Mineral nutrition during wheat grain development has large effects on wheat flour protein content and composition, which in turn affect flour quality and immunogenic potential for a commodity of great economic value. However, it has been difficult to define the precise effects of mineral nutrition on protein composition because of the complexity of the wheat flour proteome. Recent improvements in the identification of flour proteins by tandem mass spectrometry (MS/MS) and the availability of a comprehensive proteome map of flour from the US wheat Butte 86 now make it possible to document changes in the proportions of individual flour proteins that result from the application of mineral nutrition.Triticum aestivum 'Butte 86' were grown with or without post-anthesis fertilization (PAF) and quantitative 2-dimensional gel electrophoresis (2-DE) was used to analyze protein composition of the resulting flour. Significant changes in the proportions of 54 unique proteins were observed as a result of the treatment. Most omega-gliadins, high molecular weight glutenin subunits (HMW-GS) and serpins as well as some alpha-gliadins increased in proportion with PAF. In contrast, alpha-amylase/protease inhibitors, farinins, purinins and puroindolines decreased in proportion. Decreases were also observed in several low molecular weight glutenin subunits (LMW-GS), globulins, defense proteins and enzymes. The ratio of HMW-GS to LMW-GS in the flour increased from 0.61 to 0.95 and the ratio of gliadins to glutenins increased from 1.02 to 1.30 with PAF. Because flour protein content doubled with PAF from 7 to 14%, most protein types actually increased in absolute amount (μg/mg flour protein). Data further suggest that flour proteins change with PAF according to their content of sulfur-containing amino acids Cys + Met.Plants of A 2-DE approach revealed changes in the wheat flour proteome due to PAF that are important for flour quality and immunogenic potential. The work forms a baseline for further studies of the effects of environmental variables on flour protein composition and provides clues about the regulation of specific flour protein genes. The study also is important for identifying targets for breeding programs and biotechnology efforts aimed at improving flour quality. Each year wheat farmers make strategic decisions about the amount and timing of fertilizer applications that influence yield, grain quality, and economic returns for this major world crop. Such decisions may also impact local water and air quality. One well-documented effect of fertilization with nitrogen (N) is the increase in protein content. However, this depends in a complex way upon genotype, environment, timing and type of N application -7. ThereWheat flour protein composition is complex . The majFractionation studies showed that additional N was differentially partitioned into gliadins and glutenins, which increased in amount per mg of flour, compared to an albumin/globulin fraction that did not increase -19. ProtA recent 2-DE MS/MS study of proteins in white flour from Butte 86, a US spring wheat cultivar, identified 157 distinct proteins in 233 spots that together comprised 93% of the flour protein . The 157The effects of PAF on grain yield and flour properties were measured for grain harvested at maturity Table . Bushel Total protein was extracted from white flour from grain produced with and without PAF and analyzed by quantitative 2-DE Figure . Gel patAmong the spots that increased with PAF were 13 spots identified as omega-gliadins, 11 spots identified as HMW-GS, eight spots identified as alpha-gliadins, seven spots identified as serpins, five spots identified as LMW-GS, and one spot identified as gamma-gliadin Figure . AdditioAmong the spots that decreased significantly with PAF were 19 identified as alpha-amylase/protease inhibitors, 11 as LMW-GS, six as gamma-gliadins, six as farinins, six as purinins, five minor spots identified as globulins, five minor spots identified as HMW-GS, two spots identified as beta-amylase, two spots as chitinase and two as puroindoline Figure . Single In the Dupont et al study , the samGli-D3 omega-gliadins to 161% for the Gli-B3 omega-gliadins. The Gli-B3 omega-gliadins are of particular interest because they have been associated with the serious food allergy wheat-dependent exercise-induced anaphylaxis (WDEIA). Omega-gliadins that contain single cysteine residues and are likely incorporated into the glutenin polymer (omega-gliadin Cys type TC_262777) also increased in SV from 0.5 to 1.2, a 148% change. Combined spots associated with each of the five HMW-GS sequences also increased significantly in volume. SVs for individual HMW-GS types ranged from 1.5 to 3.9 in the absence of PAF and 2.3 to 5.0 with PAF , one involved in glycolysis (triose phosphate isomerase) and one involved in defense (chitinase). Our results are more detailed and precise but are in general agreement with previous studies that distinguished among protein types based on protein fractionation, banding patterns in SDS-PAGE and retention times in RP-HPLC. Most studies separated glutenin, gliadin and albumin/globulin or amphiphilic fractions before quantifying them, as it has been difficult to distinguish among these proteins in total flour extracts. However, protein fractionation introduces a trade-off between the purity of each fraction and the amount of protein recovered. Also, a single protein may partition into more than one fraction, and environmental treatments may alter partitioning of proteins among fractions. In contrast, the present study measured the effects of PAF on individual, genetically identifiable proteins without subjecting them to fractionation and thereby provides a valuable overview of the effect of PAF on the major components of the flour.Gli-B3 omega-gliadins  and the serpins while those that decreased include the defense proteins LTP, chitinase, the alpha-amylase inhibitor WTAI-CM3, and thaumatin-like protein. Globulin-2 also has substantial similarity to known food allergens in other plants [It is notable that a number of the proteins that changed in proportion with PAF are likely food allergens. Proteins designated as food allergens in the Allergome