The intake of meat, particularly processed meat, is a dietary risk factor for diabetes. Meat intake impairs insulin sensitivity and leads to increased oxidative stress. However, its effect on postprandial gastrointestinal hormone (GIH) secretion is unclear. We aimed to investigate the acute effects of two standardized isocaloric meals: a processed hamburger meat meal rich in protein and saturated fat  and a vegan meal rich in carbohydrates . We hypothesized that the meat meal would lead to abnormal postprandial increases in plasma lipids and oxidative stress markers and impaired GIH responses.In a randomized crossover study, 50 patients suffering from type 2 diabetes (T2D) and 50 healthy subjects underwent two 3-h meal tolerance tests. For statistical analyses, repeated-measures ANOVA was performed.The M-meal resulted in a higher postprandial increase in lipids in both groups (p<0.001) and persistent postprandial hyperinsulinemia in patients with diabetes (p<0.001). The plasma glucose levels were significantly higher after the V-meal only at the peak level. The plasma concentrations of glucose-dependent insulinotropic peptide (GIP), peptide tyrosine-tyrosine (PYY) and pancreatic polypeptide (PP) were higher  and the ghrelin concentration was lower (p<0.001) after the M-meal in healthy subjects. In contrast, the concentrations of GIP, PYY and PP were significantly lower after the M-meal in T2D patients (p<0.001). Compared with the V-meal, the M-meal was associated with a larger increase in lipoperoxidation in T2D patients (p<0.05).Our results suggest that the diet composition and the energy content, rather than the carbohydrate count, should be important considerations for dietary management and demonstrate that processed meat consumption is accompanied by impaired GIH responses and increased oxidative stress marker levels in diabetic patients.NCT01572402ClinicalTrials.gov  The current guidelines for the treatment of diabetic patients focus primarily on carbohydrate counts Dietary saturated fat is known to impair insulin sensitivity and to enhance hepatic glucose production Epidemiological studies suggest a positive association between high consumption of total and red meat and incident T2D In contrast, some intervention trials demonstrated a greater improvement in insulin sensitivity, glycemic control and a reduction in oxidative stress markers in T2D patients consuming a vegetarian diet compared with those consuming a traditional diabetes diet http://clinicaltrials.gov/show/NCT01572402). The authors confirm that all ongoing and related trials for this intervention are registered.The protocol for this trial and supporting CONSORT checklist are available as supporting information; see 2. Patients diagnosed with T2D had been treated with diet and/or oral hypoglycemic agents (metformin and/or sulfonylureas) for at least one year. Healthy volunteers exhibiting normal glucose tolerance and without metabolic syndrome or diagnosed diabetes among first-line relatives were included. Subjects exhibiting renal, liver or thyroid disease, drug abuse, including alcoholism, and pregnant or breastfeeding women were excluded.The inclusion criteria were men and women aged between 30–70 years with a body mass index (BMI) between 27–50 kg/mAll measurements were performed on an outpatient basis after a 10- to 12-h overnight fast with only tap water allowed ad libitum. The diabetic patients did not take any of their diabetes medication the evening or the morning before the assessments. The postprandial state was measured after intake of a standard breakfast – one of two isocaloric test meals in a random order consisting of either a processed meat burger meal  or a plant-based burger meal . For each intervention visit, the meals were delivered fresh from the manufacturer. The study nurses generated the random sequence of the meals and assigned participants to the interventions in one-week intervals. The participants ate the meals under the nurse’s control in the laboratory. Neither the study staff nor the participants were blinded to the content of the meals. The compositions of the meals are presented in The plasma concentrations of glucose, immunoreactive insulin, C-peptide, triglycerides (TG), free fatty acids (FFA), oxidative stress markers and GIH were measured during the fasting state (time 0) and then 30, 60, 120 and 180 min after the meal. In this single-center study the samples were collected in the Laboratory of Clinical Pathophysiology in Institute for Clinical and Experimental Medicine.Plasma glucose was analyzed via the glucose-oxidase method using a Beckman Analyzer . Serum immunoreactive insulin and C-peptide concentrations were determined using Insulin and C-peptide IRMA kits , respectively. Plasma lipid concentrations were measured via enzymatic methods .Protease and dipeptidyl peptidase-4 inhibitors were added to two samples at each time point. The concentrations of glucagon-like peptide −1 (GLP-1), gastric inhibitory peptide (GIP), pancreatic polypeptide (PP), peptide YY (PYY), leptin and ghrelin were determined via multiplex immunoanalyses based on xMAP technology using a MILLIPLEX MAP Human Gut Hormone Panel  and a Luminex 100 IS analyzer  The levels of lipid peroxidation were determined via a home-made method using thiobarbituric acid reactive substances (TBARS) For statistical analysis, repeated-measures ANOVA was performed. An estimate of the number of subjects that we recruited was carried out using a power analysis of repeated measurements via PASS 2005 statistical software . The power analysis was completed for the most important variables and the number of subjects should provide the power >0.8 for all these variables. The factors of subject, group, meal and time were included in the analyses. The interactions between meal and time  were calculated for each variable. ANOVA was followed by least significant difference (LSD) multiple comparisons post-hoc analysis. The original dependent variable was subjected to a power transformation to obtain constant variance and a symmetric distribution of the data and residuals The plasma concentrations of glucose, IRI, C-peptide and lipids during the fasting state and their postprandial profiles are illustrated in The basal concentrations of nearly all of the GIHs were significantly increased in the T2D patients compared with the healthy controls. There was a considerable difference in the GIH response after ingestion of the M-meal compared with the V-meal in both healthy subjects and the T2D patients . The V-mThe concentrations of ghrelin and leptin differed significantly between the T2D patients and the healthy controls throughout the entire meal assessment . In the We have added a subanalysis by adding a “baseline ghrelin” factor to the model. The baseline ghrelin was the most important factor when included in the model with F = 187, p<0.0001. It means that the higher the baseline level of ghrelin is, the larger the drop. The difference between the two meals remained significant with F = 19.43, p<0.001.The fasting concentrations of all of the oxidative stress markers differed between the groups. . In the During the postprandial phase, consumption of the M-meal was associated with a significantly greater increase in the TBARS levels in diabetic patients than that of the V-meal. In the healthy controls, no postprandial dynamics of the TBARS levels were detected. The SOD activity in the healthy controls was significantly increased after the V-meal compared with the M-meal. There was no significant change in the plasma concentration of SOD during the postprandial phase in diabetic subjects after either meal. The plasma concentrations of reduced glutathione and ascorbic acid did not change significantly in either patients with diabetes or healthy subjects after either meal.Our results suggest that after two isocaloric meals containing different carbohydrate contents , the postprandial plasma glucose levels during the first 180 min after meal ingestion differed significantly only with respect to the peak glucose levels. Some human studies suggest that other macronutrients can modify the postprandial insulin demand and the glycemic response and therefore exert an additional impact on the postprandial increase in the glucose levels, as shown in healthy subjects Concerning the lipid levels, the significantly higher postprandial increase in the triglyceride levels and the smaller decrease in the free fatty acid levels detected in both groups after the M-meal is not surprising. Free fatty acids are known to impair insulin sensitivity and to enhance hepatic glucose production We initially detected significantly lower IRI and C-peptide responses after the M-meal than the V-meal in healthy subjects. However, these levels decreasing more slowly, and after 180 min, the level of IRI was significantly higher in both groups after the M-meal. This result indicates that after the M-meal, hyperinsulinemia persisted longer in both groups.This finding is in accordance with a glucose clamp study of non-diabetic human subjects indicating that increases in the FFA levels lead to insulin resistance within several hours Carbohydrate, fat and protein in the lumen of the gut have been demonstrated to stimulate the secretion of a broad range of GIHs The incretin effect, the postprandial augmentation of insulin secretion by gut hormones, has been primarily associated with the secretion and insulinotropic effect of two GIHs – GIP and GLP-1. The incretin effect is thought to mediate approximately 50–70% of the overall insulin response after a mixed meal or glucose ingestion in healthy subjects It is well understood that in patients with diabetes, the incretin effect is diminished secondarily The mechanisms underlying the loss of incretin activity remain incompletely understood, but several hypotheses have been proposed, including hyperglycemia- and hyperlipidemia-associated receptor desensitization Studies that have examined the GIP and GLP-1 responses after meals differing with respect to caloric and macronutrient content report inconsistent results regarding the contribution of meal composition to the stimulation of these hormones. One study reported a higher GIP response after a mixed meal than after an oral glucose load Our results suggest that the intake of each type of isocaloric meal consumed in amounts typical of normal eating was associated with different postprandial changes in the GIP and GLP-1 plasma levels. In the T2D patients, the V-meal resulted in a larger release of GLP-1 and GIP than the M-meal. This result is in accordance with a study suggesting that the GLP-1 response after fat intake is impaired in T2D patients Our data suggest that carbohydrates appear to represent the strongest activator of GLP-1 and GIP secretion among T2D patients, whereas healthy subjects display higher GLP-1, GIP, PYY and PP responses to the mixed meal containing higher fat and protein contents.In our study, we demonstrated increased basal and postprandial levels of GIP and GLP-1 in the T2D patients compared with the healthy subjects. This finding is in accordance with a study that evaluated the GLP-1 secretion levels and indicated relatively unchanged or even elevated secretion in response to oral nutrition intake in subjects suffering from diabetes Additionally, a recent meta-analysis revealed that T2D patients do not