Circulating cell-free microRNAs (miRNAs) are potential biomarkers of cancer. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is widely used in miRNA expression studies. The aim of this study was to identify suitable reference genes for RT-qPCR analyses of miRNA expression levels in pleural effusion. The expression levels of candidate reference miRNAs were investigated in 10 benign pleural effusion (BPE) and 10 lung adenocarcinoma-associated malignant pleural effusion (LA-MPE) samples using miRNA microarrays. The expression levels of candidate reference miRNAs, together with those of U6 small nuclear RNA (snRNA), RNU6B, RNU44 and RNU48 small RNAs, in 46 BPE and 45 LA-MPE samples were validated by RT-qPCR, and were analyzed using the NormFinder and BestKeeper algorithms. The impact of different normalization approaches on the detection of differential expression levels of miR-198 in BPE and LA-MPE samples was also assessed. As determined by the miRNA microarray data, five candidate reference miRNAs were identified. Following RT-qPCR validation, U6 snRNA, miR-192, miR-20a, miR-221, miR-222 and miR-16 were evaluated using the NormFinder and BestKeeper software programs. U6 snRNA and miR-192 were identified as single reference genes and the combination of these genes was preferred for the relative quantification of miRNA expression levels in pleural effusion. Normalization of miR-98 expression levels to those of U6 snRNA, miR-192 or a combination of these genes enabled the detection of a significant difference between BPE and LA-MPE samples. Therefore, U6 snRNA and miR-192 are recommended as reference genes for the relative quantification of miRNA expression levels in pleural effusion. MicroRNAs (miRNAs) constitute a class of small non-coding RNAs 19–24 nucleotides in length that function as post-transcriptional regulators of gene expression ,4. CircuPleural effusion is a common clinical manifestation of the various types of non-small cell lung cancer, particularly adenocarcinoma, and the diagnosis of pleural effusion in these patients is of particular clinical importance. According to the Cancer Staging system, the existence of malignant pleural effusion (MPE) at stage IV indicates systemic disease; these patients cannot be treated with local therapeutic methods, such as surgical resection or radiotherapy . CurrentmiRNA expression levels have been analyzed using traditional semi-quantitative methods, including northern blotting, bead-based flow-cytometry and microarray technology; however, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the most sensitive, reproducible and widely used approach for the evaluation of miRNAs . To prevAlthough several miRNA expression profiling studies in pleural effusion specimens have been performed –9,16, vaPleural effusion samples were derived from 111 patients who visited Chungbuk National University Hospital  or Kangwon National University Hospital  between February 2009 and September 2012. The pleural effusions were diagnosed as benign, as determined by the clinical context and the absence of malignant cells in at least two separate samples from the same patient. All lung adenocarcinoma-associated MPE (LA-MPE) samples were obtained from patients with tumors that were histologically and clinically diagnosed as primary adenocarcinoma of the lung, and that contained adenocarcinoma cells, as confirmed by pathologists. All samples were transported to the laboratory within 30 min of collection. The samples were then centrifuged at 11,300 × g for 5 min, and the supernatant and sediment fractions were aliquoted into separate microcentrifuge tubes and stored at −80°C prior to use. All patients provided written informed consent, and the study was reviewed and approved by the Institutional Review Board of Chungbuk National University Hospital.RNA was extracted from 500 μl of each sample by the use of a Genolution urine miRNA purification kit , according to the manufacturer’s instructions. The quantity of RNA extracted from each sample was examined using a NanoDrop ND-1000 spectrophotometer .TM miRNA expression profiling kit  was used to identify the candidate reference miRNAs. This kit is a peptide nucleic acid (PNA)-based microarray, which uses a novel miRNA labeling method to examine the expression profiles of cancer- and stem cell-related miRNAs.Candidate reference miRNAs were selected from 10 BPE samples and 10 LA-MPE samples using data from miRNA microarray analyses. The BPE and LA-MPE miRNA expression profiles were generated by hybridizing small RNAs extracted from the pleural effusion samples to locked nucleic acid probes targeting 160 human miRNAs. A PANArray® MicroRNA Assay kit (Applied Biosystems). RT-qPCR was performed using the Applied Biosystems 7,500 Fast Real-Time PCR system along with a TaqMan® MicroRNA Assay, TaqMan® Universal PCR Master mix and No AmpErase® UNG (Applied Biosystems). All reactions were performed in triplicate and Cq data were determined using the default threshold settings. The expression levels of the candidate reference miRNAs and small RNAs included in the final analysis were calculated using the 2−ΔΔCt method.The expression levels of the candidate reference miRNAs in 46 BPE samples and 45 LA-MPE samples were validated using RT-qPCR. The expression levels of two small nuclear RNAs  and two small nucleolar RNAs  were also measured. Reverse transcription of 100 ng isolated miRNA was conducted using a high-capacity cDNA reverse transcription kit , according to the manufacturer’s instructions, and a specific miRNA primer provided with a TaqManhttp://moma.dk/normfinder-software) and BestKeeper (http://www.gene-quantification.de/bestkeeper.html) software programs were used to analyze the expression stabilities of the reference miRNAs. NormFinder is a Microsoft Excel add-in that uses an ANOVA-based model to calculate stability values from a panel of candidate genes in different subgroups by combining the intra- and inter-group expression variation (rel) calculated as follows: Qrel = 2test sample − Cqnormalizer)−Δ−Δ with Dunn’s multiple comparisons correction] were used to determine any statistically significant differences between independent groups. Spearman’s correlation coefficients were used to calculate the associations between the clinical variables and the expression levels of the candidate reference genes. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using SPSS software for Windows, version 15.0 . The NormFinder  between the BPE and LA-MPE samples was detected. To avoid artifacts resulting from normalization of the microarray data, raw microarray expression data were also used. Of the 160 human miRNAs included in the PANArray miRNA expression profiling kit, 59 were identified in all pleural effusion samples. Five of these miRNAs  exhibited mean fold changes of <1.1-fold, with expression levels that did not differ significantly between the BPE and LA-MPE samples.The baseline characteristics of the patients in the validation cohort are listed in Variations in the expression stabilities of the reference genes were further assessed using the NormFinder and BestKeeper algorithms. The ranking of the genes by these software programs is summarized in Cell-free miR-198 expression levels have been previously demonstrated to be significantly lower in LA-MPE samples than in BPE samples . HoweverTo the best of our knowledge, the present study is the first systematic investigation of suitable reference genes for the relative quantification of miRNAs in pleural effusion. In the present study, four strategies were used to identify suitable reference genes. miRNA microarray analyses of BPE and MPE samples were performed to identify the invariant miRNAs that are stably expressed. These candidate reference miRNAs, as well as the U6 snRNA, RNU6B, RNU44 and RNU48 small RNAs, which are the most frequently used reference genes in miRNA expression studies ,11,26, wThe selection of appropriate reference genes as normalizers for the relative quantification of miRNA expression levels is required to avoid erroneous results and to improve the comparability of miRNA expression level data amongst studies . The conNon-human synthetic miRNA also frequently serves as a spike-in control to normalize for technical variability . In two In the current study, the candidate reference genes identified by the microarray and subsequent RT-qPCR experiments were analyzed using the NormFinder and BestKeeper algorithms. NormFinder calculated the intra- and inter-group variations and identified U6 snRNA as the single most stable reference gene with a stability value of 0.096. The use of more than one reference gene is generally considered to increase the accuracy of miRNA quantification, and the combination of U6 snRNA and miR-192 expression exhibited an improved NormFinder stability value of 0.081. NormFinder and BestKeeper did not produce the same reference gene ranking order for normalization; this difference may be attributable to the different calculation models used in the tools. However, NormFinder and BestKeeper ranked U6 snRNA and miR-192 as the top two reference genes.The impact of different normalization strategies on the accuracy of RT-qPCR results was examined by analyzing the expression levels of cell-free miR-198 in pleural effusions. In a previous study, the levels of miR-198 expression were found to be significantly lower in LA-MPE samples than in BPE samples, indicating that this miRNA may have diagnostic potential for differentiating BPE and MPE  were screened using the PNA-based microarray. Furthermore, the number of candidate reference genes examined was small; the use of multiple reference genes is critical to achieve more reliable expression data . AnotherIn conclusion, the use of the expression levels of U6 snRNA, miR-192 or a combination of these genes for the relative quantification of miRNA expression levels in pleural effusion is recommended, as determined by the results from the present study.