database  that incr plants . Thus, tr plants . InteresWhen considered in terms of absolute protein amounts, it is clear that the μg protein per mg flour increased for all gluten proteins as well as for many of the other protein classes. The gliadins and glutenins have a high content of Gln and Pro and serve as efficient reservoirs for storing seed N. As the N-supply increased, the synthesis of gliadins and glutenins increased, particularly ones that were low in S. This is consistent with the observed increase in the ratio of N to S in the flour. The response of the farinins to PAF was very different from that of the major gluten proteins. The farinins have moderately high levels of Gln and Pro (31-35%) and structural similarities to the major wheat gluten proteins. Yet their absolute amounts did not increase with PAF. Instead, their response to PAF was similar to that of the high S alpha-amylase/protease inhibitors.The mechanisms by which wheat flour proteins respond to PAF are undoubtedly complex. Overall, it appeared that the response to PAF was related to the S content of the proteins with low S proteins showing the greatest increase with PAF. However, individual proteins within each class were accumulated to different levels, suggesting different basal levels of expression of the corresponding genes. Additionally, there was a differential response to PAF among individual proteins within certain classes, most notably the alpha-gliadins and LMW-GS. For the omega-gliadins, changes in protein levels are in accord with the results of transcript studies in Butte 86 ,32. In pWhile 2-DE is a powerful method for evaluating changes in flour protein profiles in response to specific treatments, the data is complex and needs to be considered on several levels. Frequently, 2-DE studies report changes in individual spots that are subsequently identified by MS/MS. This approach reveals only part of the overall picture. Recent studies indicate that the same protein sequence can be distributed among several different 2-DE spots ,36-39 anMillers and bakers encourage scientists to produce cultivars with increased stability of flour quality across years and growth environments. However, to select targets for breeding programs or biotechnology efforts it is important to first identify the genes and proteins that contribute to variability in quality. The current study used quantitative 2-DE to address the effects of PAF on the entire array of abundant flour proteins. The results indicate that omega-gliadins are very sensitive to PAF and could be a major source of environmental variability. HMW-GSs, serpins, certain alpha-gliadins and LMW-GSs also appear to contribute to this variability. The study is essential for predicting the effects of agronomic inputs on flour composition and establishes the foundation for future work aimed at deciphering the complex effects of temperature and drought on the wheat flour proteome.Triticum aestivum 'Butte 86' were grown in a climate-controlled greenhouse with 16 hr days and 8 hr nights at 24°C maximum daytime and 17°C minimum night-time temperatures [-1 Plantex fertilizer , at approximately 0.1 g each of N, P and K per pot per day. Plants were grown in 36 pots of 25 cm diameter at a density of seven plants per pot. At anthesis, pots were divided into six groups. Three sets of pots were flushed with water to remove remaining NPK and subsequently were hand-watered without fertilizer. The other three sets of pots received drip irrigation with NPK fertilization, at approximately 0.3 g each of N, P and K per pot per day. Sets with and without fertilizer were alternated spatially in the greenhouse, and pots within a set were rotated weekly. Pots were weighed daily and water added to maintain the pots at 80% of water capacity. Grain was harvested at maturity and samples were milled to white flour with a Brabender Quadrumat Junior  at the Hard Winter Wheat Quality Laboratory  and tested for mixing and baking quality as previously described [Plants of the US hard red spring wheat eratures . Prior tescribed . SubsequThree separate samples of flour from each biological replicate were extracted and analyzed by 2-DE  as described in detail previously ,40,41. B® 9.2. Cary, NC: SAS Institute Inc.). Means and standard deviations for all proteins were similar among biological replicates. Probabilities from an F-test comparing means of the two treatments were calculated, both assuming that the variances were similar enough to be pooled for both treatments (4 error degrees of freedom) or differed enough between the treatments that pooling would not be appropriate (2 error degrees of freedom). An F-test was used comparing the two error terms to determine which mean comparison test was more appropriate. A Bonferroni adjustment to the F probability was also made to account for the increased likelihood of chance significance in a large set of tests. Similar calculations were made for the sums of the SVs for specific protein sequences and specific protein types, in which the average SVs for each spot for a biological replica were summed and then these sums for the three biological replicas were used in computing the analysis of variance for each protein sequence or protein type. Appropriate calculations are shown in Additional files The experimental plan included two treatments and three biological replicas, obtained from the three sets of six individual pots for each treatment. For each of the resulting six flour samples, 2-DE was performed in triplicate. The 2-DE analysis software computed the SV for each spot as the average of the three replicate gels. These average SVs for the three biological replicates and two treatments were then subjected to analysis of variance for each spot with SAS software (SAS Institute Inc. 2009. SAS OnlineDocThe absolute amount of each protein per mg of flour was estimated by multiplying SV by total protein per mg of flour. Proportions of Cys, Met, Lys, Pro and Gln were calculated as the percentage of total amino acids in the mature proteins using published sequences from Butte 86 -30 or se2-DE: 2-dimensional gel electrophoresis; HMW-GS: high molecular weight glutenin subunit; LMW-GS: low molecular weight glutenin subunit; MS/MS: tandem mass spectrometry; N: nitrogen; NPK: nitrogen, phosphorus, potassium 1:1:1 fertilizer; PAF: post-anthesis fertilization; RP-HPLC: reverse phase-high pressure liquid chromatography; S: sulfur; SDS-PAGE: SDS polyacrylamide gel electrophoresis; SV: normalized spot volume.The authors declare that they have no competing interests.FD and SA designed the study, analyzed and interpreted the data and drafted the manuscript; CT carried out the quantitative 2-DE gel analysis; WH participated in the 2-DE gel analysis; LW advised on experimental design and performed statistical analysis and interpretation; WV provided the original MS/MS analysis that was essential to the project. All authors read and approved the manuscript.Statistical analysis for all individual 2-DE spots.Click here for fileStatistical analysis for summed spot volumes for each unique protein sequence.Click here for fileStatistical analysis for summed spot volumes for each protein class.Click here for fileProportions of key amino acids in flour protein types based on the sequences of the mature proteins.Click here for file


Behavioral, anatomical, and gene expression studies have shown functional dissociations between the dorsal and ventral hippocampus with regard to their involvement in spatial cognition, emotion, and stress. In this study we examined the difference of the multisynaptic inputs to the dorsal and ventral dentate gyrus (DG) in the rat by using retrograde trans-synaptic tracing of recombinant rabies virus vectors. Three days after the vectors were injected into the dorsal or ventral DG, monosynaptic neuronal labeling was present in the entorhinal cortex, medial septum, diagonal band, and supramammillary nucleus, each of which is known to project to the DG directly. As in previous tracing studies, topographical patterns related to the dorsal and ventral DG were seen in these regions. Five days after infection, more of the neurons in these regions were labeled and labeled neurons were also seen in cortical and subcortical regions, including the piriform and medial prefrontal cortices, the endopiriform nucleus, the claustrum, the cortical amygdala, the medial raphe nucleus, the medial habenular nucleus, the interpeduncular nucleus, and the lateral septum. As in the monosynaptically labeled regions, a topographical distribution of labeled neurons was evident in most of these disynaptically labeled regions. These data indicate that the cortical and subcortical inputs to the dorsal and ventral DG are conveyed through parallel disynaptic pathways. This second-order input difference in the dorsal and ventral DG is likely to contribute to the functional differentiation of the hippocampus along the dorsoventral axis. The hippocampus, well known for its critical involvement in episodic memory and spatial navigation The main connections of the hippocampus have been reported to show a topographical organization that is in line with these behavioral dorsoventral differences. The best documented are the reciprocal connections of the hippocampus with the entorhinal cortex, where it has been shown that a dorsolateral to ventromedial entorhinal origin of projections maps onto a dorsoventral termination of those projections. This longitudinal organization of these inputs is characteristic of the projections to all subfields of the hippocampus Viral tracers that can be transferred retrogradely across synapses, such as rabies virus or pseudorabies virus, allow this question to be addressed efficiently and with a high level of precision in vivo infectious experiments. All experiments were approved by the Center for Laboratory Animal Research, Tohoku University, and all experiments were conducted according to the Guidelines of the National Institutes of Health and the Tohoku University Guidelines for Animal Care and Use.A total of 19 young adult male Wistar rats weighing 200–230 g were used in this study. All experiments with injections of recombinant rabies virus vectors were carried out in a special laboratory (biosafety level 2) designated for Rats were deeply anaesthetized with ketamine  and xylazine  and were mounted in a stereotaxic frame. The skull was exposed, and a small burr hole was drilled above the injection site. The viral vectors we used as tracers were injected at different dorsoventral levels of the dentate gyrus, levels based on the atlas of Paxinos and Watson 8 FFU/ml) or 100–200 nl of rHEP5.0-CVSG-EGFPx2 8 FFU/ml) into the dorsal DG. Seven rats received injection of either 100 nl of rHEP5.0-CVSG-mRFP (7.0×108 FFU/ml), 100–200 nl of rHEP5.0-CVSG-EGFPx2 (5.0×108 FFU/ml), or 200 nl of rHEP5.0-CVSG-LynVenusx2 8 FFU/ml) into the ventral DG. In another five rats, 100–200 nl of rHEP5.0-CVSG-mRFP (7.0×108 FFU/ml) was injected into the dorsal DG and 100–200 nl of rHEP5.0-CVSG-EGFPx2 (5.0×108 FFU/ml) was injected into the ventral DG. In two rats, 200 nl of rHEP5.0-CVSG-mRFP (7.0×108 FFU/ml) and 200 nl of rHEP5.0-CVSG-EGFPx2 (5.0×108 FFU/ml) were injected into adjacent sites (AP −3.7 and AP −5.2) of the dorsal DG. Each virus was injected along with 1% of pontamine sky blue in order to mark the injection site. After the injection, at 20 nl per minute, the pipette was left in place for another 30 minutes before it was withdrawn. After all injections were completed, the wound was sutured and the animal was monitored for recovery from anesthesia before being returned to its home cage. Throughout the survival times the rats were kept inside a small safety cabinet.Nine rats received injection of either 100 nl of rHEP5.0-CVSG-mRFP After survival periods ranging from 3 to 5 days, the animals were deeply anaesthetized with sodium pentobarbital  and perfused transcardinally with 10% sucrose in 0.1 M PB followed by 4% paraformaldehyde in 0.1 M phosphate buffer. The brains were removed from the skulls, postfixed in 4% paraformaldehyde in 0.1 M PB for 4 hours at 4°C and then cryoprotected in PB containing 30% sucrose for at least 48 hours at 4°C. The brains were cut into 50 µm coronal sections on a freezing microtome, and sections for processing were collected in four equally spaced series.In order to visualize the infected neurons in the single-virus injection samples, one series of