exhibit reduced GLP-1 secretion in response to an OGTT or a meal test Other gastrointestinal peptides play a role in the regulation of energy intake, appetite and overall energy homeostasis in humans. They are secreted by different cells in the intestine and from the pancreas, and their release is modulated by food ingestion.PYY is co-secreted predominantly from the endocrine L cells in the ileum together with GLP-1. PYY plays an important regulatory role in GIT function PP is secreted from endocrine cells in the pancreas. Both PYY and PP, when infused intravenously, reduce appetite and energy intake in healthy humans The course of PYY secretion consisted of two phases. The initial rise in the PYY level is likely due to a link to the proximal small intestine, and the continuous rise reflects the direct contact of lipids with the distal small intestine Ghrelin stimulates appetite and food intake and is secreted in the stomach. In healthy individuals, the ghrelin plasma concentrations increase during fasting and are suppressed by meal intake We detected postprandial suppression of ghrelin secretion during the first 60 min after meal ingestion. The postprandial decrease depended significantly on the baseline level of ghrelin, which means that the higher the baseline level of ghrelin is, the larger the drop. Furthermore, it was significantly greater after the M-meal in healthy subjects. This finding is in accordance with a study that demonstrates that fat digestion is required for the suppression of ghrelin The postprandial state, characterized by hyperglycemia and hyperlipidemia, is associated with increased oxidative stress in patients suffering from diabetes The strength of our study was that we took a physiological approach to measure the postprandial state. We selected two simple isocaloric meals at amounts corresponding to those ingested during a typical meal. The M-meal was rich in both saturated fat and protein; thus, we cannot separate the effect of each macronutrient.This study also had several limitations. First, the T2D patients exhibited a significantly higher body weight and BMI compared with the control subjects, and these differences may have affected some of the responses reported. The object of our study was to compare the effect of the two different meals within each group . In healthy subjects, no effect of adiposity on the postprandial GIP and GLP-1 levels has been reported Finally, the 3-h study duration might be insufficient for complete absorption of the nutrients, and this limitation may have led to an underestimation of the measured parameters.In conclusion, according to our hypothesis, our results suggest that a processed meat meal is accompanied by an impaired GIH response and increased oxidative stress marker levels in diabetic patients. Our results revealed comparable glycemic responses to isocaloric meals containing different carbohydrate contents, despite the distinct peak glucose concentrations. This finding illustrates that the diet composition and the energy content, rather than the carbohydrate count or the glycemic load, should be important considerations for the dietary management of diabetes.Checklist S1CONSORT Checklist.(DOC)Click here for additional data file.Protocol S1Trial Protocol in English.(DOC)Click here for additional data file.Protocol S2Trial Protocol in Czech.(DOC)Click here for additional data file.


Saccharomyces cerevisiae glycolysis pathway. Metabolic fluxes were quantified by the COPASI tool using the kinetic models of Hynne and Teusink at varied concentrations of external glucose. The enzyme sequences were taken from the UniProtKB and the average amino acid (AA) properties were computed using the set of Georgiev's uncorrelated scales that satisfy the VARIMAX criterion and specific AA indices that show the highest correlations with those. Multiple linear regressions  were found between the values of metabolic fluxes and the selected sets of the average AA properties. The hydrophobicity, α-helicity, and net charge were pointed out as the most influential characteristics of the sequences. The results provide an evidence that metabolic fluxes of the yeast glycolysis pathway are closely related to certain physical-chemical properties of relevant enzymes and support the view on the interdependence of catalytic, binding, and structural AA residues to ensure the efficiency of biocatalysts and, hence, physiologically adequate metabolic processes.Metabolic fluxes are key parameters of metabolic pathways being closely related to the kinetic properties of enzymes, thereby could be dependent on. This study examines possible relationships between the metabolic fluxes and the physical-chemical/structural features of enzymes from the yeast  Saccharomyces cerevisiae glycolytic enzymes and the AAC of corresponding sequences has been found [The general concepts and methods of systems biology become increasingly important in modern microbiology research. In terms of “systems microbiology,” this approach allows to analyze and describe as a whole the molecular interactions that occur within a microbial cell or community –3. The cen found , as wellen found . At the en found .However, the possible solutions have been defined as rather difficult task, arising from the sequence metrics problems , 15. In  Saccharomyces cerevisiae glycolysis pathway and the physical-chemical/structural features of enzyme sequences.Therefore, following our previous line of research, the goal of the present study is to test the possible relationship (QSAM) between the flux distribution through the yeast Saccharomyces cerevisiae glycolysis pathway: low-affinity glucose transporter , hexokinase , glucose-6-phosphate isomerase , 6-phosphofructo-2-kinase , fructose-biphosphate aldolase , triose-phosphate isomerase , glyceraldehyde-3-phosphate dehydrogenase , 3-phosphoglycerate kinase , phosphoglycerate mutase , enolase , pyruvate kinase , pyruvate decarboxylase , and alcohol dehydrogenase , as well as the main branches that include glycogen synthase  and glycerol-3- phosphate dehydrogenase  together with enzymes involved into the turnover of ATP: plasma membrane ATPase 1 , adenylate kinase 1 .The data set consisted of the amino acid (AA) sequences, representing the enzymes/carriers for the core reactions of the yeasthttp://www.uniprot.org) database under accession numbers as indicated in the brackets above. The AA composition (frequencies of AA occurrence) of sequences (AAC) was computed using ExPASy/ProtParam (http://web.expasy.org/protparam/) tool.The protein AA sequences were taken from the UniProtKB ( Saccharomyces cerevisiae glycolysis pathway were estimated using the kinetic models of Hynne [http://www.ebi.ac.uk/biomodels/)—BIOMD0000000061 and BIOMD0000000064, respectively. Simulation experiments were performed for both models using the COmplex PAthway Simulator tool  at perturbed initial concentrations of external glucose .Metabolic fluxes within the yeastof Hynne  and Teusof Hynne  from thePave(i) is the average AA property for each sequence and P(j) is the property value for jth residue and the summation over N, the total number of residues in a protein. The transformation of sequences into fixed-size numerical feature vectors was performed in two steps. First, the average AA property for each sequence was estimated using the generalized numerical scores in respect of individual AA [http://dx.doi.org/10.1155/2014/817102. Second, the average AA property for each sequence was computed using the narrower sets of specific AA indices from generalized AA property groups with a highest correlation to the VARIMAX-derived scales [The enzyme AAC was converted into a feature-based numerical representation using the average AA property for each sequence and computed according to the standard formula  (see 1)1):(1)Paidual AA . Appropridual AA . This led scales . The relThe accession numbers (AAindex database), description, and designation of specific AA indices which were used for this purpose are represented in R-square value besides keeping the variance inflation factor (VIF) below the threshold value of 3.3 [R-square criterion of regression models. This process was repeated to obtain four-variable and larger models until no variables could increase the adjusted R-square value.The data representing metabolic fluxes, as the dependent variables and the sets of numerical property vectors for respective enzymes, as the independent variables  were processed by correlation analysis (parametric and nonparametric) using the Statgraphics Plus  and SPSS 11.0 for Windows  and subjected to the multiple linear regression analysis using the same software. Explanatory variables in the models were subsequently checked by stepwise forward selection procedures thus finding the significant one-variable models as well as significant two-variable models to arrange all the variables in groups of 2 at a time for each model. The best three-variable models were formed by adding another variable one by one from the remaining variables, and the variables that yielded the greatest increase in the adjusted e of 3.3  or, in eF-test for analysis of variance (ANOVA) was employed to evaluate the statistical significance of regression models and Student's t-test was used to check the significance of regression coefficients. The leave-one-out cross-validation (LOOCV) procedure was used to validate developed regression models [R-square values.In addition, the corrected Akaike's (AICc) information criterion  was usedn models . The linP values < 0.05 were considered to be statistically significant for parametric and nonparametric tests.The  Saccharomyces cerevisiae glycolysis pathway and the values of individual average amino acid (AA) properties for respective enzymes expressed according to VARIMAX scales [Statistical analysis of the data set  revealed a number of significant pair correlations  between X scales  and, theR-square values  caused a marked decrease in actual R-square levels, as well as an increase of AIC c, which indicates a certain decline in the quality of extended models , and the measure of linker propensity (group 5) [Thus, in the present case seven (model I) or eight (model II) variables proved to be adequate to provide statistically robust multiple linear regression models linking the values of metabolic fluxes predicted by different kinetic models , 21 withgroup 5) .R-square values of corresponding Ridge regression, as described above, led to statistically robust multiple linear regressions  of the metabolic fluxes estimated by kinetic models against those predicted by regression models Tables . The higs Tables  adequateR-square values (Tables P < 0.00001). Besides, the observed values of the variance inflation factor (VIF)   showed o) Tables  and 3 in Saccharomyces cerevisiae glycolysis pathway appear as closely related to the sequence-derived properties of implicated enzymes.The obtained results indicate that the metabolic fluxes determined by Hynne's and Teusink's full-scale models for the yeastThe relevant multivariate regression models Tables  and 3 shOn the other hand, such an approach meets the general lines for the multiscale nature and, consequently, the scale-space representation of real-world objects , which iDifferent sets of the predictor variables in the regression models Tables  and 3, aThis study in the most direct way continues