Heart valve diseases are common with an estimated prevalence of 2.5% in the Western world. The number is rising because of an ageing population. Once symptomatic, heart valve diseases are potentially lethal, and heavily influence daily living and quality of life. Surgical treatment, either valve replacement or repair, remains the treatment of choice. However, post-surgery, the transition to daily living may become a physical, mental and social challenge. We hypothesize that a comprehensive cardiac rehabilitation program can improve physical capacity and self-assessed mental health and reduce hospitalization and healthcare costs after heart valve surgery.VR, aims to investigate whether cardiac rehabilitation in addition to usual care is superior to treatment as usual after heart valve surgery. The trial will randomly allocate 210 patients 1:1 to an intervention or a control group, using central randomization, and blinded outcome assessment and statistical analyses. The intervention consists of 12 weeks of physical exercise and a psycho-educational intervention comprising five consultations. The primary outcome is peak oxygen uptake (VO2 peak) measured by cardiopulmonary exercise testing with ventilatory gas analysis. The secondary outcome is self-assessed mental health measured by the standardized questionnaire Short Form-36. Long-term healthcare utilization and mortality as well as biochemistry, echocardiography and cost-benefit will be assessed. A mixed-method design will be used to evaluate qualitative and quantitative findings, encompassing a survey-based study before the trial and a qualitative pre- and post-intervention study.This randomized clinical trial, CopenHeartThis randomized clinical trial will contribute with evidence of whether cardiac rehabilitation should be provided after heart valve surgery. The study is approved by the local regional Research Ethics Committee (H-1-2011-157), and the Danish Data Protection Agency (j.nr. 2007-58-0015).NCT01558765).Trial registered 16 March 2012; ClinicalTrials.gov ( VR trial study protocol, a randomized clinical trial comparing comprehensive cardiac rehabilitation after heart valve surgery, including physical exercise and psycho-education, with usual care. This update should be read in conjunction with the original protocol publication [This update relates to the CopenHeartlication .The plan for this trial was to include patients having either open heart surgery or percutaneous heart valve procedures. However, because of administrative reasons and other local ongoing clinical trials including patients having percutaneous procedures, we were only able to include patients having open heart surgery.Our original recruitment target was 210 with the recruitment period defined as two years. At the end of the two-year recruitment period in May 2014, we were only able to include 147 patients. Therefore, the power calculation has been repeated, based on this revised sample size.2 peak) measured by cardiopulmonary exercise testing. Based on the assumptions defined in the protocol , a sample size of 147 would provide 86% power to be able to reject the null hypothesis, with a type I error of 5%. The secondary outcome measure is mental health measured on the continuous variable mental health component scale on the Short Form-36 questionnaire. Based on the assumed minimum important difference of seven points and an updated standard deviation of nine points [The primary outcome is the continuous variable peak oxygen uptake VO peak meaWhile the primary analysis essentially remains the same as defined in the protocol, we propose slight revisions to the analysis model and handling of missing data, and also provide further specification of the handling of multiplicity of outcome testing.The level of significance is set at 5%. The intention-to-treat analysis using a mixed model with repeated measures (MMRM) for continuous outcome measures will be adjusted by the trial stratification variable ‘left ventricular ejection fraction <45%, yes/no’ .P ≤0.05), we will undertake sensitivity analyses that use the method of multiple imputation [As stated in the protocol, using MMRM ensures that missing data values will not create bias, as long as the values are missing at random . For theputation  and also2 peak and mental health component scale of Short Form-36) will be analyzed as stated above. The various exploratory outcome measures pre-defined in the protocol [P-values due to multiplicity. Instead, the interpretation of each exploratory outcome measure will be assessed in the light of multiple testing; that is, statistically significant effects will be interpreted in the context of increased risk of type I error.The primary and secondary outcomes  of the exercise sessions.The psycho-educational intervention is based on the theories of RR Parse  and compThe study was approved by the regional Danish Research Ethics Committee in the Capital Region of Denmark (H-1-2011-157) and the Danish Data Protection Agency (j.nr. 2007-58-0015). All participants have given full written and oral informed consent.


Manual measurement of 4-meter gait speed by a stopwatch is the gold standard test for functional assessment in older adults. However, the accuracy of this technique may be biased by several factors, including intra- and inter-operator variability. Instrumental techniques of measurement using accelerometers may have a higher accuracy. Studies addressing the concordance between these two techniques are missing. The aim of the present community-based observational study was to compare manual and instrumental measurements of 4-meter gait speed in older individuals and to assess their relationship with other indicators of physical performance.One-hundred seventy-two  non-disabled community-dwellers aged ≥65 years were enrolled. They underwent a comprehensive geriatric assessment including physical function by Short Physical Performance Battery (SPPB), hand grip strength, and 6-minute walking test (6MWT). Timed usual walking speed on a 4-meter course was assessed by using both a stopwatch  and a tri-axial accelerometer . Correlations between these performance measures were evaluated separately in men and women by partial correlation coefficients.In both genders, 4-MA was associated with 4-MM , handgrip strength  and 6MWT . 4-MM was associated with handgrip strength and 6MWT in both men and women. Considering gait speed <0.6 m/s as diagnostic of dismobility syndrome, the two methods of assessment disagreed, with a different categorization of subjects, in 19% of men and 23% of women. The use of accelerometer resulted in 29  additional diagnoses of dismobility, compared with the 4-MM.In an older population, the concordance of gait speeds manually or instrumentally assessed is not optimal. The results suggest that manual measures might lead to misclassification of a substantial number of subjects. However, longitudinal studies using standardized and validated procedures aimed at the comparison of different techniques are needed before recommending the use of accelerometers in comprehensive geriatric assessment. Epidemipulation , hospitapulation , disabilpulation  and mortpulation .In particular, the 4-meter gait speed test is one of the most widely used assessment tools in the clinical geriatric settings . This meMobility Working Group have identified the timed 4-meter usual gait speed as the main tool to diagnose dismobility, a condition characterized by poor mobility and defined by gait speed slower than 0.6 m/s [Recent recommendations from the  0.6 m/s –8.However, the current standard methodology of assessment of the walking time during the test by a stopwatch can be biased by high inter- and intra-operator variability ,10. ThesHowever, studies investigating the correlation between the assessment of gait speed, when conducted with a traditional stopwatch and by using accelerometers, are missing. Scarce information is also available on the relationship between the results obtained with these two assessment methods and other objective tests of physical performance . In a reGiven the clinical and research relevance of slow gait speed, we sought to investigate the correlation between its manual  and technological  assessment in older individuals. The study was also aimed at evaluating the correlation of 4-MM and 4-MA with other measures of physical performance, namely hand grip strength and the 6-minute walking test (6MWT).This community-based observational study is an ancillary project of the MED&SANO study, an epidemiological study conducted in a representative sample of non-disabled older persons living in the Medesano geographic area . The current analysis used cross-sectional data from the baseline comprehensive geriatric assessment conducted between November 2012 and April 2013 as part of the Provide multicenter European study, whose aims and design are detailed elsewhere .A total of 455 non-disabled older community-dwellers aged 65 years or older were eligible for study enrolment after a screening evaluation by general practitioners of the Medesano area, according to the absence of physical disability. Among them, 205 were randomly selected to undergo a second-level specialist evaluation including comprehensive geriatric assessment. All subjects with overt disability (Barthel index score <65), with severe chronic diseases or cancer were excluded from the study at this second step (n = 8). For the present analysis, 172 participants (69 men and 103 women) with complete data for the measures of interest were considered. Thus, 33 subjects were excluded since they did not complete the battery of physical tests included in this study.The study protocol was approved by the Ethics Committee of Parma Province. All participants were informed about the study procedures, purposes, and known risks, and all gave their written informed consent.The battery of physical evaluation tests included the assessment of gait speed by