sections was immunohistochemically processed for the expressed fluorescent protein . Floating sections were washed in phosphate-buffered saline (PBS), soaked with PBS containing 5% normal goat serum and 0.1% Triton-X 100 for an hour at room temperature, and then incubated overnight at 4°C with rabbit anti-RFP antibody  or rabbit anti-GFP antibody  dissolved in PBS containing 5% normal goat serum and 0.1% Triton-X 100. The sections were then incubated for 2 hours at room temperature in biotinylated goat anti-rabbit IgG antibody  diluted in PBS containing 0.1% Triton X-100 (PBT), after which they were reacted with the avidin-biotin-peroxidase complex  for another 4 hours. For visualization of the antigen, the sections were reacted in PBS containing 0.04% diaminobenzidine (DAB) and 0.002% hydrogen peroxide.After washes in PBS the sections were mounted on gelatin-coated glass slides, air-dried, dehydrated in ethanol, cleared in xylene, and coverslipped with mounting medium . The second series of sections was Nissl-stained with thionin and used to establish cytoarchitectonic borders. Sections were examined under an Olympus Provis AX70 microscope (Olympus) and photographed using an AxioCam MRc 5 Zeiss digital camera (Carl Zeiss) and Axiovision image processing software (Carl Zeiss).A double-labeling immunofluorescence procedure was used to visualize mRFP- and EGFP-labeled neurons in the dual-virus injection samples. After being washed in PBS, floating sections were immersed in PBS containing 5% normal goat serum and 0.1% Triton-X 100 for an hour at room temperature and then incubated overnight at 4°C with rabbit anti-mRFP IgG  and mouse anti-GFP IgG  dissolved in PBS containing 5% normal goat serum and 0.1% Triton-X 100. They were then washed and permeabilized in PBT and incubated for 2 hours at room temperature in Cy3-conjugated goat anti-rabbit IgG  and Alexa488-conjugated goat anti-mouse IgG  diluted in PBT. The sections were counterstained with Hoechst 33258 , coverslipped, examined under a Zeiss Axiovert 200 M microscope, and photographed using an AxioCam MRm digital camera (Carl Zeiss) and Axiovision image processing software (Carl Zeiss). To examine the dual-labeled neurons, images were captured at a fixed Z-level by using a laser scanning confocal microscope .To compare the topographical labeling patterns of neurons innervating either the dorsal or ventral DG, the center of mass of the labeled neurons was obtained by averaging their positions. The results from different animals were superimposed onto a coronal atlas by using anatomical landmarks to normalize the positions of the labeled neurons. To examine the center of mass of labeled neurons in the piriform cortex and endopiriform cortex along the rostrocaudal axis, the positions of labeled neurons from four coronal sections  were averaged.All numerical data are expressed as mean values ± the SEM. The statistical significance of differences between means along the dorsoventral, mediolateral or rostrocaudal axes was evaluated by using a two-tailed Student's t-test performed using Prism .To investigate multi-synaptic input to dorsal and ventral levels of the DG, we injected recombinant rabies virus vectors at different dorsoventral levels of the DG. The locations of the injections in four representative cases are shown in For the dorsal DG injections the virus was injected either at AP −3.7  or at APTo determine the first- and second-order projection area, we first injected recombinant rabies virus vectors  into either the dorsal or the ventral DG and examined the distribution of labeled neurons at different post-inoculation survival times. With these viral vectors the optimal survival time for labeling first-order projection neurons is three days. It takes another 2 days for the vector to cross one synapse and infect the second-order projection neurons Three days after an injection into the dorsal (N = 4) or ventral DG N = 3), many labeled neurons were seen in CA3, the entorhinal cortex, the medial septum, the diagonal band, and the supramammillary nucleus , 3. In t, many laTo compare the topographical organization of mono- and disynaptic inputs to the dorsal and ventral DG, we injected two recombinant rabies virus vectors expressing different fluorescent proteins into the brains of two animals. The mRFP expressing virus (rHEP5.0-CVSG-mRFP) was injected into the dorsal DG, and the EGFP expressing virus (rHEP5.0-CVSG-EGFPx2) was injected into the ventral DG. After a survival time of five days, the distribution of labeled neurons was similar to what was seen in case of single injections in either the dorsal or ventral DG . SimilarTo statistically evaluate whether these topographical labeling patterns are consistent in the single- and dual-virus injection samples, we performed a center of mass analysis and determined the center of the inputs to the dorsal and ventral DG . OverallSecond-order labeling was prominent in a number of cortical and subcortical brain areas. The distribution patterns of the two differently labeled populations of neurons in case of all second-order inputs showed striking conservation of the topographical organization described for the first-order inputs. This was similar to what was noted in case of the single-virus injections into the DG. In the anterior part of the brain, many labeled neurons were seen in the anterior piriform cortex, the posterior piriform cortex, the endopiriform nucleus, the claustrum, and in the posterior cortical nucleus of amygdala, all thought to be second-order input areas of the DG , Fig. 8.At these anterior levels we further saw retrogradely labeled neurons of both colors in the medial prefrontal cortex . In the Finally, large numbers of labeled neurons were seen in the medial raphe nucleus, the interpeduncular nucleus, the medial habenular nucleus, and in the lateral septum. In the median raphe nucleus, neurons in medial positions were labeled by GFP, while more laterally positioned neurons were labeled by RFP . In the Similar to what was seen in the first-order projection areas, only few double-labeled neurons were seen at the border of the two populations of GFP- and RFP-labeled neurons in these brainstem areas. The relatively sparse presence