the overexpression of c-Met and 8 (25%) articles using RT-PCR, FISH, RPPA and MIP respectively. All the articles included were retrospective. The study quality was assessed using the Newcastle-Ottawa quality assessment scale, generating scores ranging from 5 to 8 with a mean of 6.625 ; high histologic grade, OR=1.547  and distant metastasis, OR=20.431 . However, high c-Met overexpression was not found to be associated with Menopausal status, OR=0.758 ; age, OR=1.072 ; ER status, OR=1.049 ; PR status, OR=1.300 ; HER-2 status, OR =1.017 ; triple negative breast cancer, OR=0.956 ; ki-67 overexpression, OR=1.677 ; lymph node status, OR=1.801 ; histologic type, OR=1.053 . All the above results could be seen in Table .153; higOS was analyzed in 18 studies with 4751 patients. Results showed that c-Met overexpression was related to poor OS, HR=1.65 1.328, 2.051) Figure . Besides28, 2.051Analysis of 12 studies with 3598 patients indicated overexpression of c-Met was related to poor RFS/DFS, HR=1.53 Figure . BesidesFunnel plot and Egger’/Begg’ test was used to evaluate publication bias. Results of Egger’/Begg’ test for OS and RFS/DFS were 0.945/0.520 and 0.270/0.131 respectively. Begg's funnel plots with pseudo 95% confidence limits of the OS and RFS/DFS were listed in Figure Results of removal of each study at a time could be seen in Figure The tyrosine kinase c-Met fosters invasive growth, a complex physiological program that signifies concerted activation of cell proliferation, survival, invasion and angiogenesis , 14. In In our meta-analysis, the results of OS showed moderate heterogeneity. Then we conducted subgroup analysis and found that data sources were the origin of heterogeneity. The HR value extracted from survival curve of 2 articles showed a favorable prognosis of c-Met overexpression while other 16 articles with HR value given by author indicated a poor prognosis. The difference is mainly because data extracted from survival curve is not as accurate as that given by author and the article quality is relatively low. Subgroup analysis of RFS/DFS was also conducted on the basis of data source. Only one study with HR value derived from survival curve and both the two subgroups showed poor prognosis of c-Met overexpression. And subgroup analysis of methods reached in same conclusion. Subgroup analysis of ethnicity showed c-Met overexpression in non-Asian group rather than Asian group had statistical difference, which might because the significant heterogeneity in Asian group. What's more, no evidence indicated publication bias for OS and RFS/DFS in regard to c-Met overexpression using Egger’/Begg’ test. And influence analysis of OS and RFS/DFS showed no big difference. All that demonstrated that our results were stable and reliable.Some studies have investigated the role of c-Met in TNBC and BLBC  and found that c-Met was related to TNBC and BLBC phenotype, which could be exploited as a potential target , 17, 18.This study has important implications in breast cancer. Firstly, it demonstrates c-Met overexpression is related to worse OS and RFS/DFS, which indicates that c-Met may be a potential therapeutic target. Secondly, c-Met is involved in malignant biological behavior, such as large tumor size, high histological grade and distant metastasis, and combination therapy with c-Met inhibitor in future will dramatically reduce mortality in invasive breast cancer. However, there are also limitations in this meta-analysis. First of all, identifications of c-Met overexpression of individual studies are not exactly same and as a dichotomous variable, cut-off value may be a source of considerable interstudy heterogeneity. Additionally, although Begg's and Egger's test were performed and there was no statistical significance. Results should be interpreted cautiously because we only include studies with available HR value or K-M survival curves with necessary data.Currently, the most promising approach for disrupting c-Met signaling is to use small molecular inhibitors to target the intracellular kinase domain . The cliTaken together, our analysis shows that overexpression of c-Met in breast cancer tissues is associated with worse prognosis in human breast cancer. Since c-Met inhibitor has already been investigated in numerous clinical trials, the future clinical application will be easier. Combination therapy of c-Met inhibitor will improve the prognosis of breast cancer patients especially invasive breast cancer and TNBC/BLBC, which are types of the poorest prognosis.This meta-analysis was conducted according to PRISMA guidelines. Studies were identified by searching PubMed and EMBASE databases from 1997 until April, 2016 by using the key words “breast cancer or breast tumor or breast carcinoma” and “hepatocyte growth factor receptor or HGFR or c-Met”. Titles and abstracts were first scanned