using a stopwatch (4-MM) and an accelerometer (4-MA), SPPB, 6MWT and hand grip strength. For each participant, all measures were collected during the same day. SPPB was assessed first, followed, respectively, by 6MWT, 4-meter gait speed and handgrip strength. A period of 2–3 minutes of rest was granted between one test and another.The manual and instrumental assessment of gait speed were performed during the same 4-meter walk. A straight clearly marked course was used. The manual measurement was made by a trained operator using a stopwatch (4-MM). A tri-axial accelerometer was instead used for the instrumental assessment (4-MA) . InstrucThe inertial triaxial sensor  consists of a small case of 78x48x20 mm weighting 48 g only, easy to use, requires no specialized equipment, does not interfere with regular walking, and could be used to analyze walking in clinical practice. The accelerometer, placed on a semi-elastic belt covering the L4-L5 inter-vertebral space, transmitted the data to a PC via Bluetooth. The sensitive axes of the sensing unit were automatically aligned along the anatomical vertical, medio-lateral, and antero-posterior axis.Lower extremity performance was evaluated using the SPPB, a strong predictor of physical disability in older adults . The SPPThe 6MWT was performed indoors, using a 30 meter-long walking course with a hard surface. The length of the corridor was marked every 3 meter and cones marked the turnaround points . A startIsometric hand grip strength was measured using a handheld dynamometer . The device measured strength in kilograms, with a precision of 0.1 kg. Participants were asked to perform the task twice with each hand. The average of the best result obtained with each hand was used for the present analyses.2. The Barthel Index was assessed for measuring the participants' functional ability. Cognitive performance was assessed using the Mini-Mental State Examination (MMSE). Depressive symptoms were assessed by the 15-item Geriatric Depression Scale (GDS-15), which is a widely used screening instrument for depressive symptoms in the elderly. The GDS-15 detects changes in depressive symptoms after a major negative life event [Weight and height, objectively measured and employing a standard protocol, were used to calculate the body mass index as kg/mfe event . Nutritife event . InformaThe prevalence of specific medical conditions was established using standardized criteria that combine information from self-reported history, medical records, and a clinical medical examination. Diagnostic algorithms were modified versions of those adopted in the Women’s Health and Aging Study .Categorical variables were expressed in numbers and percentages, and continuous variables were reported according to gender as means  for normally distributed parameters or as median and interquartile range (IQR) for those non-normally distributed.Since reference values for some muscle function and gait parameters in non-disabled older individuals are different between males and females , a gendeStatistical significance was defined as p≤0.05. SAS 8.2 statistical package  was used for all analyses.Characteristics of the study population  are summarized in Scatterplots of correlations between 4-MM and 4-MA in men and women are shown in Figs Calculation of unadjusted Pearson correlations confirmed that 4-MM was significantly and positively correlated with 4-MA in both genders, as highlighted in The correlations of 4-MM and 4-MA with other measures of functional performance are also shown in 4-MA showed a significant positive correlation with handgrip strength , as also 4-MM .In a cohort of community-dwelling older individuals, we found a significant correlation between the assessment of gait speed using a manual  and technological  technique. However, our results suggest that the concordance of two tests is less strong than anticipated and might be suboptimal in the classification of single subjects. This is the first study investigating the correlation between these two assessment modalities of gait speed.There is a wide range of methods available for assessing physical function in both research and clinical practice . The finThe present study shows that both 4-MM and 4-MA were correlated to other tests of physical performance like 6MWT and hand grip strength. The correlation found between the 4-MM and the 4-MA highlights that further studies are needed to investigate the role of accelerometer-measured gait speed assessment. Accurate, standardized and reproducible techniques of measurement are in fact needed, privileging those techniques less affected by methodological issues, and inter- and intra-operator variability that may represent a relevant bias ,10.Four-meter gait speed depends on lower limb muscle function and is a good predictor of mobility impairment and adverse outcomes ,25,26. T4-MM remains the easiest and commonest way to assess physical performance . NeverthThe problem of insufficient accuracy of manual measurements of walking speed is well known in medical literature ,10,27. SOur study has some limitations. The relatively small sample size and the cross-sectional nature of the study do not allow definitive conclusions. The capacity of 4-MA to predict functional outcomes in older community-dwellers is in fact still poorly investigated. The studies available in the literature, and performed with accelerometers, did not use the same position to place the device. In our study, the accelerometer was placed on a semi-elastic belt covering the L4-L5 intervertebral space. Thus, our results cannot be completely comparable with other studies using different positions . A standDespite these limitations this is the first study having assessed the correlation of 4-MM with 4-MA. The association between these two techniques of gait speed assessment should be better investigated in larger populations, with longitudinal design, including also subjects with known dismobility syndrome. On this regard longitudinal studies comparing the two tests are needed to validate 4-MA as a gold standard. Meanwhile, 4-MM remains the easiest approach to be used in the clinical practice. The implementation of technological methods of gait speed assessment in second-level geriatric care services or for clinical research purposes should however be considered although based on our findings it becomes hard to make recommendations on the better usefulness of automatic than manual measurement.In a population of elderly community-dwellers without known dismobility syndrome, we found a significant correlation between the assessment of gait speed using manual and technological techniques. The concordance of gait speed manually or instrumentally assessed is not optimal and manual measures might lead to misclassification of a substantial number of subjects. However, there is need of future longitudinal studies comparing the two techniques, using standardized procedures and taking quantitative gait analysis as gold standard reference, before to recommend the usefulness of accelerometers in the comprehensive geriatric assessment.


Euonymus lectin (EUL) domain(s). The carbohydrate-binding specificity of EUL proteins from a monocot, a dicot and a lower plant has been compared. Furthermore, modeling of the different EUL domains revealed a similar ß-trefoil fold consisting of three bundles of ß-sheet organized around a pseudo three-fold symmetry axis. Despite the sequence similarity and the conserved amino acids in the binding site, glycan array analyses showed that the EUL domain has a promiscuous carbohydrate-binding site capable of accommodating high mannose N-glycans, blood group B related structures and galactosylated epitopes.Plants synthesize small amounts of carbohydrate-binding proteins on exposure to stress. For example, on exposure to drought, high salt, wounding and by treatment with some plant hormones or by pathogen attack. In contrast to the ‘classical’ plant lectins that are mostly located in the vacuolar compartment, this new class of inducible lectins is present in the cytoplasm and in the nucleus. Taking into account that any physiological role of plant lectins most likely relies on their specific carbohydrate-binding activity and specificity, the discovery of these stress-related lectins provides strong evidence for the importance of protein-carbohydrate-interactions in plant cells. Hitherto, six families of such nucleocytoplasmic lectins have been identified in plants. This review will focus on the nucleocytoplasmic lectins with one or more  Lectins are carbohydrate-binding proteins that recognize and bind well-defined simple sugars or more complex carbohydrates in a reversible way. These carbohydrate-binding domains are widespread but have been studied most intensively within the plant kingdom. Plant lectins are a very diverse group of proteins with obvious differences in their biochemical/physicochemical properties, molecular structure, carbohydrate-binding specificity and biological activities . AlthougN- and O-glycans the idea was gradually developed that most of these lectins represent a special class of aspecific defense proteins that help the plant to cope with attacks from phytophagous invertebrates and/or herbivorous animals. This concept is further supported by the high expression levels of most lectins , their accumulation in a developmentally regulated manner and the toxicity of multiple lectins for insects and fungi . Binding assays carried out at different lectin concentrations allowed the conclusion that EEA has a much higher affinity for blood group B oligosaccharides than for high mannose N-glycans [N-glycans and for the blood group B substances. However, no final conclusion could be drawn with respect to the possible occurrence of two distinct carbohydrate-binding sites in EEA [Detailed analyses of the interaction of the fluorescently labeled EEA with the array revealed a high affinity of EEA especially for glycans