or even absence of double-labeled cells following injections of the two rabies vectors in the dorsal and ventral hippocampus is taken to be caused by a topographical organization of both first- and second-order inputs. However, the lack of double-labeled neurons may result from competition between the two vectors In this study we not only identified brain areas that provide second-order inputs to the dentate gyrus (DG) but, more important, found that these disynaptic inputs to dorsal and ventral levels of the DG showed a topographical organization maintaining that of the monosynaptic inputs to the DG In both first- and second-order input areas, we mostly observed single-labeled neurons, even if the two populations showed some overlap. The relative sparseness of double-labeled cells is interpreted as supporting the topographical organization of both first- and second-order inputs. Although two viral vectors may fail to double-infect neurons because of competition, thus resulting in a false impression of topography The retrograde cell labeling that was noted in CA3, the entorhinal cortex, the medial septum, the diagonal band, and the supramammillary nucleus three days after injection in either the dorsal or ventral DG probably represents direct inputs to the DG. This conclusion is consistent with the results of tracing studies using more conventional  anterograde or retrograde tracers Other subcortical areas that according to our data send second-order projections to the DG are the lateral septum and the medial habenula, both structures that project to the medial septum Our most striking observation with respect to the second-order inputs to the DG is that they all show a clear topographical organization that seems to reflect that of the first-order projections . The firIn the medial septum, medially located neurons project to the dorsal DG and laterally positioned neurons project more ventrally. In the diagonal band this projection pattern is reversed and the neurons that project ventrally are ventromedial to those that project dorsally. The second-order inputs mediated through the medial septum-diagonal band complex include those originating in the lateral septum In the supramammillary nucleus, laterally located neurons project to the dorsal DG, while medially positioned neurons project more ventrally. This topographical projection patterns is in accordance with a previous study The connectional differences along the long axis of the dentate gyrus are complemented by connectional gradients in the connectivity of the other hippocampal subfields as well as differences in intrinsic connections For some of those multisynaptic routes, differences have been reported along the axes observed in the present study. These functional differences seem to focus around two main themes, and for both the topographies observed in first- and second-order input regions are in accordance with each other. First, the dorsal and ventral hippocampus differ with respect to the firing characteristics of neural populations in that theta power decreases from dorsal to ventral Second, dorsal and ventral parts of the hippocampus differ with respect to the overall quality of the information they process. The dorsal hippocampus apparently interacts with brain structures that are part of the realm that processes exteroceptive information. The ventral hippocampus, in contrast, through its first order connected structures, is strongly implicated in motivational and reward systems of the brain. The present data indicate that this focus is also reflected in the second-order inputs. The medial part of posterior piriform cortex receives strong input from the amygdaloid complex The topographically distributed labeling in the lateral septum mirrors the dorsoventral distributions of hippocampal projections to the lateral septum. This organization has been suggested to reflect a functional relationship with functionally different hypothalamic domains, such that the ventral hippocampus is connected with hypothalamic nuclei involved in the modulation of endocrine and autonomic responses, whereas the dorsal hippocampus is connected with hypothalamic domains relevant for exploratory behavior and behavioral arousal In conclusion, we show that the topographical organization of the two different connectional chains into the dorsal and ventral DG is consistent for both first-order and second-order inputs and fits with reported and hypothesized functional differences along the hippocampal dorsoventral axis. Recent findings that the dorsoventral differentiation in the hippocampus is established early during development


Gene-gene interactions may be partly responsible for complex traits such as obesity. Increasing evidence suggests that the renin-angiotensin system (RAS) contributes to the etiology of obesity. How the epistasis of genes in the RAS contributes to obesity is still under research. We aim to evaluate the contribution of RAS-related gene interactions to a predisposition of obesity in a Chinese population.AGT), angiotensin converting enzyme (ACE), angiotensin type 1 receptor (AGTR1), MAS1, nitric oxide synthase 3 (NOS3) and the bradykinin B2 receptor gene (BDKRB2), and genotyped them in 324 unrelated individuals with obesity (BMI ≥28 kg/m2) and 373 non-obese controls (BMI 18.5 to <24 kg/m2) from a large scale population-based cohort. We analyzed gene-gene interactions among 6 polymorphic loci using the Generalized Multifactor Dimensionality Reduction (GMDR) method, which has been shown to be effective for detecting gene-gene interactions in case-control studies with relatively small samples. Then we used logistic regression models to confirm the best combination of loci identified in the GMDR. It showed a significant gene-gene interaction between the rs220721 polymorphism in the MAS1 gene and the rs1799722 polymorphism in the gene BDKB2R. The best two-locus combination scored 9 for cross-validation consistency and 9 for sign test (p = 0.0107). This interaction showed the maximum consistency and minimum prediction error among all gene-gene interaction models evaluated. Moreover, the combination of the MAS1 rs220721 and the BDKRB2 rs1799722 was associated with a significantly increased