our previous research , 12 therThe results thus provide further evidence that the enzyme operation and hence the metabolic fluxes are directly dependent on the general physical-chemical and structural properties of the full enzyme sequences. Therefore, it might be useful for structure/function studies to look more beyond the active centre composition of the enzyme but carefully assess the overall physical and chemical properties of biocatalysts. The more so that the modest success of creating artificial enzymes also points to currently unknown, probably crucial, parameters that could significantly affect enzyme catalysis . In part Saccharomyces cerevisiae kinetic model [It is clearly understood that the estimates of metabolic fluxes currently obtained by the kinetic models represent an approximation, albeit realistic enough, of their “true” values which could involve multiple regulatory mechanisms including gene expression and posttranslational modification , 25, 35.The obtained sequence-activity relationships in the form of quantitative models (QSAM) will allow to assess the contribution of individual AA properties to the overall compositional features of proteins and thus to specify potential targets within enzyme sequences in order to attain a purposeful modification of biocatalysts and, hence, the metabolic fluxes in microorganisms, particularly if there is a need to include or replace  any additional enzyme currently not represented in a given metabolic pathway, which is essential for metabolic engineering and synthetic biology.Supplementary information 1:Table S1-1: The dataset used in multivariate analysis which comprises metabolic fluxes and numerical vectors representing the average amino acid (AA) property for each enzyme sequence .Table S1-2: The dataset used in multivariate analysis which comprises metabolic fluxes and numerical vectors representing the average AA property for each enzyme sequence .Table S1-3: The dataset used in multivariate analysis which comprises metabolic fluxes and numerical vectors representing the average AA property for each enzyme sequence .Table S1-4: The dataset used in multivariate analysis which comprises metabolic fluxes and numerical vectors representing the average AA property for each enzyme sequence .Supplementary information 2:Figure S2: The linear plots for the metabolic fluxes of the yeast Saccharomyces cerevisiae glycolysis pathway estimated by kinetic models against those predicted by linear regression models I-IV and by the leave-one-out cross-validation (LOOCV) of the models.Table S2-1: The variance analysis of the regression models.Table S2-2: Standard errors and confidence intervals for the linear regression models.Supplementary information 3:Table S3: Elements and the statistical indices for multiple linear regression model which links the values of metabolic fluxes and the average AA properties of the yeast Saccharomyces cerevisiae enzyme sequences expressed according to the specific AA indices.Figure S3: The linear plot for the metabolic fluxes of the yeast Saccharomyces cerevisiae glycolysis pathway estimated by kinetic models against those predicted by linear regression model.


The Prion Laboratory Section of the Public Health Agency of Canada supports heath care professionals dealing with patients suspected to have Creutzfeldt-Jakob disease (CJD) by testing cerebrospinal fluid (CSF) for protein markers of CJD. To better serve Canadian diagnostic requirements, a quaking-induced conversion (QuIC)-based assay has been added to the test panel. The QuIC tests exploit the ability of disease-associated prion protein, found in the CSF of a majority of CJD patients, to convert a recombinant prion protein (rPrP) into detectable amounts of a misfolded, aggregated form of rPrP. The rPrP aggregates interact with a specific dye, causing a measurable change in the dye's fluorescence emission spectrum. Optimal test and analysis parameters were empirically determined. Taking both practical and performance considerations into account, an endpoint QuIC (EP-QuIC) configuration was chosen. EP-QuIC uses a thermo-mixer to perform the shaking necessary to produce the quaking-induced conversions. Fluorescence readings are obtained from a microwell fluorescence reader only at the beginning and the end of EP-QuIC reactions. Samples for which the relative fluorescence unit ratio between the initial and final readings represent a ≥4 increase in signal intensity in at least two of the three replicates are classified as positive. A retrospective analysis of 91 CSF samples that included 45 confirmed cases of CJD and 46 non-CJD cases was used to estimate the performance characteristics of the EP-QuIC assay. The diagnostic sensitivity and specificity of the EP-QuIC test of this set of samples were 98 and 91%, respectively. Subacute encephalopathies, including Creutzfeldt-Jakob disease (CJD), constitute a large and heterogeneous group of brain diseases that can be diagnostically challenging . Among tThe diagnostic approach for the most common form of CJD, sporadic CJD, has to date relied upon a standardized clinical presentation and supporting investigations such as the presence of increased amounts of indirect protein markers of disease found in patients' cerebrospinal fluid (CSF) , 4. The 8–in vivo represent a major advance in the diagnoses of sporadic CJD. In quaking-induced conversion (QuIC)   or real-n (QuIC) , the priThe high degree of sensitivity and specificity exhibited by RT-QuIC based tests in a growing number of laboratories strongly supports the inclusion of such a test in the clinical investigation of suspected cases of sporadic CJD and genetic CJD –18. In o–12–This project, involving the use of human specimens of the CJD biobank housed at the National Microbiology Laboratory, was reviewed and approved by the Health Canada and Public Health Agency of Canada's Research Ethics Board (HC-PHAC REB 2014-0033). In total 91 CSF specimens, sent to the Prion Laboratory Section for testing due to suspicion of CJD, were used: 45 pathologically confirmed cases of CJD (43 for sporadic CJD and 2 for genetic CJD) and 46 clinically confirmed non-CJD cases. The final diagnoses of all samples are provided in Table S1 in the supplemental material. All sample handling was performed in a biological safety cabinet inside a biosafety level 3 containment laboratory.The substrate used was Syrian hamster recombinant prion protein , provided by the Bristol Institute for Transfusion Sciences. Previously confirmed negative and positive CSF samples were run on each plate as controls. CSF analyses were performed in triplicate using black 96-well optical-bottom plates with 100-μl reaction volumes. Each well had 15 μl of CSF added to 85 μl of QuIC reaction buffer, resulting in a final composition of 300 mM NaCl, 10 μM EDTA, 10 μM ThT, and 0.1 mg/ml Syrian hamster rPrP in 10 mM phosphate buffer (pH 5.8). All the reagents were made fresh with mass spectrometry-grade water and passed through a 0.22-μm-pore size filter after preparation.Sealing tape was applied and both RT-QuIC and EP-QuIC reaction mixtures were incubated at 42°C with intermittent shaking cycles consisting of 90 s of shaking at 900 rpm and 30 s of rest. Fluorescence readings were taken with an excitation wavelength of 450 nm and emission wavelength of 480 nm by a FLUOstar Omega plate reader. In RT-QuIC reactions the plate remained in the FLUOstar Omega plate reader with continued cycles of double orbital shaking and relative fluorescent unit (RFU) readings every 45 min for the duration of the 90-h experiment. In EP-QuIC, the plates were placed in an Eppendorf Thermomixer C equipped with a Smart Block plate and ThermoTop with shaking cycles consisting of 90 s of circular shaking at 900 rpm and 30 s of rest at 42°C. After 20 min, the plates were removed from the Eppendorf Thermomixer C, and an initial RFU reading was taken on a preheated FLUOstar Omega plate reader using the same excitation and emission spectra used for the RT-QuIC reactions . The plates were then returned to the Eppendorf Thermomixer C, and the intermittent shaking cycles continued for 90 h. An endpoint reading was then taken on the FLOUstar Omega using the excitation and emission spectra described above.At the end of the experiment, the RFU final/RFU initial ratio of each well was calculated. Based on receiver operating characteristic curve analyses, individual wells were scored as positive in RT-QuIC reactions when the RFU final/RFU initial was ≥2.0 . Positive EP-QuIC wells were determined to have a RFU final/RFU initial ratio of ≥4.0 . A sample was considered positive when at least two of the three assay replicates for a sample were positive. In instances where one of the three assay replicates for a sample was positive, the sample was reanalyzed using three different volumes of CSF in the reaction mixture . Samples consistently exhibiting one of the three assay replicate wells as positive were scored as indeterminate. The detailed method is described in in the supplemental material.In the development of any diagnostic test a balance must be struck between its sensitivity and specificity. Too much emphasis on sensitivity increases the odds of false positives. Too much emphasis on specificity increases the odds of false negatives. In this respect EP-QuIC and RT-QuIC were slightly different. As shown in The reliability of the assay was assessed by having 8 CSF samples tested by two analysts on the same day, as well as the reanalysis of 41 samples by EP-QuIC on two separate dates. Both experiments demonstrated 100% agreement between all samples.The results in Indeterminate samples did not return a defined positive or negative result and are therefore excluded from being considered true positive or true negative results. Instead the indeterminate samples known to be CJD positive are included in the false negatives and indeterminate samples known to be non-CJD are included in false positives in the calculation of sensitivity and specificity, as shown in It had been previously determined by an EU Joint Programme–Neurodegenerative Disease Research (JPND) CSF QUIC consortium that the optimal shaking cycles for RT-QuIC reactions consisted of 90 s of shaking at 900 rpm, followed by a 30-s rest . The higTime is obviously another important factor to consider when using such assays in a clinical setting. EP-QuIC throughput could be greatly increased by having multiple thermomixers and a single plate reader. Nonetheless, the 90-h run time discussed is relatively long in comparison to the enzyme-linked immunosorbent assays used to measure 14-3-3 and other surrogate markers of CJD in CSF. Normally, the Prion Laboratory Section runs samples received during the preceding week on Monday and Tuesday, with reports being issued on Wednesday. In order to ensure adherence to this schedule expected by referring physicians, the EP-QuIC reactions are started on Friday and read on Tuesday. Other laboratories are exploring the use of different substrates and run parameters that are reported to reduce the RT-QuIC run time to hours as opposed to days . These aThe lower cost and potentially higher throughput, coupled with sensitivity and specificity comparable to that of established RT-QuIC protocols, strongly support the inclusion of the EP-QuIC protocol described in the clinical investigation of suspected cases of CJD.