to exclude irrelevant articles and final inclusion of the articles was determined by reading the full text. The references from identified articles were manually searched for additional relevant records.All studies in this meta-analysis satisfied the following inclusion criteria: 1) full-text studies published in English; 2) proven diagnosis of breast cancer by pathology; 3) considering the relation between c-Met overexpression and OS, RFS/DFS or clinicopathological features among breast cancer patients; 4) provided the HRs and 95% CIs, or Kaplan-Meier survival curves that provided sufficient data to extract HRs and 95% CIs. Exclusion criteria: 1) no data on survival or clinicopathological features and inability to calculate from Kaplan-Meier survival curve; 2) with previous cancer history.Two reviewers (Zhao XX and Qu JK) performed the search and assessed the studies independently. The following items were extracted from each eligible study, including first author, year, patients source, type of patients, protein location, median age, patients number, technique, c-Met overexpression (%), analysis, median follow up, OS/DFS and clinicopathological features. When the univariate and multivariate analysis were both available, the multivariate results were used. If the above-mentioned data was not reported, items should be treated as “NA (not available)”.The Newcastle-Ottawa Scale was used to assess the quality of each study . The NOShttp://sourceforge.net) and extracted survival data HRs and 95%CI [2 statistics and P value, and if I2 > 50% or P< 0.1, the results were considered statistically significant and random effects models were employed; otherwise, fixed effects models were employed. Sensitivity analysis, also named influence analysis, was carried out to evaluate the effect of single study on the whole results and meanwhile try to find the origin of heterogeneity. Publication bias was assessed graphically using funnel plots, and funnel plot Symmetry was evaluated by Begg's and Egger's linear regression method. P<0.05 was considered statistically significant. Statistical analyses were performed using Stata 13.0 .HRs and 95% CIs were used to study the association between c-Met overexpression and OS/DFS. If data were only available in the form of figures, we read Kaplan-Meier curves by Engauge Digitizer version 4.1 (free software downloaded fromnd 95%CI . Data of


Antibiotics are the ‘wonder drugs’ to combat microbes. For decades, multiple varieties of antibiotics have not only been used for therapeutic purposes but practiced prophylactically across other industries such as agriculture and animal husbandry. Uncertainty has arisen, as microbes have become resistant to common antibiotics while the host remains unaware that antibiotic resistance has emerged. The aim of this review is to explore the origin, development, and the current state of antibiotic resistance, regulation, and challenges by examining available literature. We found that antibiotic resistance is increasing at an alarming rate. A growing list of infections i.e., pneumonia, tuberculosis, and gonorrhea are becoming harder and at times impossible to treat while antibiotics are becoming less effective. Antibiotic-resistant infections correlate with the level of antibiotic consumption. Non-judicial use of antibiotics is mostly responsible for making the microbes resistant. The antibiotic treatment repertoire for existing or emerging hard-to-treat multidrug-resistant bacterial infections is limited, resulting in high morbidity and mortality report. This review article reiterates the optimal use of antimicrobial medicines in human and animal health to reduce antibiotic resistance. Evidence from the literature suggests that the knowledge regarding antibiotic resistance in the population is still scarce. Therefore, the need of educating patients and the public is essential to fight against the antimicrobial resistance battle. Antibiotics, either are cytotoxic or cytostatic to the micro-organisms, allowing the body’s natural defenses, such as the immune system, to eliminate them. They often act by inhibiting the synthesis of a bacterial cell, synthesis of proteins, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), by a membrane disorganizing agent, or other specific actions . AntibioTo combat against infections or microbes, undoubtedly antibiotics are a blessing to human civilization that has saved millions of people . MultiplMillions of metric tons of newer classes of antibiotics have been produced in last 60 years since its inception. Increased demand for antibiotics across many sectors has allowed for less expensive and off-label uses of drugs. Conversely, due to the enormous and irresponsible use of the antibiotics, has contributed significantly to the advent of the resistant strains .Resistance to an antibiotic