risk of obesity .We selected six single nucleotide polymorphisms (SNPs) located in angiotensin (These results suggest that the SNPs from the RAS-related genes may contribute to the risk of obesity in an interactive manner in a Chinese population. The gene-gene interaction may serve as a novel area for obesity research. It has been argued that gene-gene interaction is a ubiquitous component of the genetic architecture of common human diseases, such as obesity Increasing evidence suggests that the renin-angiotensin system (RAS) contributes to the etiology of obesity. The renin-angiotensin system is involved in adipocyte growth and differentiation and possibly in adipose tissue metabolism. Hyperactivity of systemic and tissue-specific RAS is related to increased propensity to obesity The study was conducted with the approval from the Ethics Committee of Beijing Tongren Hospital, Capital Medical University. Written informed consent was obtained from each participant.2 were defined as obese, while 373 subjects with BMI from 18.5 to <24 kg/m2 were chosen as the group of control individuals. Both groups had comparable blood pressure, serum creatinine, fasting plasma glucose (FPG) and age. Subjects with secondary obesity, coronary heart disease and chronic kidney disease were excluded from the study.Study subjects were selected from a completed large scale population-based cohort (the Beijing Community Pre-Diabetes Study) designed to facilitate the conduct of genetic epidemiology investigations and clinical trials on individuals originating from the settled community of Nanfaxin, a satellite rural town of Beijing. Demographic data were depicted in details in our published paper Biochemical parameters were measured by Beckman-Unicel Dxc800 biochemistry analysator  including FPG, total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL) cholesterol, alanine aminotransferase (ALT) and creatinine (Cr).Genomic DNA was extracted from peripheral blood leukocytes using standard methods. This set of SNPs was chosen based on the following criteria: HapMap validation status, functional relevance and importance The genotyping was performed using primer extension of six loci. Detection was completed by a matrix-assisted laser desorption/ionisation time-of-flight mass spectroscopy using a MassARRAY platform . 5% of the samples were duplicated to evaluate the genotyping concordance rate. Genotypes between duplicates were concordant for all six SNPs, and no evidence of departure from Hardy-Weinberg equilibrium was observed in control subjects using chi-squared-test analysis.Statistical analysis was performed using SPSS version 13.0 for Windows . Data are presented as numbers or means ± SD as appropriate. The between-group data were compared with Student's unpaired t-test for continuous data and with the chi-squared-test for categorical data. Allele frequencies were calculated from the genotypes of the subjects. Differences in allele and genotype frequencies between cases and controls were compared with a chi-squared-test or a Fisher's exact test.http://www.multifactordimensionalityreduction.org). All possible two-, three-, four- and five-way SNP interactions were tested using 10-fold cross-validation in an exhaustive search. The MDR method is described in detail elsewhere To identify higher order gene-gene interactions in our samples, we used the MDR software 2.0-beta , the maximum cross-validation consistency, and scored 9 for the sign test at the 0.0107 level. The two-locus genotype combinations were classified into high- or low-risk groups. In the chi-squared test, the OR of high-risk combination of the two-locus model increased the risk of obesity by 1.82 times .The GMDR was used to assess the contribution of combinations of the six variants to the etiology of obesity including the age and sex as the covariates. MAS1 rs220721 and BDKRB2 rs1799722, as well as a marginal significant three-locus model involving MAS1 rs220721, AGT rs699 and NOS3 rs1799983. Overall, the two-locus model had 10 cross-validation consistency, the highest testing balance accuracy of 0.5681 and 10 for the sign test (p = 0.001), which was the same best model as that which was identified with BMI as two-classified variable. The two-locus model was thus chosen as the best model.In addition, BMI as the continuous variable was analyzed to explore the relationship with the combination of the six variants . With coSince the GMDR was based on the MDR, analysis from the MDR was also performed. The accuracy of the two-locus model using the same SNPs without co-variable adjustment was similar to that with adjustment see .MAS1 rs220721 variant was significantly and positively associated with the predisposition of obesity under both the allelic genetic model , and the recessive genetic model  even after adjusting for other confounders (age and sex or plus smoking and FPG) . Comparison of the genotype combinations of MAS1 rs220721 GG and BDKRB2 rs1799722 CC, an increased number of risk alleles correlated with a significantly increased risk of obesity . There was a non-significant gene-gene interaction of deviation in a multiplicative manner in the logistic regression model.To further classify the interactions of genetic variants on the disposition of obesity, the following analysis was explored on the basis of two variants identified by GMDR. and FPG) . Under bMAS1 and BDKRB2 conferred the genetic susceptibility to obesity in the Chinese population. We consider the present study to be explorative and hypothesis-generating and thus find the results intriguing. However, a more convincing conclusion can only be reached by further independent replication of the results reported in this investigation.Obesity is a complex disease, which is influenced by genetic and environmental factors. Family and twin studies have estimated that 40–70% of the variance in obesity-related traits is attributed to genetic factors MAS1 rs220721 and BDKRB2 rs1799722 was the best model, irrespective of whether a dichotomous (obesity) or continuous (BMI) variable was measured, adjusting for the covariates. This SNPs combination increased the risk of obesity by 1.82 times, which inferred that the gene-gene interaction play a role in