Several types of colorectal cancers are associated with a prominent lymphoid component, which is considered a positive prognostic factor.We report a case of a dome-type carcinoma of the cecum in a 57 year old female.The sessile, non-polypoid lesion histologically consisted of a tubulovillous adenoma with low-grade dysplasia.The submucosal invasive component showed low-grade architectural features that included cystically dilated glands containing eosinohilic debris. Immunohistochemical studies displayed retention of the four mistmach repair proteins, consistent with a stable phenotype. After 3 years, the patient remains free of recurrence.A literature review highlighted striking similarities between dome-type carcinoma and the gut-associated lymphoid tissue carcinoma, the two sharing an intimate association with the gut associated lymphoid tissue.The two variants might therefore be grouped into a unified category. The term “dome-type carcinoma” (DC) was first applied by De Petris in 1999 when describing the macroscopic appearance of a dome-shaped elevation of the mucosa of a colon carcinoma associated with gut associated lymphoid tissue [Since then 11 cases of this distinct variant have been reported –9. ThereWe report an additional case of so-called DC of the colon along with a literature review.The histological and immunohistochemical diagnostic features are presented as well the differential diagnosis with similar variants.A 57 year old female with no family history of CRC was hospitalized because of fever and abdominal pain. The abdominal computed tomography detected a cecal mass and colonoscopy showed two lesions: a 30 mm sessile mass in the cecum Fig.  and a 40At definitive histology the rectal polyp was a tubulovillous adenoma with high-grade dysplasia.The cecal mass was a moderately differentiated adenocarcinoma evolving from a tubulovillous adenoma, invading the submucosa Figs.  and 3.FiThe submucosal tumour was surrounded by a prominent lymphoid tissue exhibiting reactive germinal centres Figs.  and 4. TImmumohistochemistry showed retained expression of the mismatch repair proteins MSH2, MSH6, MLH1 and PMS2.The patient remains recurrence free after three years of follow-up.Since the original report in late 90’s by De Petris , 11 caseAll of these cases were presented matching the macroscopic and histopathological features as described by De Petris: The macroscopic dome-like, non polypoid appearance and the architecture encompassing dilated malignant glands lined by columnar epithelium with eosinophilic cytoplasm on a prominent lymphoid background .The earliest report of carcinoma of the colon originating in lymphoid-associated mucosa was in 1984, describing the lesion in a patient with ulcerative colitis . A few aTables As with DC, GALT-carcinoma has a plaque/sessile macroscopic appearance; it is limited to the submucosa, is associated with GALT and shows a low differentiation grade.Clinical presentations of DC and GALT-carcinoma are reported either as sporadic-type colon cancer or in association with ulcerative colitis, familial adenomatous polyposis, Lynch syndrome and other positive family histories of colorectal cancer, in both right and left colon Table  thereforAnother similarity is that almost all tumors reported are early-invasive, limited to the submucosa (T1) except in cases of DC reported by Amussen in 2008 (T2) [On immunohistochemistry, all DCs studied for mismatch repair protein expression showed retention of all 4 proteins. One case of GALT-carcinoma arose in a patient with Lynch syndrome and accordingly showed microsatellite instability .Despite an overlap in key histological features between DC and GALT-carcinoma, there is variability in some ancillary aspects including tumor infiltrating lymphocytes, intraacinar necrosis, the presence of a preexisting adenoma remnant and foci of usual-type adenocarcinoma.The mucosal-associated lymphoid tissue (MALT) in the intestine is termed gut-associated lymphoid tissue (GALT). It consists of isolated and aggregated lymphoid follicles .The discrete lymphoid aggregates form dome-like masses that bulge into the gut lumen .A follicle-associated epithelium (FAE) overlies the aggregated lymphoid follicles. Such epithelium is a single cell layer composed of enterocytes and specialized epithelial microfold cells, so called “M-cells”, and is devoid of goblet and enteroendocrine cells .A very small minority of CRC is thought to derive from M-cell. In 1999, De Petris described the dome-shaped elevation of the mucosa corresponding to a small submucosal adenocarcinoma composed of dilated malignant glands lined by columnar epithelium with eosinophilic cytoplasm and hyperchromatic nuclei on a prominent lymphoid background [De Petris speculated that that DC is the malignant counterpart of the lymphoglandular complex and it may represent a precursor of LELC in view of the tumor-associated lymphoid stroma and the presence of less well differentiated areas .There are three arguments for a linkage between the lymphoglandular complex and DC thus supporting the concept that this tumor may originate from M-cells of the FAE.Firstly the malignant epithelium of such a tumor present an intimate relationship with a conspicuous lymphoid tissue showing the characteristic organization of GALT .Secondly, there is the observation that DC, as in FAE, is described lacking goblet cells .Finally, there are morphological similarities between the neoplastic changes observed in rat experimental models and the morphology of DC observed in reported human cases .In this regard, studies of colon tumors in experimental carcinogen-treated rats showed a significant association between the location of lymphoid aggregates, the location of sessile-type non polypoid tumors and an early appearance of malignant glands inside the aggregates , 18.Regarding histological and diagnostic features, DC show a constellation of morphological features other than the prominent lymphoid component which are variably present Table , thus acThe presence of tumor infiltrating lymphocytes is reported in half of the cases of DC Table .Despite M-cells normally exhibit numerous intercellular lymphocytes, this may not always the case: accordingly the amount of lymphocytes is related to the state of maturity of these cells , 19.A preexisting associated adenoma has been described in almost half of both DC and GALT carcinoma Table .An explanation for the absence of an adenomatous component could be that some of these tumors arise from submucosal FAE or from herniated glands in the submucosa, typically in UC, as described by one of us , 14.When present, an adenomatous transformation may in turn be induced by the lymphoid follicles themselves in the colonic mucosa/submucosa .The presence of usual type adenocarcinoma observed within some DC could explain the relative rarity of DC as an entity, by which the DC component is obliterated by overgrowth of the usual type carcinoma .The only difference among the DC and the GALT-carcinoma is the unique cyto-architectural features described by De Petris. Evidence of the cystically dilated glands and the cytological features typical of DC are reported in only one case of GALT-carcinoma by Rubio in 2002 .Medullary carcinoma and lymphoepithelioma-like carcinoma differs from DC and GALT-carcinoma in several ways. In cyto-architectural features, they show high-grade-undifferentiated features  and theyThe medullary phenotype is associated with MSI-H CRC  and accoThe most important morphological features serving as diagnostically useful markers of MSI-H-CRC occurring sporadically and in the context of Lynch syndrome are lymphocytic infiltration, mucin secretion and poor differentiation .Patterns of lymphocytic infiltration in MSI-H CRC include a nodular or Crohn’s-like peritumoural lymphocytic reaction and the presence of tumour infiltrating lymphocytes (TILs) .The stromal lymphoid component associated with DC and GALT carcinoma organized with lymphoid follicles and germinal centres differs from the lymphoid infiltrates encountered in medullary carcinoma, lymphoepithelioma-like carcinoma and in MSI-H CRC; most probably it represent remnants of pre-existing lymphoid nodules rather than an adaptive immune response , thus acSince any kind of lymphocytic infiltration  in CRC is considered a positive prognostic factor , it is nWe report an additional case of so-called DC of the colon showing typical staging, morphological and immunohistochemical features: An early and low grade lesion, associated with a conspicuous lymphoid tissue showing the characteristic organization of GALT, lacking features of biological aggressiveness and with retained expression of 4 mismatch repair proteins.The association of morphological features, in particular the pattern of lymphocytic infiltration with the immumohistochemical mismatch repair status allows the distinction from other types of CRC associated with a prominent lymphoid component.We highlighted the similarities between DC and the GALT-carcinoma, the two showing an intimate relationship with lymphoid tissue with the characteristic organization of GALT.Despite heterogeneity in tumour presentation (arising in the context of both sporadic and syndromic cases), and some variable ancillary morphological features both DC and GALT-carcinoma could be grouped in a single category.Since the term “dome” is an endoscopic descriptor lacking in diagnostic histopathological specificity we consider the term GALT carcinoma more appropriate for categorization purposes.Written informed consent was obtained from the patient for publication of this Case Report and any accompanying images.