containing the B-antigen structure, but also other blood group B-related structures such as glycans containing the H-antigen structure are recognized by EEA. These data are in agreement with earlier results that proved high affinity of EEA for Galα1-3Gal and Galα1-3Galß1-4GlcNAc carbohydrate epitopes, and binding to the blood group B  and O epitopes  as well as some types of glycosphingolipids ,32,33. O-glycans . It was -glycans . In an as in EEA .Arabidopsis revealed that this EUL lectin interacts preferably with complex N-glycans, since especially binding to galactosylated structures was observed. ArathEULS3 recognizes glycans containing one or more Lewis X GlcNAc-), Lewis Y GlcNAc-) or lactosamine  motifs. A comparative analysis of the glycan array data obtained for the recombinant full length ArathEULS3 protein  as well as for the EUL domain revealed no major differences, confirming that the N-terminal domain of the chimeric protein does not interfere with glycan binding , but it also recognizes glycans containing Lewis A, Lewis X and Lewis Y structures. The domain has only weak affinity for the blood group B related structures such as the glycans carrying the B-antigen, A-antigen and H-antigen structures . Although this result is in agreement with the lower agglutination activity of these recombinant proteins, it cannot be excluded that the recombinant proteins expressed in the fungal host C-terminal domain of the HA33/A protein from Clostridium botulinum [Bacillus sphaericus (RCSB PDB code 2VSE) [Sclerotinia sclerotiorum agglutinin (RCSB PDB code 2X2S) [In an attempt to unravel which amino acids are required for the carbohydrate-binding activity of the EUL domain, three-dimensional models were made for the EUL domains in EEA, ArathEULS3, OrysaEULS2 and PhypaEULS3. Molecular modeling of the EUL domains was performed with the YASARA Structure program  and tookotulinum , the holde 2VSE) , the Sclde 2X2S)  as well de 2X2S) . The useN-terminal end of the polypeptide chain it could create a disulphide bridge with the corresponding Cys residue of another ß-trefoil domain, resulting in the dimeric structure of EEA [in vitro. In view of the biosynthesis of the lectin in the cytoplasmic compartment the bridging of cysteine residues is unlikely to occur in vivo since the required enzyme resides in the endoplasmic reticulum. The EUL sequences for OrysaEULS2 and PhypaEULS3 contain three and four Cys residues, respectively . Judging from the three-dimensional models, these Cys residues are too far from each other to create intra-chain disulphide bonds. However, these Cys residues are also sufficiently exposed to participate in inter-chain disulphide bonds with another lectin-like monomer to create homodimers. The EUL sequences encoding EEA and ArathEULS3 both contain 1 cysteine (Cys) residue  [C-terminal end of the four studied EUL proteins    a putatipaEULS3)  and putaOryza sativa), a dicot  and a lower plant (Physcomitrella patens), were compared with EEA, the prototype of the EUL family.Evidence is accumulating that shows that the EUL domain represents a conserved structural unit of a family of carbohydrate-binding proteins that is widespread among plants. In this review the biological activity of EUL proteins from three plant species, in particular a monocot  and a homologous protein from maize which shares 64% sequence similarity and has conserved residues in the carbohydrate-binding sites, showed important differences in their specificity. While GNA binds strongly to high mannose N-glycans the GNA homologue from maize has a high affinity for more complex glycans [N-glycans [C-terminal Nictaba domain of the so-called F-box Nictaba proteins, glycan array analysis clearly demonstrated that the Arabidopsis F-box protein exhibits a substantially different carbohydrate-binding specificity [N- and O-glycans containing LacNAc structures as well as Lewis A, Lewis X, Lewis Y and blood type B motifs were recognized by the Nictaba domain of the Arabidopsis F-box protein. All these data show that similar carbohydrate-binding motifs can accommodate unrelated oligosaccharides.Analyses of the protein sequences of different members of this family showed that the EUL domain is strongly conserved in different plant species, whereas the epitopes ,18,28. Sepitopes ,41. Simi glycans . Recentl-glycans . Althougcificity . N- and Molecular modeling of the EUL domains of EEA, ArathEULS3, OrysaEULS2 and PhypaEULS3 demonstrated that they all exhibit a similar three-dimensional structure which is a ß-trefoil fold consisting of three bundles of ß-sheet. Although the EUL proteins show a very similar overall fold, there are clear differences in the overall structure and the charge distribution on the protein surface. It was shown before that small changes in the amino acids building the carbohydrate-binding site or surrounding the site can lead to changes in their specificity . For insUlex europaeus (UEA-II), a legume lectin, has a binding site which is capable of binding both N-acetylglucosamine and galactose. It was shown that hydrophobic interactions are important in the protein–carbohydrate complexes [Similarly, a single variable loop determines the exact shape of the promiscuous monosaccharide binding site of the legume lectins and is responsible for discrimination between galactose, mannose or glucose. Lectin II from omplexes .N-glycans  as wradation ,47, but N-glycans. As a consequence high mannose containing free N-glycans as well as glycosylated proteins are putative targets for these lectins. Free N-glycans are reported to occur ubiquitously at micromolar levels in various cells or tissues like in elongating hypocotyls of seedlings, developing seeds or maturated fruits, indicating that these free N-glycans play an important role during cell differentiation, plant growth or fruit ripening [N-glycans, a high mannose type and a complex type, were found. A quantitative analysis of high mannose type free N-glycans using cotyledons of pumpkin seedlings as model cells showed that the high mannose free N-glycans accumulate in the cytosol [N-glycans act as signaling molecules, it appears likely that the N-glycosylation mechanisms working in plants are associated with plant development, plant growth or fruit ripening. It was shown that EEA and OrysaEULS2 have affinity for high mannose type ripening ,49,50,51 cytosol . AlthougN-glycans, galactosylated structures were recognized by several EUL proteins. EEA strongly binds to blood group B related structures. Glycan array analysis revealed that the EUL protein from the lower plant Physcomitrella patens has a high affinity for glycans containing lactosamine structures. These LacNAc structures were also recognized by the EUL from the dicot Arabidopsis thaliana and the monocot Oryza sativa. PhypaEULS3 binds also Lewis A structures while ArathEULS2 recognizes Lewis X and Lewis Y structures. Next to high mannose EULs are not the only lectins that recognize galactosylated structures. The family of human galectins for instance also has specificity towards the disaccharide LacNAc, poly-LacNAc and internal LacNAc present in poly-LacNAc . MoreoveN-glycans [N-glycans of a glycoallergen from mountain cedar pollen [N-glycans linked to glycoproteins in rice cells revealed that most of them had the plant complex type structure including a Lewis A epitope-harboring type whereas the high mannose type structures were low abundant [N-glycans could also be secreted [et al. [N-glycans in the intracellular and the extracellular spaces of a rice cell culture system. It was observed that the sugar chain profile differed between the soluble fraction and the cell culture medium of rice-cultured cells. The intracellular fraction contained mainly high mannose type N-glycans with one GlcNAc residue while in the culture medium complex free N-glycans containing the Lewis A epitope and high mannose type free N-glycans with the N-diacetylchitobiosyl unit were found [Although Lewis X and Lewis Y structures can be found in higher animals they have not yet been identified in plants. Until now only Lewis A motifs have been reported at the non-reducing end of biantennary complex type plant -glycans ,56,57. Tr pollen ,59. Struabundant . It was secreted . In 2010 [et al. , analyzere found . Secretire found .Calystegia sepium rhizomes is classified as a mannose-binding jacalin-related lectin [Calystegia sepium lectin is just a cytoplasmic storage and/or defence related protein [Next to the carbohydrate-binding properties of the EUL proteins, their presence in low concentrations in the plant cell under specific stress conditions also urges for a specific role of these lectins within the plant cell. However, in contrast to ArathEULS3, OrysaEULS2 and PhypaEULS3 EEA is an abundant protein in the arillus where it accounts for up to 80% of the total protein content. Besides in the arilli, the lectin is also found in leaves and bark tissue, though at much lower concentrations . No lectd lectin . In cont protein .This evidence shows that the EUL domain can be considered a universal lectin domain within the plant kingdom. However, it is clear that the specificity of the binding site has evolved during evolution. It can be concluded that it is difficult to make a prediction on the specificity of the EUL domain based on the sequence and on the conservation of the amino acids constituting the sugar-binding site. Surely, a detailed analysis of the carbohydrate-binding specificity of more members of the EUL family will provide more insight as to their specificity. Since these carbohydrate-binding properties most likely are a determining factor for their physiological role in the plant cell, this knowledge will also help to unravel the biological significance of these proteins. Future work will focus on the identification of the interacting partners of the EUL lectins with the ultimate goal of unraveling their physiological importance for plant growth and development.