the vulnerability of obesity in our study population. In this research, differences were observed in the distribution of polymorphic variants of MAS1 and BDKRB2 between obesity patients and controls. The results of the logistic regression further show that an increased number of risk alleles correlates with a significantly increased risk of obesity, in comparison with the genotype combinations of MAS1 rs220721 GG and BDKRB2 rs1799722 CC. However, the susceptibility interaction was not confirmed by the logistic regression analysis. The possible reason for these inconsistent results is that GMDR did not detect the interaction defined by “deviation from the multiplicative” in the logistic regression model. The significant results from GMDR only showed that the combination of different loci may increase or decrease the risk of obesity and the interaction among the loci may refer to either multiplicative, deviation from the multiplicative or departure from additivity In recent years, more and more studies have focused on gene-gene interactions Activation of the RAS in adipose tissue has been implicated in the regulation of adiposity and overt obesity through its ability to increase fat cell growth and differentiation, increase synthesis, uptake and storage of fatty acids and triglycerides MAS1 and BDKRB2 is associated with the predisposition of obesity in our Chinese population. We believe these findings will guide further investigations in gene detection and may shed light on the genetic architecture of obesity.In conclusion, our study is the first to show evidence that the interaction between


Although the prevalence of arthritis dramatically increases with age, the great majority of preclinical studies concerning the mechanisms that drive arthritic joint pain have been performed in young animals. One mechanism hypothesized to contribute to arthritic pain is ectopic nerve sprouting; however, neuroplasticity is generally thought to be greater in young versus old nerves. Here we explore whether sensory and sympathetic nerve fibers can undergo a significant ectopic nerve remodeling in the painful arthritic knee joint of geriatric mice.Vehicle  or complete Freund's adjuvant (CFA) was injected into the knee joint of 27- to 29-month-old female mice. Pain behaviors, macrophage infiltration, neovascularization, and the sprouting of sensory and sympathetic nerve fibers were then assessed 28 days later, when significant knee-joint pain was present. Knee joints were processed for immunohistochemistry by using antibodies raised against CD68 (monocytes/macrophages), PECAM , calcitonin gene-related peptide , neurofilament 200 kDa , tyrosine hydroxylase , and growth-associated protein 43 .+ macrophages, density of PECAM+ blood vessels, and density and formation of neuroma-like structures by CGRP+, NF200+, and TH+ nerve fibers in the synovium and periosteum.At 4 weeks after initial injection, CFA-injected mice displayed robust pain-related behaviors , whereas animals injected with vehicle alone displayed no significant pain-related behaviors. Similarly, in the CFA-injected knee joint, but not in the vehicle-injected knee joint, a remarkable increase was noted in the number of CD68Sensory and sympathetic nerve fibers that innervate the aged knee joint clearly maintain the capacity for robust nerve sprouting and formation of neuroma-like structures after inflammation/injury. Understanding the factors that drive this neuroplasticity, whether this pathologic reorganization of nerve fibers contributes to chronic joint pain, and how the phenotype of sensory and sympathetic nerves changes with age may provide pharmacologic insight and targets for better controlling aging-related joint pain. The prevalence of arthritis generally increases with age and is frequently accompanied by significant joint pain -5. In inArthritic joint pain most frequently occurs with movement and/or loading of the affected joint but can also be present at rest ,16. MoveOne potential mechanism that may explain the dissociation between disease progression and pain in arthritis is that an active and ectopic sprouting of sensory and sympathetic nerve fibers plays a role in driving pain due to arthritis. For example, a previous study performed in young rats suggested that after injection of complete Freund adjuvant (CFA) into the temporomandibular joint of rats, significant sprouting of sensory nerve fibers occurred in the painful arthritic joint . HoweverIn this study, we examined the ability of sensory and sympathetic nerve fibers to undergo ectopic sprouting. The model used here was generated by unilaterally injecting CFA into the articular space of the knee joint of geriatric (27- to 29-month-old) female mice. With this model, we demonstrate that even in the arthritic knee joint of these geriatric mice, robust sprouting and formation of neuroma-like structures by both sensory and sympathetic nerve fibers was observed in all CFA-injected animals and in none of the vehicle-injected mice. These newly sprouted sensory and sympathetic nerve fibers were not only present in a higher density per unit area than was ever found in the normal aged joint, but were also found in areas of the joint that are normally poorly or not innervated.ad libitum. The Institutional Animal Care and Use Committee at the Minneapolis VA Medical Center approved all procedures.Experiments were performed on a total of 12 geriatric female B6/C3H mice . Mice were received at our animal care facilities 2 months before experiments were performed. At the time of initiation of the experiments, mice were 27 to 29 months old. The mice were housed in accordance with the National Institutes of Health guidelines under specific pathogen-free conditions in autoclaved cages maintained at 22°C with a 12-hour alternating light-and-dark cycle and were given autoclaved food and water A modified version of a previously validated model of arthritic inflammation of the knee joint  was prod+ monocytes/macrophages into the synovial membrane as well as tissues outside the joint, including tendons, ligaments, and surrounding muscle (data not shown). Taken together, these results suggest that the present model involves an inflammation in the synovium as