Plasmodium falciparum. To date, this pathway has not been exploited in anti-malarial drug development. Herein a novel high throughput approach for the identification of chemically diverse inhibitors of the CoA synthesis pathway from chemical compound libraries is reported.Drug resistance has rendered previous generation anti-malarials ineffective and is also rapidly emerging against the current therapeutics of choice, artemisinin and its derivatives, making the discovery of new anti-malarials with novel mechanisms of action a priority. The Coenzyme A (CoA) synthesis pathway is a well-known anti-microbial drug target that is also essential for the malaria parasite P. falciparum imaging assay. In short, a test compound was considered likely to inhibit the P. falciparum CoA synthesis pathway if addition of the pathway’s end product, CoA, was able to negate the compound’s growth-inhibitory action on P. falciparum asexual parasites.To identify novel CoA synthesis pathway inhibitors, a chemical rescue screening approach was developed by modifying a well-established 50) of these compounds was determined for both P. falciparum and human embryonic kidney cells. Seven compounds showed sub-micromolar activity against the parasite, with selectivity indices ranging between 6 and over 300 times. CoA supplementation was confirmed to alleviate the effects on parasite growth and cell viability in a dose dependant manner. Microscopic investigation of the stage of effect and phenotype of treated parasites was performed on a selection of the active compounds.The chemical rescue approach was employed to screen the Medicines for Malaria Venture (MMV) malaria box and another small focussed compound library. This resulted in the identification of 12 chemically diverse inhibitors. To ascertain accurate potency and selectivity, the half maximal inhibitory concentration (IC50 values ranging between 120 nM and 6 μM. The chemical rescue approach for the CoA pathway yielded hit compounds with demonstrated anti-plasmodial action and defined species selectivity. An additional benefit of such an approach is the first step in the determination of the mode of action of novel anti-malarial compounds, namely inhibition of the CoA synthesis pathway. Collectively, we propose the CoA rescue approach as a valuable novel tool for future anti-malarial drug discovery.Investigation of two medium-sized prioritised compound libraries resulted in the identification of a set of chemically diverse CoA synthesis pathway inhibitors with IC


Quality of life (QoL) is considered an important outcome in health research. It can be rated by the patient, or by an external assessor. We wished to identify the predictors of any discrepancies between these two approaches in people with schizophrenia.Patients with DSM schizophrenia and related disorders (N = 80) completed both patient-rated  and assessor-rated  measures of QoL.r = 0.38). In a regression analysis, independent predictors of subjectively-rated QoL being higher than objectively-assessed QoL in the same patient, were low insight score (BIS), negative symptoms (PANSS), absence of depression (CDSS), and less positive attitude toward prescribed treatment (DAI).Patient-rated (LQOLP) and assessor-rated (QLS) measures showed a modest correlation (In people with schizophrenia, scores on objectively- and subjectively-rated measures of quality of life can differ markedly. When comparing subjective to objective assessments, patients with depressive symptoms will value their QoL lower, and those with low insight will value their QoL higher. This has important implications for the utility and interpretation of QoL measures in schizophrenia. Quality of life (QoL) is an important outcome in schizophrenia research and was the primary outcome of the UK CUtLASS trials (Cost Utility of the Latest Antipsychotics in Severe Schizophrenia) -3. Self-Existing literature converges on extensive differences between the predictors of subjective and objective QoL. Previous researchers have found, for example, with the use of factor analysis, that subjective and objective QoL components cluster separately . The majTwo major QoL measures used for schizophrenia are the objective QLS   and the It is not enough, however, to know that the two types of measure differ. An important issue to examine is what makes them differ. QoL measurement discrepancy serves to highlight fundamental differences between the priorities of the patient and those of the clinician, which may drive patient dissatisfaction with treatment. In addition, knowledge of the drivers of QoL discrepancy can inform research using QoL as an outcome measure.Despite a large QoL evidence base, few researchers have specifically examined predictors of discrepancy between the two methods of QoL measurement in the same individual. One such study did suggest that patients with QoL measurement concordance were more likely to be young, male and have high symptom scores . PatientDuring the UK CUtLASS trials -3 QoL ofThe study was conducted in parallel to the CUtLASS trials -3, whichIndividuals with DSM schizophrenia, schizoaffective disorder, or delusional disorder and aged 18–65 years, whose treating clinicians were considering a change of antipsychotic due to lack of response, or intolerable side effects, were eligible for the study. The referring clinician discussed the CUtLASS trials with the participant and supplied them with an information sheet. The Trial Support Clinician checked their eligibility from case notes, assigned a study number and visited within 3 days to obtain written consent [Inclusion criteria for the first trial were at least one month since the first onset of positive psychotic symptoms and psychiatrist electing to change current treatment because of inadequate clinical response or intolerance . InclusiParticipants were assessed using the Positive and Negative Syndrome Scale (PANSS) ; the CalTreatment-related side effects were measured using the Simpson-Angus scale ; the BarThe primary outcome for the study was quality of life measured on an observer-assessed measure: the Quality of Life Scale (QLS) . The QLSFor the purposes of the current study, 80 participants at the Manchester trial site also completed the widely-used, patient-rated Lancashire Quality Of Life Profile (LQOLP)  and the z scores, or standard scores (with a mean of zero and unit variance), to allow for an analysis of the statistical determinants of discrepancy between the two measures. Correlations were calculated for z score difference between subjective and objective measures (LQOLP minus QLS) and baseline variables. Forwards multiple linear regression analyses of predictor variables  on patient-rated and assessor-rated QoL and on QoL discrepancy (LQOLP minus QLS) were performed. A p value of less than 0.2 was chosen as we did not wish to exclude confounders with trend significance with the potential to affect the relationship between the predictors of interest and the outcome variable. Data were analysed using SPSS for Windows, version 20.Scores on each QoL measure were converted into Characteristics of the sample are set out in Table r = 0.384 (p = 0.001).At baseline assessment correlation between the two measures of quality of life in those 80 participants with scores on both QoL measures was Associations between patient-rated (LQOLP) and assessor-rated (QLS) quality of life and baseline variables were examined  and between PANSS subtotal scores  multi-collinearity was not present in the dataset (tolerance of above 0.5 and VIF less than 2.0 for each predictor).Possible predictor variables were, again, entered into a forwards multiple linear regression on the basis of a univariate relationship with a p value of less than 0.2 (see Table The following variables were entered into a forwards multiple linear regression on the basis of a univariate relationship with a r = 0.38) was found between patient-rated and assessor-rated measures of QoL in patients with schizophrenia (N = 80) participating in a clinical trial. This confirms previous findings [A significant but moderate correlation  predicted low concurrent patient-rated QoL but not assessor-rated QoL nor QoL measurement discrepancy. Other research has suggested that direct links between adherence and QoL are weak or absent ,53, althAs in previous studies, greater negative symptomatology predicted lower assessor-rated QoL ,39,40,55Increased experience of non-neurological side effects was predictive of higher self-reported QoL but of neither assessor-rated QoL nor the discrepancy. Patients with a higher QoL rating may report side effects as more severe; these experiences may have a greater impact on the lives of better-functioning individuals. This relationship was only observed for non-neurological side effect scores, not extrapyramidal side effects (EPS); others have found an association between patient-rated QoL and EPS . A signiThe study had a number of limitations. Data were collected during baseline assessments of a RCT; the cross-sectional design therefore means that assumptions of causality in the statistical models must be viewed with caution. In addition, the sample analysed here was comprised of patients entered into a RCT, because of poor clinical response or intolerance, although the trial did have broad inclusion criteria. Although conclusions were derived from a relatively small sample (N = 80) QoL predictive factors highlighted by this study are consistent with the existing evidence base.In terms of measures used, a number of variables found to be associated with QoL in previous studies, for example, neurocognitive deficits, social support, coping style and anxiety, were not assessed in this sample. It is not known whether the statistical determinants of QoL measurement discrepancy observed here will apply to discrepancies between other patient- and assessor-rated QoL measures. Analysis did include demographic variables previously associated with QoL, such as gender, although other demographic data, which may also be associated with QoL, such as social class and educational level, were not available. In terms of the possible effects of medication, the main outcome paper of the CUtLASS study reported no significant effect of antipsychotic drug class on QoL . The CUtdiscrepancy is insight. This explains 20% of its variance (with less insight associated with better subjective but not objective QoL) but its relationship may be mediated by depression and medication concordance so it is not a significant predictor in a multivariate model. Greater negative symptoms predict discrepancy as high scores predict low objective but not subjective ratings of quality of life.Our primary aim was to examine predictors of discrepancy between two widely-used patient-rated and assessor-rated quality of life measures in schizophrenia. There was only moderate correlation between them and their predictors differed. Patient-rated QoL appears mainly to reflect depression, whilst assessor-measured QoL reflects negative symptoms. The largest single predictor of QoL measurement These findings have relevance to both the clinical and the research setting. In terms of practical management, it appears that the alleviation of depressive symptoms rather than the alleviation of negative symptomatology is more likely to lead to the patient rating their quality of life as improved. For studies using QoL as the primary outcome measure it is worth bearing in mind that the inclusion of patients with low insight scores, low depression scores and high negative symptom scores will serve to increase the degree of discrepancy between patient-rated and assessor-rated QoL.