For both bacteria and fungi, the addition of PAHs led to a shift in community composition, but thereafter the major factor determining the fungal community composition was whether it was in the planktonic phase or attached to filters. In contrast, the major determinant of the bacterial community composition was the nature of the PAH serving as the carbon source. The main bacteria enriched by HMW-PAHs were Pseudomonas, Bacillus, and Microbacterium species. This report demonstrates that OSPW harbors microbial communities with the capacity to transform HMW-PAHs. Furthermore, the provision of suitable surfaces that encourage PAH sorption and microbial adhesion select for different fungal and bacterial species with the potential for HMW-PAH degradation.High-molecular-weight polycyclic aromatic hydrocarbons (HMW-PAHs) are natural components of fossil fuels that are carcinogenic and persistent in the environment, particularly in oil sands process-affected water (OSPW). Their hydrophobicity and tendency to adsorb to organic matter result in low bioavailability and high recalcitrance to degradation. Despite the importance of microbes for environmental remediation, little is known about those involved in HMW-PAH transformations. Here, we investigated the transformation of HMW-PAHs using samples of OSPW and compared the bacterial and fungal community compositions attached to hydrophobic filters and in suspension. It was anticipated that the hydrophobic filters with sorbed HMW-PAHs would select for microbes that specialize in adhesion. Over 33 days, more pyrene was removed (75% ± 11.7%) than the five-ring PAHs benzo[ PAHs wicinogens . The Eurcinogens . Generalcinogens . These pcinogens .−1 of PAHs, a value 1,000-fold greater than the limit specified in the Canadian Environmental Quality Guidelines , and benzo[b]fluoranthene (BbF), were selected based on differences in physico-chemical characteristics and toxicity , benzopyrene, and benzo[b]fluoranthene. All compounds were obtained from Sigma-Aldrich, Gillingham, United Kingdom .Three high-molecular-weight polycyclic aromatic hydrocarbons (HMW-PAHs) were used in this study: pyrene, benzopyrene) and metabolite 4   . Metabolypyrene) , were teP < 0.05 by analysis of similarities [ANOSIM]) in the bacterial communities was observed with respect to the control   region between the 5.8S and 28S rRNA genes, respectively . For enry stage) . The bioy stage) .For the bacterial communities derived from sample 2m, a pattern similar to that for sample TPW was observed, with 62% similarity in composition between the control communities  and those enriched on HMW-PAHs . The bioFor enrichments derived from both samples TPW and 2m, the fungal communities were less diverse than the bacterial communities based on the number of DGGE bands observed . For TPW-derived fungal communities, there was 68% similarity between the control community and those communities enriched on HMW-PAHs . In contCommunities derived from TPW enrichments were characterized by 454 pyrosequencing of the bacterial 16S rRNA gene , in ordePseudomonas stutzeri increased 4-fold in the BbF planktonic community and 13-fold in the BbF filter community, constituting 76% of the OTUs in the latter  Arthrobacter (99%), and Phenylobacterium (98%). The Pyr filter community contained one specific DGGE band, which had a high 16S rRNA gene sequence identity (98%) to Bacillus lentus. Both the filter and planktonic communities enriched on either BaP or BbF contained two bands, which had high 16S rRNA gene sequence identities to Pseudomonas stutzeri (99%) and a Microbacterium sp. (99%), respectively .Sequence analysis of the DGGE bands that differed in abundance gave a broad comparison of the differences in the main taxa between treatments . Bacterial 16S rRNA gene sequence analysis from the TPW enrichment  revealed that the planktonic community grown on Pyr had three specific DGGE bands, which had high 16S rRNA gene sequence identities to Streptomyces sp. (99%) and a Micromonospora sp. (99%), while two bands specific to the Pyr planktonic community had a high 16S rRNA gene sequence identity to Paracoccus aminovorans (98%) and Pseudomonas spp. (98%) .A number of different species were identified in the 2m bacterial community. Two bands specific to the Pyr filter community had a high 16S rRNA gene sequence identity to a Cladosporium cladosporioides . In the 2m fungal community, one band, specific to the BbF filter community, also had ITS sequence identity to Cladosporium spp. (94%) .Sequence analysis of selected fungal DGGE bands showed that all the TPW PAH-enriched filter communities (but not the no-PAH controls) contained one specific DGGE band, which had a high ITS sequence identity (99%) to Streptomyces. Strains A3 and A7, isolated from the culture broth from enrichments containing Pyr and BaP sorbed to filters, had a high 16S rRNA gene sequence similarity to Microbacterium hydrocarbonoxydans (99%). No growth was observed with any of the isolates on plates supplemented with acetone only.Five pure bacterial cultures were isolated  from HMW-PAH-transforming communities. Strains A1, A6, and A13 from filters containing Pyr, BaP, and BbF, respectively, had 99% 16S rRNA gene sequence similarity to members of the genus Actinobacteria, Firmicutes, Bacteroidetes, or Alpha-, Beta-, or Gammaproteobacteria. Within the Actinomycetales, Streptomyces-related sequences from isolates, DGGE bands, and pyrosequences all derived from attached bacteria. Cluster 2942 (Streptomyces) represented 3% of the BbF filter community and <1% of the BbF planktonic and no-PAH control communities. The isolates related to Microbacterium spp., although cultured from the planktonic phase, were also attached to the filters as judged by sequences being detected from filters by DGGE. Furthermore, the highly abundant pyrosequence cluster 2134  and noncultured bacteria from the HMW-PAH enrichments: 20 representatives of the most abundant pyrosequence OTUs  and 17 DGGE bands . All seqter 2942   than microbes in the planktonic phase. Bacterial communities were also influenced by the specific HMW-PAH used for enrichment, while fungal communities were not. To date, there is little information on the composition of the microbial communities involved in the biotransformation of HMW-PAHs in aquatic environments, specifically with regard to freshwater fungal species. Despite the fact that PAHs are known to be abundant in oil sands , 6, attea]pyrene (metabolite 3) was produced. It has previously been detected in cultures of Mycobacterium vanbaalenii PYR-1 degrading BaP . A number of bacterial biotransformation products of BbF have also been previously identified, including hydroxylated intermediates  metabolism by Sphingobium sp. strain KK22  were significantly higher in detached cells than in either planktonic cells or biofilms  was detected only on filters and was enriched in the presence of all HMW-PAHs but was not detected in the no-PAH control . Cladosporium species from a number of terrestrial environments have been shown to degrade a variety of PAHs with more than three rings  but not in the no-PAH control. Penicillium spp. from a variety of terrestrial environments have been isolated and shown to degrade Pyr and BaP to various degrees  had a preference for growing on filters, the bacterial community composition was selected primarily by the PAH serving as the carbon source. This suggests that diverse bacterial communities would be required to degrade a range of HMW-PAHs. Future work should focus on elucidating the interactions between bacterial and fungal members of these consortia to improve the biodegradation rates of HMW-PAHs, an important suite of pollutants that are often overlooked in tailings ponds containing oil sand waste.In conclusion, we demonstrated that OSPW harbors microbial communities with the capacity to metabolize HMW-PAHs. During the transformation of HMW-PAHs, a number of metabolites were produced and tentatively identified as the hydroxylated intermediates of the parent compound. In addition, we demonstrated that the provision of suitable surfaces that encourage PAH sorption and microbial adhesion select for fungal and bacterial communities with the potential for HMW-PAH metabolism that may have been overlooked previously by the use of liquid enrichments. We showed that a major factor determining the fungal community composition was whether it was present in the planktonic phase or attached to filters. In contrast, despite the fact that some bacteria (e.g.,


Lactobacillus species is possible, despite their taxonomic complexity. Here we describe a combined approach involving both cultivation and genetic methods to ascertain and characterize the diversity and abundance of the Lactobacillus population in the oral cavities of children with severe early childhood caries (S-ECC). Eighty 3- to 6-year-old children (40 S-ECC and 40 caries free) who were seeking dental care at the Pediatric Dental Clinic of Bellevue Hospital in New York City were invited to participate in this study. Clinical data on socio-demographic information and oral health behavior were obtained from the primary caregiver. The data included a detailed dental examination, children’s medical history, and a questionnaire survey. Combined non-stimulated saliva and supra-gingival plaque samples were collected from each child and cultivated on selective media for quantitative measures of lactobacilli levels. The procedure for Lactobacillus species screening will include the random selection of 50 colonies per plate, extraction of DNA from each colony, and genotyping by arbitrarily primed polymerase chain reaction (AP-PCR). Each unique Lactobacillus AP-PCR genotype will be selected for taxonomic assessment by 16S rRNA gene sequencing analysis. Lactobacillus species will be identified by comparing the 16S rRNA sequences with the Ribosomal Database and the Human Oral Microbiome Database. Meanwhile, the same set of clinical samples will be independently subjected to genomic DNA isolation, 16S rRNA amplification with Lactobacillus genus-specific