develops in no time and hence, is a big matter of concern . With thThe present review is one such way to educate the public by showing the development and plausible future of antibiotic resistance and existing regulation to reduce the antibiotic resistance crisis.Origin of antibiotic resistanceAntibiotic resistance was reported to occur when a drug loses its ability to inhibit bacterial growth effectively. Bacteria become ‘resistant’ and continue to multiply in the presence of therapeutic levels of the antibiotics . BacteriAntibiotics are usually effective against them, but when the microbes become less sensitive or resistant, it requires a higher than the normal concentration of the same drug to have an effect. The emergence of antimicrobial resistance was observed shortly after the introduction of new antimicrobial compounds . AntibioAntibiotics used in agriculture are often the same or similar to antibiotic compounds used clinically , this ovDevelopment of antibiotic resistanceAntibiotics fight to eliminate bacteria. Hence, bacteria tend to have a natural process that encourages resistance. The resistance process occurs via gene level mutations. . AntibioAntibiotics can generally eliminate the majority of bacteria in a colony. However, there may exist a different colony of bacteria that are genetically mutated which can lead to resistance . The levConsequence of antibiotic resistanceStaphylococcus aureus (S. Aureus) infections as waves of clonal dissemination. Methicillin-resistant Staphylococcus aureus (MRSA) strains are rapidly spreading globally [Antibiotic resistant organisms are known as superbugs. These are not only a laboratory concern but have become a global threat responsible for high death tolls and life-threatening infections . Consequglobally . Estimatglobally . It has globally . Evidencglobally . Furtherglobally . Antibioglobally . For exaglobally .Regulatory issues related to antibiotic resistanceCongruent international management guidelines for daily antibiotic practices are yet unavailable. Hence, regulatory guidelines vary in different countries. Some countries have acted swiftly offering guidance e.g. United Kingdom, while other nations have yet to move toward interventions. The WHO has offered recommendations such as children in developing countries that antibiotics should only be used for the treatment of severe bloody diarrhea and cholera -35. SincWith an abundance of evidence, there is no scope to ignore global antibiotic resistance. Antibiotic resistance can be more prevalent where antibiotic consumption is found to be higher. Lack of regulation and control in using antibiotics is prominent and needs to be targeted at a global capacity. Developing nations are at the greatest risk. Low prices of antibiotics, ease of availability and unnecessary use of antibiotics are causing more burden in developing countries . AntibioWe are all affected by this multi-faceted public health issue. An all-encompassing problem that doesn’t just pertain to clinical personnel and microbiologists, but service personnel, industry stakeholders, specialists and the general public. We have to take necessary steps to tackle this complicated challenge. Social awareness, motivation, commitment in responsible sectors, stringent rules and regulation have to be prioritized. Further, we need the coupled action for the proper utilization of antibiotics, best management practices, and behavioral shifts across all industries that we can then combat against this public health burden. Application of modern technology can help the patient to take the antibiotic timely . At presTreatments for bacterial infections are becoming intensified every day. Infections remain as antibiotics gain resistance; treatment failure is common due to antibiotic resistance and multi-drug resistance, for e.g. tuberculosis. Newer and effective antibiotics that have no known resistance to bacteria are in high demand. Alternative treatment procedures are under consideration to fight bacterial infection. Passive immunization or administration of antibodies to non-immunized to prevent bacterial infections have been found effective . AnotherAntibiotic resistance is at all time high in all the parts of the world. Despite measures taken by some member states of WHO, antibiotic use in humans, animals, and agriculture is increasing. The high economic burden in the healthcare sector has become a burning issue, due to extended hospital stays, isolation wards, stringent infection control measures and treatment failures. The public health leaders should establish a pan surveillance system coordinated at national and international levels, ongoing analysis and a mandatory reporting system for antibiotic resistance. Both domestic and global policies need to be conventional and adhered-to to stop the overuse and misuse of antibiotics.