Randomized controlled trials (RCTs) were conflicting to support whether unstable angina versus non-ST-elevation myocardial infarction (UA/NSTEMI) patients best undergo early invasive or a conservative revascularization strategy. RCTs with cardiac biomarkers, in MEDLINE, EMBASE, and Cochrane Central Register of Controlled Trials from 1975–2013 were reviewed considering all cause mortality, recurrent non-fatal myocardial infarction (MI) and their combination. Follow-up lasted from 6–24 months and the use of routine invasive strategy up to its end was associated with a significantly lower composite of all-cause mortality and recurrent non-fatal MI  in UA/NSTEMI. In NSTEMI, by the invasive strategy, there was no benefit . In the shorter time period, from randomization to discharge, a routine invasive strategy was associated with significantly higher odds of the combined end-point among UA/NSTEMI  and NSTEMI  patients. Therefore, in trials recruiting a large number of UA patients, by routine invasive strategy the largest benefit was seen, whereas in NSTEMI patients death and non-fatal MI were not lowered. Routine invasive treatment in UA patients is accordingly supported by the present study. Two strategies have been used in managing patients with unstable angina/non-ST-elevation myocardial infarction (UA/NSTEMI). Patients may undergo an early invasive strategy of coronary angiography and revascularization by percutaneus coronary intervention (PCI) or a conservative, “ischemia guided” strategy in which hemodynamic procedures are performed only if there is evidence of recurrent ischemia or high risk featuresClinical trials offered conflicting evidence to support one strategy over the other. As a result, there was considerable interest in summating the available information from large-scale clinical trials by using meta-analyses and systematic reviews that can provide a more robust estimate of the effect of a specific therapy. Yet, even a number of meta-analyses have led to contradictory results regarding the efficacy of a routine use of an invasive strategy to reduce both nonfatal myocardial infarction (MI) and mortality123456789There are a number of potential limitations of these studies, mainly heterogeneity, i.e. the extent to which different trials may give similar or different results123456789In the current analysis, we explored the hypothesis that trials including a substantial number of patients with UA may provide evidence of a reduced rate of death and/or recurrent MI when treatment was by an early invasive strategy as compared to trials recruiting participants with just NSTEMI.To test this hypothesis we compared trials enrolling patients with both positive and negative biomarkers (UA/NSTEMI) with those only recruiting participants with positive cardiac biomarkers (NSTEMI).The literature search yielded 3896 hits. From these, 24 studies were selected for closer attention. Of these 16 studies were excluded according to explicit inclusion/exclusion criteria111213141516171819202122232425Eight prospective randomized placebo-controlled clinical trials (RCTs) involving 10412 patients  were included in the analysis. The primary characteristics of the eight included trials are listed in Compared with the conservative strategy, the OR for death with an invasive strategy was 0.93 , the OR for recurrent nonfatal MI was 0.86 , and the OR for death or MI was 0.92  .Based on the pooled results, the use of routine invasive strategy in studies that randomized UA/NSTEMI patients was associated with a dramatic reduction for the composite ischemic events with 20–30% lower odds based on the definitions of death and MI used in these trials . In contA routine invasive strategy was associated with significantly higher odds of the combined endpoint in both UA/NSTEMI  and NSTEMI  . There wQ:12.43, P:0.087, I2:43.7%), death  or MI  when the period of time from randomization to the end of follow-up was considered  included only patients with elevated cardiac biomarkers; the other five studies  recruited participants with both positive and negative cardiac biomarkers. The percentage of biomarker-positive patients in the selected studies ranged between 17–58% .Studies that only randomized biomarker-positive patients (NSTEMI) were analyzed separately and showed a 1.6-fold increase in the relative risk of death and MI in the invasive arm at a mean follow-up of 13.7 months. On the opposite, the composite end point was significantly decreased by an invasive strategy in those studies that did not require positive cardiac biomarker status as an inclusion criterion (UA/NSTEMI). The observed effects were consistent among most evaluated trials except MATE among UA/NSTEMI and VINO among NSTEMI trials, respectively.An exception to the potential hazards of an early invasive strategy in NSTEMI patients was the VINO trial. It should be noticed, however, that the VINO trial enrolled high-risk individuals with 53% of the patients having baseline Killip class II or III. Observational data have shown that Killip class II and III patients have significant mortality benefit from an invasive strategy while patients with Killip class I do not33An exception to the benefits of an early invasive strategy among trials recruiting a large number of UA patients was the MATE trial. Yet, in this trial, approximately 30% of the patients enrolled presented with acute ST-elevation myocardial infarction (STEMI) and only 23% of the study population had segment depression on the initial ECG. Thus, differently from the other UA/NSTEMI studies in our analysis, MATE randomized patients with borderline selection criteria, in whom the overall prognosis was partially affected by a late revascularization of the index MI. According to the Occluded Artery Trial no discernible benefit at four year follow-up was found among patients with occlusion of the infarct-related artery following a strategy of routine PCI late after acute MIOur data are concordant with prior doctrine that early or in-hospital coronary revascularization is fraught with hazard in patients with non-ST elevation acute coronary syndromes537Many old meta-analyses on this topic did not include the most recent trial, the ICTUS study, published in 20054678The findings from our analysis differ substantially from those of previous studies, as an early invasive approach was not of benefit at a mean follow-up of 13.7 months in those trials that included only NSTEMI patients, but benefited those including UA patients.Observations on the natural history of UA have shown an alarming incidence of death (6%) and the need for coronary revascularization by either PCI or coronary artery bypass graft in 27% of patients at 1-year follow-upCurrent literature indicates that an invasive strategy had favorable effect on mortality at 5 year follow-upWhen we performed a PubMed search in April 2013, we found no recent trials on the subject of invasive versus conservative treatment in patients with unstable coronary syndromes. The AHA/ACC treatment guidelines for UA/NSTEMI recommend (class I) invasive treatment in patients with UA/NSTEMI who have an elevated risk for clinical events as defined by risk scores and additional risk factors We used the same trials included in the preceding meta-analyses that so far offer background to guidelines’ recommendations. There are limitations in the published meta-analyses456789444543Today, the evidence base for the most appropriate treatment of UA/NSTEMI is still limited, and there are differences between the European and the American guidelines. The current study supports class I recommendation of routine invasive treatment for patients diagnosed as UA. These results emphasize the need for further randomized trials to provide a better evidence base for the invasive versus conservative treatment of patients after UA/NSTEMI.acute coronary syndrome, unstable coronary syndrome, unstable angina, non-st-elevation myocardial infarction, early invasive therapy and selective invasive therapy.We searched RCTs in English language, from 1975 to October 2013, through MEDLINE, EMBASE, and the Cochrane Central Register of Controlled Trials databases using the keywords: Eligible studies for inclusion were RCTs comparing a routine invasive versus a selective invasive strategy in patients presenting with UA/NSTEMI. Only studies reporting data on cardiac biomarkers were considered for inclusion. Trials were excluded if the majority of patients had STEMI or stable ischemic disease .The primary end-points were mortality, recurrent non-fatal MI and their combination identified in two periods of time: from randomization to discharge and from randomization to the end of follow-up. The follow-up was extracted from each original article and was calculated as the mean of all studies included. The mean follow-up was 16 months .Data were extracted independently by two authors of the present study onto data extraction sheets. Disagreement was resolved first by consensus and then by consultation with another two co-authors.We analyzed trials recruiting participants with positive cardiac biomarkers versus those that did not specify cardiac biomarker status as an inclusion criterion.Q statistic and 2I index were used to assess statistical heterogeneity across trials. The Q statistic was considered significant if the p < 0.10, and heterogeneity was considered high if the 2I index >50%.To standardize the reporting of our results we calculated relative risk (RR) and 95% confidence intervals (CI) from multiple standard 2 × 2 tables built based on the number of events among the participants for each group in every trial. We used fixed effect model meta-analysis to assess the effect of selective invasive treatment versus routine invasive treatment on the outcomes of interest. The We also used a random effect model when the statistical test of heterogeneity showed significance. However, the results did not differ qualitatively from those of the primary analysis. Furthermore the results were double-checked performing analysis on the risk ratios’ log scale, using the log of the ratio and its corresponding standard error for each study, calculated according to Woolf’s method. Results were displayed as Forest Plots using fixed-effects summary estimates.To examine the influence of individual studies on the summary effect estimate, we performed an influence analysis, in which the meta-analysis estimates are computed omitting one study at a time. Finally, to analyze associations between treatment effect and study characteristics we used meta-regression for the possible confounders. Analyses were performed using STATA software version 11 and all tests were 2-sided.How to cite this article: Manfrini, O. et al. Early Invasive Strategy for Unstable Angina: a New Meta-Analysis of Old Clinical Trials. Sci. Rep.6, 27345; doi: 10.1038/srep27345 (2016).