primers, sequencing, and taxonomic identification, both at genus and species levels with a customized pipeline. The distribution and phylogenetic differences of these Lactobacillus species will be compared between children with or without S-ECC. One of the main objectives of this study is to establish a study protocol for the identification and characterization of lactobacilli in the oral cavity. Future caries risk assessments can include lactobacilli counts (quantitative) and the presence/absence of specific cariogenic genetic signatures of a Lactobacillus species  associated with S-ECC.Lactobacilli have been consistently associated with dental caries for decades; however, knowledge of this group of bacteria in the etiology of the disease is limited to quantitative elucidation. Nowadays, explicit identification of oral  Severe early childhood caries (S-ECC) is a particularly aggressive form of dental caries associated with a history of maternal malnutrition and illness, tooth developmental defect  2], exc, exc2], Lactobacillus have been isolated from various human body sites, plants, foods and the environment , on, onLactoobacilli . Other sironment  22] and andLacton others  23]. Th. ThLactoLactobacillus colonization, identify lactobacilli to the species level, and determine the constellation of genetic elements associated with the cariogenicity of this group of bacterial species.The lack of knowledge about the genetic characteristics and diversity of lactobacilli is, in part, hindered by the taxonomic challenges from the initial step of clinical sample collection to the final step of species identification. Traditionally, studies of caries-associated lactobacilli are based on laboratory cultivation, but results vary due to the lack of reliable protocols for screening and characterizing clinical isolates that enable the identification of lactobacilli. Recently, advancements in molecular genetics and comparative genomics have been applied to microbiology, largely facilitated by 16S rRNA sequencing and sophisticated analytical methods  25]. It. It25]. i.e., genomic signature of Lactobacillus species/strains associated with S-ECC. The outcome variables can be included in future caries risk assessments, which, at present, rely mainly on lactobacilli counts in saliva. Monitoring the caries-associated genetic signature of some of the Lactobacillus species can also be clinically relevant for developing new caries treatment modalities.Previously, we reported a pilot study to demonstrate methods for determining the diversity of lactobacilli in the oral cavity . The proLactobacillus abundance relative to other microbes; 3) determination of lactobacilli diversity at the species level in an aggressive S-ECC group and a caries-free (CF) group; 4) comparison of whole ge-nome sequences from the clinical Lactobacillus species/strains. The overall study design is illustrated in Lactobacillus isolates. The final Lactobacillus species identification will be based on 16S rRNA gene sequence analysis. Furthermore, whole genome intra-species and inter-species comparisons will be conducted to identify niche-specific genetic elements (This exploratory study consists of four components: 1) subject recruitment, clinical and bacterial sample collection; 2) assessment of elements .The study is conducted at two sites: New York University College of Dentistry and the Pediatric Dental Clinic of Bellevue Hospital Center. Applications for this study have been approved by the Institutional Review Boards of the New York University School of Medicine, New York University College of Dentistry , and the New York City Health and Hospital Corporation  for human subjects participating in research activities.The study recruitment began in February 2012 and ended in May 2014. A total of 80 children, 3 to 6 years of age, were recruited for this study from the Bellevue Hospital Pediatric Dental Clinic. Forty of them were diagnosed with S-ECC according to the criteria set forth by the American Academy for Pediatric Dentistry . They weS-ECC is defined as caries in children younger than 3 years of age with one or more non-cavitated or cavitated lesions, filled or missing (due to caries) smooth surfaces in primary maxillary anterior teeth (dmfs), or a dmfs score ≥ 4 at age 4, ≥ 5 at age 5, or ≥ 6 at age 6 . All of To be eligible for this study, children must be 3 to 6 years old, healthy without chronic diseases other than dental caries, and not having taken antibiotics within 3 months prior to bacterial sample collection. Children with chronic disease other than dental caries or a history of antibiotic use within 3 months prior to the dental treatment or missing more than 6 primary teeth are excluded.Lactobacillus population was 43.4% (SD ± 17.7%) for S-ECC children and 58.4% (SD ± 14.3%) for CF children, respectively (unpublished data). The proposed study hypothesis is that the Lactobacillus population between S-ECC and CF children is the same if the criterion for significance  is set at 0.05 for 2-tailed tests and the proposed sample size is 40 for each group. To test the null hypothesis, the study will achieve a power of >85% to yield statistically significant results.The study is designed to recruit 80 children divided into an S-ECC group (n = 40) and a CF group (n = 40). The sample size of estimation is based on data previously reported that the composition of oral microbiota of S-ECC children exhibits less diversity than that of CF children . In an eBefore undergoing dental examination, all primary caregivers who agreed to participate in this study were interviewed by a trained project coordinator to obtain the child’s current and past medical history, nursing practices, dietary habits, oral hygiene habits, and prescribed medications. Both English and Spanish versions of the questionnaire survey were available. The entire interview took no more than 20 minutes. Minimum or no risks result from participation in this study, and all information related to the project will be kept confidential following state laws and regulations governing confidentiality of medical records.A standardized clinical examiner performed the dental examination for each child in the Bellevue Pediatric Dental Clinic using standard dental mirrors and established criteria and scoring system. Using a sterilized soft toothbrush , saliva and plaque samples were collected from all S-ECC children in the operating room by the standardized attending dentist. The same samples were collected from the CF children by a trained dental hygienist. A second bacterial sample was also obtained from caries lesions of the S-ECC children by the same dentist. More specific procedures of bacterial sample collection are described in the following sections.Subject-specificFor S-ECC children scheduled for treatment under general anesthesia in the operating room: before comprehensive restorative treatment, the dentist brushed the child’s teeth for 1 min using a sterile soft toothbrush .For CF children in the dental clinic: the hygienist brushed a child’s teeth for 1 min using a sterile soft toothbrush without swallowing saliva.ProcedureThe dentist/hygienist slowly brushed the surfaces of the teeth, and then mixed with saliva.The toothbrush was immediately placed into a pre-labeled glass tube containing 9 mL of a pre-reduced liquid dental transport (LDT) medium .The bacterial sample was removed from the toothbrush by shaking the toothbrush vigorously for 30 sec (approximately 30 strokes) in the LDT medium; the toothbrush was then discarded.The bacterial sample was transported on ice to the microbiology laboratory within 2 h for processing and cultivation.Caries lesion site-specific sample for S-ECC groupBacterial sample was collected from 1 caries lesion at the most severe site.A sterile regular No. 6 dental round bur  was used with a slow-speed handpiece to remove caries lesion for 5 sec.Bacterial sample along with the bur was immediately placed into a pre-labeled glass tube containing 1 mL of LDT medium , and the tube was capped.The sample was transported on ice to the microbiology laboratory within 2 h for processing and cultivation.Fissure site-specific sample for CF groupBacterial sample was collected from 4 sound occlusal fissures .A sterile 30-gauge extra-short dental needle  was held in a hemostat and gently passed across the 4 occlusal fissures to collect as much bacterial sample as possible.The needle was immediately placed into a pre-labeled glass tube containing 1 mL of the LDT medium, and the tube was capped.The sample was transported on ice to the microbiology laboratory within 2 h for processing and cultivation.Lactobacillus genotypes are present in S-ECC children, we conducted another pilot study and found that, on average, children with S-ECC harbored 4 distinct genotypes, ranging from 1 to 6 . Th. Th37].  