Checklists are increasingly used to measure quality of care. Recently we implemented an antibiotic checklist in nine Dutch hospitals and showed that use of the checklist resulted in more appropriate antibiotic use. While more appropriate antibiotic use was associated with a reduction in length of stay, use of the checklist in itself was not. In the current study we explored discrepancies between reported and actually performed checklist items at the patient level to test the validity of checklist answers, to evaluate whether discrepancies between reported and actually performed checklist items could explain the lack of effect of checklist use on length of stay, and to identify missed opportunities for performance per checklist item.Checklist answers represented reported performance. Actual performance was assessed by data from the patients’ medical files. Reported and actually performed checklist items could be ‘both YES’; ‘both NO’; ‘YES reported, NOT actually performed’; or ‘NO reported, YES actually performed’. We determined an overall ‘both YES’ score per checklist, and used mixed models to evaluate whether an association existed between this overall score and patient’s length of hospital stay. Finally, we analysed whether the items that were not actually performed, could have been performed.Between January and October 2015 physicians filled in 1207 checklists. In total 7881 items were checked. Most items were ‘both YES’  or ‘both NO’ . The number of ‘YES reported, NOT actually performed’ items was 1628/7881 (20.7%) compared to 260/7881 (3.3%) ‘NO reported, YES actually performed’ items. The level of discrepancy between reported and actually performed items differed per checklist item. The item ‘prescribe antibiotic treatment according to the local guideline’ had the highest percentage of ‘YES reported, NOT actually performed’ items, namely 45.1%. A higher overall ‘both YES’ score of the checklist was significantly associated with a shorter length of hospital stay. Of all checklist items 21.8% were not performed while they could have been performed.Checklist answers do not accurately assess actual provided care. As actual performance of the antibiotic checklist items is associated with length of stay, efforts to increase actual performance appear to be justified.The online version of this article (10.1186/s12879-017-2878-7) contains supplementary material, which is available to authorized users. Checklists have been embraced in the medical field. Since the impressive success of the WHO surgical safety checklist in 2009 , more thPreviously we developed and introduced an antibiotic checklist to improve the quality of antibiotic use by reminding physicians of the most important steps in recommended appropriate antibiotic use , 6. SuchAlthough previous studies have suggested that variation in actual compliance with checklists exists , 11, 12,This observational study was performed alongside a stepped wedge cluster randomised trial evaluating the effectiveness of introducing an antibiotic checklist . PatientThe antibiotic checklist was meant as a supporting tool to remind physicians of the most important steps in recommended appropriate antibiotic use . It consPhysicians completed the checklists for hospitalized adults (≥18 years old) or adults at the emergency department who were admitted at a participating ward with a suspected community-acquired and/or hospital-acquired bacterial infection and were prescribed intravenous antibiotics. The participants were both residents and specialists with different levels of experience. One month preceding the start of checklist use in each hospital, introduction of the antibiotic checklist was prepared. The checklists were displayed in printed form at all working places at the participating departments. Stimulating activities to use the checklist, such as education, feedback and reminders, were organised. It was the physicians’ responsibility to use the checklist each time an antibiotic was started. Physicians were informed that the effect of checklist use on the appropriateness of antibiotic use and on length of hospital stay would be measured. They did not know that their answers on the checklist would be checked against the electronic medical records.We included checklists used for eligible patients with at least one item checked.  The answActual performance of the checklist items was assessed by collection of data from the patients’ electronic medical records (EMR), including medication charts and laboratory results. Data were recorded on the collection of blood cultures, the collection of cultures of suspected sites of infection, antibiotic use including dosage, route of administration and duration of treatment, relevant laboratory parameters, suspected type of infection and, if relevant, changes in diagnosis.14 For example -concerning the checklist item ‘prescribe antibiotic treatment according to the local guideline’- when a patient was treated with ceftriaxone because of a suspected community-acquired pneumonia, an algorithm determined whether that treatment was according to the local guideline.The ticks on the checklist directly assessed the reported performance. For the actual performance, the data of the patients’ EMRs were evaluated using previously developed algorithms.Both YES. E.g., both the checklist answer “YES, I prescribe antibiotic treatment according to the local guideline” and the algorithm indicated that the antibiotic treatment was according to the local guideline;Both NO. E.g., both the checklist answer “NO, I do not prescribe antibiotic treatment according to the local guideline” and the algorithm indicated that the antibiotic treatment was not according to the local guideline;YES reported, NOT actually performed. E.g., the checklist answer “YES, I prescribe antibiotic treatment according to the local guideline” indicated that antibiotics were prescribed according to the guideline, while the algorithm indicated that the antibiotic treatment was not according to the local guideline;NO reported, YES actually performed. E.g., the checklist answer “NO, I do not prescribe antibiotic treatment according to the local guideline” indicated that antibiotics were not prescribed according to