To describe the domain structure and calibration of the Spinal Cord Injury Functional Index for samples using Assistive Technology (SCI-FI/AT) and report the initial psychometric properties of each domain.Cross sectional survey followed by computerized adaptive test (CAT) simulations.Inpatient and community settings.A sample of 460 adults with traumatic spinal cord injury (SCI) stratified by level of injury, completeness of injury, and time since injury.NoneSCI-FI/ATConfirmatory factor analysis (CFA) and Item response theory (IRT) analyses identified 4 unidimensional SCI-FI/AT domains: Basic Mobility (41 items) Self-care (71 items), Fine Motor Function (35 items), and Ambulation (29 items). High correlations of full item banks with 10-item simulated CATs indicated high accuracy of each CAT in estimating a person's function, and there was high measurement reliability for the simulated CAT scales compared with the full item bank. SCI-FI/AT item difficulties in the domains of Self-care, Fine Motor Function, and Ambulation were less difficult than the same items in the original SCI-FI item banks.With the development of the SCI-FI/AT, clinicians and investigators have available multidimensional assessment scales that evaluate function for users of AT to complement the scales available in the original SCI-FI. Generic functional outcome measures developed for the general population often lack the sensitivity needed to detect meaningful differences in the spinal cord injury (SCI) population, and they also contain irrelevant and sometimes offensive questions that lack validity in this population.6 An extensive item pool was developed across several hypothesized functional domains and administered to 855 adults with a traumatic SCI. Investigators applied factor analysis and item response theory (IRT) methods to identify five distinct functional domains . Computer adaptive test (CAT) and short form methodologies were employed to allow the SCI-FI to achieve measurement precision while reducing participant burden.9In recognition of these limitations, the Spinal Cord Injury Functional Index (SCI-FI) was developed by a team from seven SCI Model Systems Centers. This successful collaboration resulted in a new comprehensive activity limitations outcome instrument  that offers unique advantages over outcome measures developed using classical test theory methods. The content of the SCI-FI is based upon an extensive review of the literature, input from patients with SCI and clinician focus groups, and grounded within the International Classification of Function, Disability, and Health (ICF) framework.10 of the SCI-FI item banks.12 A SCI-FI pilot study in 269 adults with recent SCI demonstrated excellent responsiveness to change during the first post-injury year. The SCI-FI achieved excellent 1–2 week test-retest reliability, with Pearson's r values ranging from 0.92 (Wheelchair Mobility) to 0.96 (Self-care).10There is a growing body of evidence to support the psychometric properties13 Unfortunately, the rehabilitation field lacks a consistent approach for assessing function when AT is used. Rust and Smith analyzed 100 rehabilitation measures and categorized how these measures addressed AT use.14 AT use was ignored in some measures, while other measures raised or lowered functional scores for items when the person used AT. Current SCI functional measures, such as the Spinal Cord Independence Measure, Quadriplegic Index of Function and the Functional Independence Measure account for the impact of AT using a hybrid approach where the use of AT is considered for some, but not all items. For items that consider AT, the item score is lower when AT is used than when it is not. However, because of the inconsistent consideration of AT in item scores, the summary scores derived from these measures do not fully reflect the impact of AT on a person's ability to function.15Assessing the impact of Assistive Technology (AT) on a person's function is a significant problem that must be addressed when developing a rehabilitation outcome measure. When ability to do an activity is scored without regard to use of AT, the functional score may improve without a concomitant change in ability, thus weakening the relationship between impairment and activity.The original SCI-FI banks contain few items in the calibrated item pool that measure a person's performance using AT. Outside of the wheelchair mobility scale, only 17 of 219 items calibrated in the SCI-FI item pool involve the use of AT. Moreover, the instructions to examinees who complete the SCI-FI item banks instruct participants to rate each item on their ability to perform the task without the use of AT or help from another person.Given the paucity of AT related items and the focus on completion of tasks without devices, the original SCI-FI is likely to be less sensitive to how people perform activities in their usual settings when they would typically use AT to complete a task. The SCI-FI banks are also not likely to be precise and sensitive to clinically relevant change for interventions where the focus is on function using usual and customary AT. For this reason, we developed the SCI-FI/AT to provide supplemental item content that is most appropriate for AT users.In this manuscript, we present analysis of the structure of the SCI-FI/AT item banks, describe the calibration and development of the SCI-FI/AT items and scales in each identified domain, discuss the initial psychometric properties of the SCI-FI/AT item banks, and compare the SCI-FI/AT item level difficulties with parallel item content in the original SCI-FI.16 The original SCI-FI sample is described in detail elsewhere.8This SCI-FI/AT sample included 460 participants with a diagnosis of traumatic SCI recruited from 9 national SCI Model Systems programs. Eligibility criteria included age of 18 or older and the ability to speak and understand English. To best test functional performance with AT, the sample was stratified to include approximately equal numbers of individuals with the following characteristics: level of injury (paraplegia vs. tetraplegia), severity of injury (complete vs. incomplete) and time since injury . The sample was representative of the key demographic variables reported by the National Spinal Cord Injury Statistical Center (NSCISC).We reviewed the previously calibrated SCI-FI item banks to retain as many items as possible from the original SCI-FI version so that similar items would be administered in the SCI-FI/AT, but with different instructions about the use of AT. The original SCI-FI items in each item bank were reviewed by two investigators  to determine if items could be retained in SCI-FI/AT. This review identified original SCI-FI items that included use of a specific type of AT or human assistance. These items were removed, as they were inappropriate for SCI-FI/AT instructions which ask participants to select a response based on their ability “without help from another person, but using the equipment or devices they normally use.” After inappropriate items were removed, the SCI-FI/AT item content for each domain was again reviewed to ensure that all important functional activities assessed in the original SCI-FI were also included in SCI-FI/AT. It was noted that items related to bathing and skin inspection had been removed from the Self Care domain, so we wrote two new items using the relevant item content from the original version, but removing the reference to AT.The final item pools administered in the SCI-FI/AT calibration study included: 47 Basic Mobility items, 35 Fine Motor Function items, 78 Self-care items, and 30 Ambulation items. The Basic Mobility item bank contains items about an individual's ability to carry out activities involving changing and maintaining body position, transfers, moving and carrying objects and moving around in different locations. The Ambulation items assess the ability to engage in walking activities in different locations that vary based on speed, time and condition and the ability to manage stairs under different conditions.without help from another person, but using the equipment or devices they normally use. Participants could skip an item if they were not able to determine a correct response. The data collection system automatically added supplemental items based on each participant's responses to screener questions .All study personnel completed a 2-hour training session and successfully completed a certification program. Each site received a detailed manual of procedures and data collectors participated in bi-weekly quality control calls. To replicate the procedure used in the original SCI-FI calibration study, all SCI-FI/AT items were presented in interview format, either over the phone or in-person. Demographic information , descriptors related to the spinal cord injury  and responses to screener questions to select appropriate sets of supplemental items  were collected for all participants. Color-coded response cards were used to direct participants to the appropriate response set for each item or set of items. In contrast to the SCI-FI instructions, SCI-FI/AT participants were asked to select the response that best describes their current ability to do each activity 2 test and multiple fit indices 18 to assess the model fit. We also examined local item dependence (LID)19 by looking at the residual correlation matrix output from Mplus.20 Any item pair with an absolute residual correlation greater than 0.221 was considered to exhibit unacceptable LID and one of the items was removed from the item pool.We employed confirmatory factor analysis (CFA) to examine the unidimensionality in each domain based on weighted least squares means and variance adjusted (WLSMV) estimation. We used the χ22 between the two models was flagged for potential DIF. For each flagged item, we examined item category frequency distributions across subgroups ; if any subgroup sample size was less than 100 