Lacto R  29] . Ep. EpStrep linkage  43]. Va. VaStrepLactobacillus species-specific 16S rRNA gene primers/probes, thus creating more obstacles against the use of advanced molecular-based techniques to determine lactobacilli diversity, abundance and taxonomical affiliation.Based on selective culture media, previous studies of caries-associated lactobacilli have been mostly quantitative. The culture-based approaches can be time- and labor-intensive, and results can vary widely based on colony morphology or counts on the particular medium or cultivation method used. On the other hand, using culture-independent methods for taxonomic differentiation of lactobacilli can be challenging as well because of a vast group of Gram-positive bacilli sharing similar morphological profiles as well as the molar percentage of guanine-cytosine content (% G + C). Furthermore, there are very few definitive Lactobacillus species associated with dental caries. Unlike most culture-based studies of the microbi-ota of S-ECC, this protocol utilizes a dual approach that utilizes both culture-based and culture-independent approaches. The culture-based component relies on a Lactobacillus selective medium that eliminates the background growth of other acid-tolerant oral bacteria, and can be completed, from sample plating to lactobacilli screening and isolation, in 10 to 12 working days for each subject. The workflow can be staggered for multiple subjects, allowing a trained team of dentists and lab technicians to process 5 – 10 subjects in one day. Lactoba-cillus strains isolated based on the selective medium can also serve as source materials for the whole genome sequencing and comparative genomics leading to the identification of niche-specific loci. The culture-independent component can be completed in 5 to 7 days from sample collection, genomic DNA purification to retrieval of the Lactobacillus 16S rRNA sequences for each subject. Up to 8 subjects can easily be processed simultaneously. Moreover, the culture-independent component relies on a new set of Lactobacillus-specific primers which allows for the detection of lactobacilli in plaque samples, even though they are present in low numbers. That all elements of the study will incorporate a custom-designed barcoding identification and tracking system and a user friendly interface will greatly enhance the efficiency and accuracy of samples process and data management.The study proposes a well-designed methodology aimed to assure that the sampling depth will be sufficient to find most Lactobacillus function- and niche-specific cariogenic variables can serve as antimicrobial targets for new therapeutic treatment for caries and help facilitate the development of new tools for future caries risk assessments.In summary, the novel culture-dependent and culture-independent approach will enable us to determine the complexity and the role of lactobacilli played in S-ECC. From a clinical perspective,


A lower frequency of positivity for anti-CCP2 was found in RA (66.2%) as compared with anti-MCV (81.0%). When comparing RA versus other CIRD, sensitivity increased when both assays were performed. This strategy of testing both assays had high specificity and LR+. We conclude that adding the assay of anti-MCV antibodies to the determination of anti-CCP2 increases the sensitivity for detecting seropositive RA. Therefore, we propose the use of both assays in the initial screening of RA in longitudinal studies, including early onset of undifferentiated arthritis.Determination of anti-citrullinated peptide antibodies (ACPA) plays a relevant role in the diagnosis of rheumatoid arthritis (RA). To date, it is still unclear if the use of several tests for these autoantibodies in the same patient offers additional value as compared to performing only one test. Therefore, we evaluated the performance of using two assays for ACPA: second-generation anti-citrullinated cyclic peptides antibodies (anti-CCP2) and anti-mutated citrullinated vimentin (anti-MCV) antibodies for the diagnosis of RA. We compared three groups: RA ( Rheumatoid arthritis (RA) is a chronic inflammatory disorder that involves synovial joints and may develop extra-articular manifestations . FrequenAssays determining antibodies against human mutated vimentin (anti-MCV) have been also proposed recently as a tool for the diagnosis of RA , 8. NeveTherefore, we evaluated the performance of using two ACPA assays: second-generation anti-citrullinated cyclic peptide antibodies (anti-CCP2) and anti-mutated citrullinated vimentin (anti-MCV) antibodies in established RA, and we correlated the titers observed of these autoantibodies with disease activity.Design. Cross-sectional study.Clinical Setting. Adult consecutive patients with RA seen in an outpatient rheumatology clinic of a secondary-care center in Guadalajara, Mexico , were invited to participate if they met at least four of the 1987 ACR criteria for RA [a for RA . They weThese patients were compared with two distinct non-RA controls selected.(i) The first comparison group was constituted by patients with other rheumatic inflammatory disorders mainly including systemic lupus erythematosus   or ankyl(ii) The second group was constituted by clinically healthy blood donors obtained from the same hospital, without history of blood transfusion or chronic infections.For these two comparison groups, similar inclusion and exclusion criteria described for patients with RA were applied.A structured assessment for patients with RA was performed including disease characteristics, evaluation of disease activity according to DAS-28 , functioA venous blood sample was taken from all included subjects at the same time of the clinical evaluation and the serum was obtained and stored at −20°C until antibodies determination. Anti-CCP2 were determined by ELISA using a commercial kit  with a cut-off value for positivity >5 U/mL and anti-MCV were determined by ELISA using also a commercial kit  with a cut-off value for positivity >20 U/mL.t-test was used to compare means between two groups. We selected as “gold standard” the 1987-ACR criteria for diagnosis of established RA. These criteria were used instead of the most recent 2010-ACR criteria because the status of positive ACPA is included within the criteria. The performance of the assays for anti-MCV and anti-CCP2, either individually or tested together, to identify RA was evaluated estimating sensitivity, specificity, and positive and negative predictive values, as well as likelihood ratios. In this study, sensitivity can be defined as the probability of positive anti-CCP2 or anti-MCV in patients with RA. Specificity was defined as the probability of negative results for these autoantibodies in patients or controls without RA. Positive predictive value (PPV+) was defined as the probability of having RA in presence of anti-CCP2 or anti-MCV. Negative predictive value was defined as the probability of not having RA in presence of a negative result for these autoantibodies. We computed 95% confidence intervals (95% CI) for the utility values for these autoantibodies. Kappa statistics was used to compute the degree of agreement in positivity between both anti-CCP2 and anti-MCV for patients with RA.Qualitative variables were expressed as frequencies and percentages and quantitative variables were expressed as means ± standard deviations. Chi-square tests were used to compare proportions among groups and Student's P value of <0.05. All analyses were done with the SPSS program (version 8).Correlation between titers of anti-CCP2 and anti-MCV and variables was examined using Spearman's correlation coefficient. The value of statistical significance was set at a One hundred and forty-two patients with RA were included and compared with 86 patients in the group of autoimmune rheumatic diseases  and 56 healthy controls.General characteristics of patients with RA are shown in Concordance between the findings of the two assays, anti-CCP2 and anti- MCV, in RA is shown in An evaluation of utility values for the strategies of testing each assay, anti-CCP2 or anti-MCV alone, or testing both assays in established RA compared with clinically healthy blood donors is shown in The utility values for the strategies of performing only anti-CCP2 or anti-MCV or both of these assays in established RA compared with other rheumatic inflammatory diseases are shown in In our study, we observed that the assay for anti-MCV antibodies showed more sensitivity and specificity than the assay for anti-CCP2 antibodies to distinguish established RA patients from other systemic inflammatory rheumatic diseases. Using the strategy of performing both assays, we obtained an increase in sensitivity in comparison with using either assay individually. In our study, the Kappa between both assays indicates that determination of both tests should be complementary and consequently increases the utility of both tests in the clinical armamentarium without decreasing specificity.Previous studies have reported, for anti-CCP2, specificities greater than 90% –25, simiNevertheless, in terms of a screening test, a higher sensitivity is extremely relevant; therefore, strategies to increase the values of sensitivity are required to establish an earlier diagnosis and opportune reference to the rheumatologist. To this regard, in the present study, the utilization of an assay for anti-CCP2 exclusively had only 66% of sensitivity, whereas when both assays, anti-CCP2 and anti-MVC, were done in the same patients, the sensitivity increased to 85%, with an improvement in the utility of these assays as a tool for clinicians. Regarding specificity of anti-CCP2, some studies have shown a wide variability ranging from 40% to 83% , 27, theSeveral factors could contribute to explaining why we observed that the anti-VCM assay was more sensitive than the anti-CCP2 assay. One of them is that vimentin contains 43 arginine residues. Each arginine residue can potentially be citrullinated by peptidylarginine deiminase (PAD) resulting in a variety of citrullinated epitopes. In contrast, in the anti-CCP2 test only a few epitopes are presented –32.Some authors reported recently that combining determinations of anti-MCV, anti-CCP2, and RF increases the sensitivity . NicaiseSome limitations of the study due to its cross-sectional nature are that we were unable to identify if controls without rheumatic disorders who depicted positivity to one or both autoantibodies will have progression to a CIRD in the future; nevertheless, this hypothesis should be tested in cohort models, increasing the number of patients. On the other side, we did not apply these tests to specific subgroups of patients, such as RA with extra-articular manifestations, undifferentiated arthritis, or early RA, where the performance of these diagnostic tests may have substantial variations to those observed in defined RA. Another limitation was that we did not include an assay for testing anti-CCP3. Anti-CCP3 assays rely upon additional epitopes not present in the anti-CCP2 antigen sequence , 34. SzeIn conclusion, using both assays, anti-CCP2 and anti-MCV, increases the sensitivity for the presence of RA as compared to performing only one assay; therefore, this strategy should be included in the clinical armamentarium to improve the value of these assays as screening test.