the guideline, while the algorithm indicated that the antibiotic treatment was according to the local guideline.Per checklist item we determined the concordance between reported performance and actual performance. We distinguished positive (“YES”) and negative (“NO”) answers. Reported and actually performed checklist items could be:First we determined the validity of the checklist answers. The items ‘YES reported, NOT actually performed’ and ‘NO reported, YES actually performed’ indicated differences between reported and actual performance. Both items disrupted the validity of the reported checklist answers. In case of ‘YES reported, NOT actually performed’ the reported checklist answer gave an overestimation of actually provided care, while in case of ‘NO reported, YES actually performed’ the reported checklist answer gave an underestimation of actually provided care.Previous studies showed an association between actual performance of the checklist items and a shorter length of hospital stay. , 15 In tFor all not actually performed items we determined the applicability to the particular patient using algorithms . A ‘missP < 0.05 was considered statistically significant. Analyses were done using IBM SPSS Statistics, version 23.0.The study was exploratory and therefore we used mainly descriptive measures. Frequencies of ‘both YES’, ‘both NO’, ‘YES reported, NOT actually performed’ and ‘NO reported, YES actually performed’ items were expressed in absolute numbers and percentages. The association between overall ‘both YES’ scores and length of hospital stay was evaluated using generalised linear mixed models. These models account for within-cluster dependencies, which is required as checklists from nine different hospitals were included. Furthermore, the models adjust for possible confounders, which is necessary because in the clinical trial several covariates appeared to influence length of stay. We included the hospitals (clusters) as a random effect, and patient and treatment characteristics -age, comorbidity, type of diagnosis, community vs hospital acquired infection, antibiotics started at the Emergency Department versus ward - as fixeIn 1207 of the 5354 eligible patients a checklist was used (22.5%). Of the 1207 included checklists 993 (82.3%) were fully and 214 (17.7%) were partly completed. In total 7881 checklist items were checked.Figure The level of concordance between reported and actually performed items differed per checklist item Table . Items wFigure Figure Table Of all ‘both NO’ items, 25.1% could have been performed according to the algorithms (654/2601)  items had a shorter length of hospital stay compared to patients with less ‘both YES’ items. This association is in line with previous results , 15, whiSince 21% of all reported items gave an overestimation of actually provided care, the antibiotic checklist cannot be considered a valid instrument to measure provided quality of care. Although much less detailed than in our study, the existence of discrepancy between reported and actual performance has been described previously –20. For According to our algorithms, 1653 opportunities for performance were missed, indicating room for improvement of performance. Several studies reported the room for improvement on performance per checklist item for (parts of) the Surgical Safety Checklist , 21–23. The second item with a relatively high percentage of missed opportunities for performance was the performance of two sets of blood cultures. The majority of these items were ‘both NO’ , meaning that the physician intentionally did not perform blood cultures. The common explanation for non-performance (84 times) was that the physician thought blood culture performance was not necessary with the suspected diagnosis. In our barrier study several physicians already mentioned these doubts, wherefore we added the option “In my opinion, not necessary with this diagnosis” in the checklist . AlthougOur study has several strengths. To our knowledge, our study is the first to evaluate the discordancy between reported and actually performed checklist items by using the ticks on the box reported by the physician him- or herself at the moment of performance. These data provide very detailed information per checklist item on the physicians’ perception of their own performances. Additionally we provided information about physicians’ considerations for resigning from recommended antibiotic care. We included a large number of checklists collected in nine representative Dutch hospitals. Finally, we illustrated that this in-depth analysis can help to better understand other results, such as, in our study, the lack of effect of checklist use on length of stay. Our findings are very relevant, since checklists are increasingly used to measure quality of care , 4, withOur study also has limitations. We did not include the overall completion rate in this evaluation. As in the majority of the eligible patients in the clinical trial a checklist was not used (77.5%) , it is pThe general limitation of algorithms is that they can do injustice to the complexity of patient care and clinical judgement . For exaIn future research the economic perspectives of using the antibiotic checklist should be considered. When the checklist is further implemented, the indicated missed opportunities for performance can guide local antibiotic stewardship teams in designing interventions. For example, 123 items were ‘YES reported, NOT actually performed’ for ‘prescribing antibiotic treatment according to the local guideline’ while the suspected diagnosis was a respiratory infection, and so education about the guidelines for respiratory tract infections could be considered. Likewise, since in 110 of the ‘YES reported, NOT actually performed’ items of ‘two sets of blood cultures’ only one set was performed, the importance of taking two sets of blood cultures should be emphasized . BuildinIn conclusion, the antibiotic checklist should be used as a supporting tool, not as an instrument to measure actual provided care. As actual performance of the antibiotic checklist items was associated with the patients’ length of hospital stay, efforts to increase actual performance appear to be justified.