or if an item category's percentage of responses was greater than 90% (i.e. a dominating response category), this item's calibration was rejected for this subgroup.24 Next, we set the non-DIF items' parameters equal across subgroups and calibrated the DIF items separately in each subgroup based on a two-group IRT model. We then examined the item parameter equality based on Wald's χ2 test25 and used the Benjamini–Hochberg (BH) procedure26 to adjust the P value. Finally, we applied the weighted Area Between the expected score Curves (wABC) to examine the DIF impact.27 The wABC was calculated by integrating the absolute difference between the expected core functions of reference and focal samples over the latent distribution. Any DIF item with wABC > 0.427 was treated as demonstrating DIF. Since our goal was to retain as many items as possible, we either calibrated the DIF item separately in relevant subgroups if the sample size in each subgroup was greater than 100 and there was no dominating response category, or only calibrated an item in one subgroup with the expectation that these items will only be administered in this subgroup in the future. We wanted to retain the maximum number of items and obtain stable item calibrations. Therefore, we adopted the least restricted requirement for Rasch models (100 subjects). Most of the subgroup sample sizes were around 200 in the examined items, which is the minimum number requirement in 2-parameter model. The analytic model was successfully converged, items showed a reasonable hierarchical difficulty order and we examined the item fit based on calibrated item parameters.Prior to finalizing the item banks, we used a three-step procedure to examine Differential Item Functioning (DIF) in group variables (sex and diagnosis—tetraplegia and paraplegia). First, each item response was fitted to two ordinal logistic regression models. The first model included the raw summed score as the single independent variable, while the second model included the three independent variables of raw summed score, group variable, and interaction between the raw summed score and group variable. Any item that exhibited an R-squared change of greater than 0.0228 as implemented in IRTPRO. The Pearson χ2 test, which examines the discrepancy between the expected and actual number of subjects in each category at each summed score level (S-X2 Test) was used to assess item fit. An item with a test statistic P value less than 0.05 was eliminated from the final item bank  and plotted the paired common item difficulty in each domain. Item difficulty is defined as the average value of threshold parameters in the Graded Response Model. The threshold parameter for response category x represents the point on the underlying concept at which the subject will have 50% probability of endorsing this category or higher. All the analyses were done in Mplus 6.0,20 IRTPRO 2.10,29 and SAS 9.13.30Subject scores on the final item banks were estimated with weighted maximum likelihood estimation. We also conducted real data simulations in SAS 9.13  to estimate person scores on 10-item fixed-length computerized adaptive tests. To compare the differences in item parameters between common items to the SCI-FI and SCI-FI/AT, we identified 41, 68, 33 and 29 common items between two versions in Basic Mobility, Self-care, Fine Motor and Ambulation domains, respectively. One item in basic mobility, 6 items in Self-care and 5 items in Fine Motor Function calibrated separately in subgroups in SCI-FI/AT. We eliminated those items in an equality test. To test item parameter invariance in the evaluation of IRT-based DIF, we would need to have the full covariance matrix of the item parameter estimations from both versions. However, SCI-FI was calibrated in PARSCALE which doesn't provide such information. Based on the similarity of sample characteristic across two calibration samples, we re-calibrated the common items across both versions at the same time using IRTPRO . We assessed invariance of the paired common items by using a Wald χ Table  Table As seen in Table As seen in Table  Table Finally, Fig. The SCI-FI instruments are multidimensional activity limitation measures appropriate for persons with SCI, regardless of level or severity of injury. The original SCI-FI scales allow for the assessment of functional capacity in the domains of Basic Mobility, Ambulation, Self-care, and Fine Motor Function. With the development of the SCI-FI/AT, we now have available scales that evaluate a person's ability to function using AT in the areas of Basic Mobility, Self-care, Ambulation, and Fine Motor Function along with the previously calibrated Wheelchair domain. In this study, the SCI-FI/AT replicated the domain structure in the original SCI-FI instrument and revealed strong psychometric properties for all domain scales in a sample of adults with paraplegia or tetraplegia. High correlations of simulated 10-item CATs with full item banks indicated high accuracy of the CAT in estimating a person's underlying functional ability. Furthermore, there was high measurement reliability for the simulated CAT scales compared with the full item bank.31 Although they are sometimes used interchangeably, clear distinctions exist between the concept of functional capacity and functional performance32 and these distinctions must be reflected in the development and selection of outcomes instruments.13 Capability refers to what a person can do without assistance while performance reflects what the person does in his or her environment using whatever means are available.33 For instance, studies examining the actual neurorecovery experienced by individuals or evaluating new physical therapy interventions designed to restore underlying functional capacity would benefit from the inclusion of SCI-FI banks. In contrast, the SCI-FI/AT would be a much more important measure and potentially more sensitive measure to include in trials of new types of adaptive equipment or of occupational therapy interventions designed to improve daily functioning at a constant level of functional capacity. Furthermore, the SCI-FI could be used to justify continued rehabilitation to payers who may require documentation of continued recovery of functional capacity (SCI-FI) or functional performance (SCI-FI/AT) as a requirement for continuity of care.SCI clinical trials, efforts to enhance our understanding about the plasticity of the nervous system and potential for neurorecovery, and studies of the efficacy of current rehabilitation interventions require functional outcome instruments that are capable of evaluating not only a person's functional capability but also one's ability using whatever AT and accommodations that are employed by the patient to accomplish the desired activity.5,15 A CAT can be used to assess selective functional domains with a common metric and the existence of SCI-FI and SCI-FI/AT versions allows for the investigator or clinician to assess either functional capacity or functional performance, or both. Instead of using multiple traditional instruments, one can employ filter questions to select the appropriate domains and items that reflect a person's clinical and demographic background, thus avoiding inappropriate functional domains and items.The CAT methodology for administering the SCI-FI assessments is ideally suited for the complexity faced when assessing functional abilities of those with SCI and overcomes many of the conceptual and methodological limitations with traditional measurement approaches.Although the SCI-FI now provides researchers and clinicians with the ability to assess functional performance across multiple domains for persons with SCI, there are several limitations to this study that should be noted. For those with paraplegia, the breadth of coverage in the Self-care and Fine Motor Function scales could be improved by including more challenging tasks in each item pool, thus reducing some of the ceiling effects that were observed in the calibration sample. Secondly, further psychometric analyses on new samples is needed to confirm the results reported in this study. In fact, a validation study of the SCI-FI/AT instrument is underway in an independent sample that will examine its validity and responsiveness to clinically meaningful change in comparison with the SCI-FI and legacy instruments. And finally, having more participants of Hispanic origin in the original SCI-FI sample (11%) as compared with the SCI-FI/AT sample (5%) could account for some of the difference in item difficulty that were observed across the two versions of SCI-FI.The findings from this study provide preliminary evidence that the SCI-FI/AT's domain structure is similar to the SCI-FI and revealed strong psychometric properties for all functional domain scales in a sample of adults with paraplegia or tetraplegia. High correlations of simulated 10-item CATs with full item banks indicated high accuracy of the CAT in estimating a person's functional ability and there was high measurement reliability for the simulated CAT scales compared with the full bank scores. SCI-FI/AT item difficulties in the domains of Self-care, Fine Motor Function, and Ambulation were less difficult than the same items in SCI-FI item banks. With the development of the SCI-FI/AT, we have available multidimensional assessment scales that evaluate function using AT in the areas of Basic Mobility, Self-care, Ambulation, Fine Motor Function, and Wheelchair Mobility to complement the domain scales already available in the SCI-FI.Contributors No other contributors.Funding This work was supported by US Department of Education, National Institute of Disability and Rehabilitation Research Grant Numbers: H133N120002, H133N060014, H133N110006, H133N110020, H133N110002, H133N110021, H133N110011, H133N110007, H133N110009.Conflicts of interest None.Ethics approval The Institutional Review Board at each site reviewed and approved this project.