Aims. The priority of Chinese herbal medicines (CHMs) plus conventional treatment over conventional treatment alone for acute coronary syndrome (ACS) after percutaneous coronary intervention (PCI) was documented in the 5C trial (chictr.org number: ChiCTR-TRC-07000021). The study was designed to evaluate the 10-year effectiveness of CHMs plus conventional treatment versus conventional treatment alone with decision-analytic model for ACS after PCI. Methods and Results. We constructed a decision-analytic Markov model to compare additional CHMs for 6 months plus conventional treatment versus conventional treatment alone for ACS patients after PCI. Sources of data came from 5C trial and published reports. Outcomes were expressed in terms of quality-adjusted life years (QALYs). Sensitivity analyses were performed to test the robustness of the model. The model predicted that over the 10-year horizon the survival probability was 77.49% in patients with CHMs plus conventional treatment versus 77.29% in patients with conventional treatment alone. In combination with conventional treatment, 6-month CHMs might be associated with a gained 0.20% survival probability and 0.111 accumulated QALYs, respectively. Conclusions. The model suggested that treatment with CHMs, as an adjunctive therapy, in combination with conventional treatment for 6 months might improve the long-term clinical outcome in ACS patients after PCI. Percutaneous coronary intervention (PCI) as well as pharmacological treatments has significantly reduced but did not eliminate the risk of major adverse cardiovascular events (MACE) in ACS patients. It is reported that approximately 10%~18% of ACS survivors after PCI ultimately suffer a second myocardial infarction (MI), stroke, or cardiovascular death despite the availability of timely and appropriate treatments. With the raising concern of recurrent cardiovascular events in ACS patients undergoing primary PCI, it is necessary to substantiate the effectiveness and outcomes of adjunctive therapies, such as Chinese herbal medicines (CHMs)  and acupuncture, when added to conventional medication.ChiCTR-TRC-07000021) showed that CHMs, Xinyue Capsule, and Fufang Chuanxiong Capsule, in combination with conventional treatment, further prevent 1-year occurrence of cardiovascular events in ACS patients after primary PCI without increasing risk of major bleeding, as compared with conventional treatment alone. Owing to the only 1-year follow-up period in this trial and other limited CHMs trial resources, the priority of CHMs in combination with conventional treatment over conventional treatment alone on long-term outcome of ACS patients has not been established.CHMs have been widely used in clinical practice for thousands of years. Previously, we published the 1-year clinical outcomes of the 5C trial [Therefore, we constructed a decision-analysis Markov model to assess the effectiveness of CHMs plus conventional treatment versus conventional treatment alone in ACS patients after primary PCI. During 10 years, whether ACS patients after PCI may benefit from reducing the risk of MACE and increasing the quality of life (QOL) when treated by additional CHMs for 6 months, as an adjunctive therapy, in combination with conventional treatment, remains unclear.To capture the short- and long-term clinical outcomes in ACS patients after PCI receiving additional CHMs for 6 months plus conventional treatment versus conventional treatment alone, a decision-analytic Markov model was developed, by following generally accepted principles of design . ReferriThe 1-year decision tree was modelled based on the clinical outcomes in 5C trial. During the first year, the patients who received additional CHMs for 6 months plus conventional treatment or conventional treatment alone could suffer a nonfatal MI, a nonfatal stroke, a nonfatal UA, or death from all causes. Those patients who experienced no events were considered as event-free.To simulate the long-term clinical outcomes in post-PCI ACS patients in 5C trial, the 1-year decision tree was extended to a long-term Markov model. A Markov model consists of a number of mutually exclusive and collectively exhaustive health states, usually named as Markov states, representing the disease progression process from entry to death or end of the time horizon of the analysis . DiseaseIn the study, the Markov model  had 8 heThe model was run up to a time horizon of 10 years. Half-cycle correction was performed in this study with assigning one-half of the state reward for simulated individuals starting in each state.Transition probabilities, which characterize how a cohort member may pass in successive cycles, vary over time and depend on patient characteristics but not on previous events as the model has no memory . In 1-yeIn Markov model, as the intervention period of additional CHMs was only 6 months and no long-term data were available after the first year of treatment, we conservatively assumed that the transition probabilities were identical for patients receiving CHMs plus conventional treatment or conventional treatment alone for year 2 and onwards. And the only difference between the two treatment strategies was caused by the different distribution of patients in the different Markov states after the first year. To obtain the transition probabilities for the Markov model, data beyond the duration of the 5C trial were required. By following the methods used to derive the transition probabilities which had been previously published and validated in some trial-based economic analyses , the traThe outcomes of each treatment strategy were quantified in terms of QALYs over a 10-year horizon, as noted previously. To calculate QALYs, the utility weights were multiplied by the duration in each health state. An annual utility was assigned for each health state in the study.Utility values describe the health-related QOL correlated with different health states on a scale of zero to one, where zero and one represent death and best imaginable health, respectively. The baseline utility values for patients of event-free during 1-year follow-up in CHM plus conventional treatment arm and conventional treatment alone arm were taken from 5C trial, in which health-related QOL was assessed at 1 year after PCI using EuroQol (EQ-5D). EQ-5D scores were derived using Japanese population tariff values . Due to For the 2 strategies, CHMs plus conventional treatment versus conventional treatment alone, we calculated QALYs and considered the strategy associated with a higher value to be preferred. Since our model was based on a number of assumptions and weighted average of published literature-derived probabilities, we performed univariate sensitivity analyses, in which we allowed any one of the variables of the model to vary at a time according to its estimates range, to determine whether and how plausible parameters in these assumptions and risks would alter our findings .The Markov model was designed and all analyses were performed with TreeAge Pro Suite 2011 software package.The Markov model predicted that the discounted survival was higher in the CHMs plus conventional treatment arm when compared with the conventional treatment alone arm by 0.20% survival probability. The survival probability over the 10-year horizon was 77.49% in the CHMs plus conventional treatment arm and 77.29% in the conventional treatment alone arm, respectively .In a cohort of 1000 patients over 10 years, the CHMs plus conventional treatment, compared with conventional treatment alone, would gain 22 patients remaining event-free, prevent 7, 9, and 5 patients further suffering from nonfatal MI, nonfatal UA, and nonfatal stroke, respectively, and avoid 20 patients dying from all causes.The model predicted that participants after PCI who received CHMs plus conventional treatment would live an average of 0.405 discounted QALYs in 1-year and 5.519 discounted accumulated QALYs over 10-year horizon. And those who received conventional treatment alone would live an average of 0.396 QALYs in 1-year and 5.408 QALYs over 10-year horizon . CompariThe priority of CHMs plus conventional treatment over conventional treatment alone for all the parameters was considered in the univariate sensitivity analysis. The analysis showed that changes of every input parameter had no impact on the interpretation of the results. Thus, CHMs plus conventional treatment remained a dominant therapy over a broad range of the input parameters. The 5 most sensitive input parameters were annual mortality risk for event-free patients, annual risk of nonfatal UA for event-free patients, annual risk of nonfatal stroke for event-free patients, annual risk of nonfatal MI for event-free patients, and disutility of UA. The annual mortality risk for event-free patients (varied from 0.014 to 0.033) had the largest influence on the QALYs .In the present study, a two-component decision-analytic model approach was used to predict the short- and long-term effectiveness of CHMs plus conventional treatment and conventional treatment alone in the treatment of ACS after PCI. The results showed that CHMs plus conventional treatment would reduce the risk of death in ACS participants after PCI over 10-year period compared to conventional treatment alone, as well as the risk of nonfatal MI, nonfatal stroke, and nonfatal UA. Comparing with conventional treatment alone, CHMs plus conventional treatment would save 0.111 QALYs for the time horizon of 10 years. Given a cohort of 1000 patients over 10 years, the CHMs plus conventional treatment, compared with conventional treatment alone, would gain 22 patients benefit from no events and prevent 20 patients from all-cause death.As far as we are concerned, this is the first study that has accounted for QOL and generated QALYs in estimating the long-term effectiveness of CHMs plus conventional treatment in ACS patients after PCI compared with conventional treatment alone. The study showed that the estimated gain with CHMs plus conventional treatment, compared with conventional treatment alone, was accrued due to an increase in survival probability and QOL as well. And the reduction in mortality and the increase in remaining event-free were the majority contributors to the survival probability. The favorable effectiveness of CHMs plus conventional treatment in our analysis was supported by earlier clinical studies, showing that CHMs plus conventional treatment were expected to reduce the risk of MI and improve myocardial reperfusion after PCI in patients with AMI during 3 months , 17, as In the present study, the input parameters were derived from 5C trial and medical literatures. Over the first year, transition probabilities and baseline utility for patients in CHMs plus conventional treatment and conventional treatment alone arms were mainly taken from 5C trial. For some parameters, however, no data in 5C trial were available, so we used international data instead. In the absence of long-term data after 1 year , the transition probabilities were obtained based on the collected data from registry-based studies, analyses of randomized controlled trials, and systematic reviews. Additionally, there were no previous published evidences of disutility values for patients experiencing MI, UA, or stroke after PCI in China; we used data from a published study  to perfoIn 5C trial, the intervention period of CHMs plus conventional treatment was only 6 months and there was no intervention difference in the subsequent 6 months between CHMs plus conventional treatment and conventional treatment alone. Our model made the conservative assumption that there was no incremental clinical benefit from CHMs plus conventional treatment versus conventional treatment alone beyond the first year of treatment; that is, the benefits of CHMs only worked in 6 months and beyond 1 year the transition probabilities were identical for both treatment arms. Advantages of using the decision-analytic model approach in long-term effectiveness evaluation are to be able to extend analyses beyond trial durations, to integrate data from a variety of sources, and to be able to explore the impact of the therapy in various treatment settings , 21. TheOur study has several limitations. First, the study was performed based on a decision-analytic Markov model which was a simplification of reality, where probability data of outcomes occurred in another population and different scenarios from that of 5C trial were inevitably employed, although we used a resource estimate adapted to our reality . The datOn the basis of the decision-analytic Markov model, the analysis suggested that treatment with CHMs, as an adjunctive therapy, in combination with conventional treatment for 6 months might improve the long-term clinical outcome in ACS patients after PCI. However, the larger long-term clinical trials are needed to prove the long-term effectiveness of CHMs plus conventional treatment in the treatment of ACS after PCI in the future.