However, several reports have described cases of other diseases that present with those features, suggesting the importance of careful differential diagnosis. Herein, we report the first case of PM with elevated serum IgG4 levels and IgG4+ plasma cells in the muscles, mimicking IgG4-RD.Polymyositis (PM) is a type of autoimmune, inflammatory myopathy. IgG4-related disease (IgG4-RD) is a recently recognized disease entity characterized by elevated serum IgG4 levels and IgG4+ plasma cells as well as CD8+ T cells. Scattered necrotic and regenerating muscle fibers with no specific findings for IgG4-RD (storiform fibrosis and obliterative phlebitis) were typical for PM. We diagnosed her condition as PM and treated her with 40 mg/day of prednisolone that decreased levels of muscle enzymes and improved muscle weakness.A 73-year-old woman visited our hospital because of proximal muscle weakness of both thighs. Her blood test showed high levels of serum creatinine kinase, aldolase, and IgG4. Magnetic resonance imaging of the thighs showed muscle edema. Needle electromyography showed findings typical of myositis. Histological analysis of her left quadriceps revealed infiltration of IgG4+ plasma-cell infiltration, mimicking IgG4-RD. Although the mechanism of IgG4 elevation in such PM is unclear, our case highlights the necessity to recognize that high serum IgG4 levels and IgG4+ plasma-cell infiltration in organs are not specific for IgG4-RD.Our case indicated that PM could present with high serum IgG4 levels and IgG4 Mononuclear inflammatory cells infiltrating affected muscles are mainly CD8+ plasma cell infiltration of various organs.,3 As IgG4-RD has been growingly recognized, cases of other diseases, such as multicentric Castleman disease–8 and small vessel vasculitis, presenting with those features have drawn attention because such cases could fulfill the diagnostic criteria for IgG4-RD.,10IgG4-related disease (IgG4-RD) is a rare lymphoproliferative disorder with elevated levels of serum IgG4 and abundant IgG4+ plasma-cell infiltration of affected muscles, mimicking IgG4-RD. Our case highlights the importance of careful assessment and differential diagnosis in a patient with elevated serum IgG4 levels and tissue IgG4+ plasma cell infiltration. An informed consent was obtained from the patient.We herein report the first case of PM with high serum IgG4 levels and IgG42A 71-year-old woman developed muscle pain and weakness in both thighs in 2014. The symptoms gradually progressed, and she was admitted to our hospital for work-up in August 2016.Physical examinations revealed normal blood pressure of 105/65 mm Hg and body temperature of 36.7°C. Manual muscle testing score of her neck flexor tendons, biceps, triceps, quadriceps, and iliopsoas muscles showed muscle weakness . Gowers’ sign was positive. The findings of ocular, lung, cardiovascular, abdominal, other neurological, joints and skin examination were normal. She had no history of statin treatment.Laboratory tests revealed elevated levels of serum creatinine kinase , aldolase , lactate dehydrogenase , IgG , IgA , and IgG4 . While serum erythrocyte sedimentation rate level was elevated , serum C-reactive protein level was within normal range . Other blood tests, including blood counts, serum levels of electrolyte, creatinine, IgE, and blood glucose were within normal range. Anti-nuclear antibody was positive with the titer of 1:1240 (homogeneous and speckled patterns). Antiaminoacyl-tRNA synthetase, anti-RNP, anti-dsDNA antibody, and anti-Sm antibodies were all negative.Fat-suppressed T2-weighted magnetic resonance imaging showed high intensity Fig. , and nee+ cells, the important findings for IgG4-RD of storiform fibrosis and obliterative phlebitis were not found .,31 Taken together, our case warrants further examinations of a role of B cells and plasma cells in PM.Our case showing massive infiltration of plasma cells as well as CD8 while a part of patients with PM are refractory,,19 and need intravenous immunoglobulins or other immunosuppressive agents like calcineurin inhibitors and cyclophosphamide in addition to high dose glucocorticoids.,32 Thus, the differentiation of PM from IgG4-RD is clinically important in the management of the disease.Moderate dose of glucocorticoid is usually effective to IgG4-RD,4+ plasma cells into affected sites, mimicking IgG4-RD. High serum IgG4 levels and tissue-infiltration of IgG4+ plasma cells are indicative but not specific for the diagnosis of IgG4-RD.We described the first case of PM that presented with elevated serum levels of IgG4 and abundant infiltration of IgG4.