well as the tendons and ligaments of the CFA-injected knee joint without signs of cartilage destruction.Currently, there is no well-accepted rat or mouse model of osteoarthritis (OA) simultaneously induces robust joint destruction and pain similar to that observed in humans with painful OA. One rodent model that has been used as a surrogate in terms of OA pain is CFA injection into the knee joint. As previous results from this model  were useBehavioral measures of arthritic joint pain included spontaneous pain (guarding and flinching), stimulus-evoked pain (limb use), and the ability of the animal to place weight on the arthritic limb versus the nonarthritic limb while ambulatory (dynamic weight bearing). In brief, the number of hindpaw flinches and time spent guarding were recorded as measures of spontaneous pain, as these measures mirror patients in a clinical setting with arthritis who have spontaneous pain (joint pain at rest) ,31. The Normal limb use during spontaneous ambulation in an open field was used as an indicator of stimulus-evoked pain and was scored on a scale of 5 to 0, as previously reported ,38, wherBehavioral measures of arthritic joint pain included spontaneous pain (guarding and flinching) and stimulus-evoked pain (limb use) were performed at day 28 after CFA initial injection, whereas dynamic weight bearing was determined at day 23 after initial injection. Not enough time was available in a single day to perform all four behaviors.Dynamic weight-bearing analysis of the arthritic limb was performed on days 0 and 23 after initial intraarticular CFA injection with the use of a Dynamic Weight Bearing device . Similar pedobarographic analysis is used clinically to determine the weight bearing of patients with arthritic knee joints . The mouM phosphate-buffered saline  followed by 30 ml of 4% formaldehyde/12.5% picric acid solution in 0.1 M PBS (pH 6.9 at 4°C). Ipsilateral and contralateral knee joints were harvested after perfusion and postfixed for at least 12 hours in the same perfusion fixative. The process of postfixing, decalcification, and sectioning of the bones/joints was performed as previously described [At day 28 after initial CFA injection, mice were deeply anesthetized with carbon dioxide asphyxiation, delivered by using a compressed gas cylinder, and perfused intracardially with 20 ml of 0.1 escribed ,41.To qualitatively assess the changes in the density and morphology of sensory nerve fibers that innervate the knee joint, the macrophage infiltration, and neovascularization of the synovium, 20 μm-thick frozen sections of the bone/joint were processed according to our previously published procedures ,40,41. FApproximately 30 separate, 20-μm-thick frozen sections were obtained from the knee joint of each mouse. Three confocal images  from different sections separated by at least 100 μm were obtained for each marker. Sections were initially scanned at low magnification (×100) to identify areas with the highest capillary or nervefiber density in the synovium. These areas in the synovium were defined as "hot spots." One image per section was acquired within the medial synovial hot spot. Whereas nerve fibers, blood vessels, and macrophages were observed throughout the inflamed synovium, neovascularization and nerve sprouting was consistently present in the synovium adjacent to the meniscus, therefore, most of the hot spots were found and quantified in this area. In the knee sections from naïve and sham mice, no evidence of "hot spots" was noted in terms of significant changes in the morphology or density of nerve fibers and blood vessels.3) [+ macrophages were quantified in each layer of the inflamed synovium from z-series images (×400 magnification) of each field of view by using Imaris Pro Software v. 6.0 . Only CD68+ cells that displayed visible nuclei, as determined by counterstaining with DAPI, were counted. Only CD68+ macrophages/monocytes located in the normal or inflamed synovium were quantified. Macrophages/monocytes were defined as CD68+ profiles that were distant from any bone surface, whereas osteoclasts were defined as CD68+ profiles that were directly apposed to mineralized bone [The volume of CFA-inflamed synovium analyzed was 315 μm (length), 315 μm (width), and 20 μm (depth). The Z-stacked images were analyzed with Image-Pro Plus v. 6.0 , and nerve fibers and blood vessels were manually traced to determine their respective lengths. To determine whether a structure in the image was a nerve-fiber profile, simultaneous observations of the image on a high-resolution computer monitor and the section viewed in the microscope were performed. The fine focus of the microscope was used to follow the fibers across the entire thickness of the knee section. Structures were considered nerve profiles only if their signal was at least 3 times greater than background signal, if their length was > 20 μm, and if they displayed the presence of dilatations (varicosities). Small dot-like structures were not counted, even though these structures likely represented cut fibers. Nerve sprouting or neovascularization was reported as density of nerve fibers or blood vessels per volume of synovium (mm/mm3) . CD68+ mzed bone ,43.+ macrophages in the synovium was reported. Total volume of the synovium (naïve and inflamed) was calculated by tracing the area of the synovium and multiplying this area by the thickness of the section (20 μm).Data from at least three slices per knee joint were recorded and averaged. The total number of CD68+ blood vessels and CD68+ monocytes/macrophages in the femoral periosteum were calculated within the distal metaphysic, as significant neural remodeling was observed in this region. The area evaluated in frozen sections was within a 1.0-mm-long region, which started 0.5 mm below the distal femoral growth plate. For frozen sections, the average volume of periosteum analyzed was 315 μm (length), 50 μm (width), and 20 μm (depth). The confocal images were viewed on a high-resolution monitor, and the length of nerve fibers was determined by manually tracing the nerve or blood vessel profile by using Image Pro Plus v. 6.0 image analysis software . Nerve sprouting or neovascularization was reported as density of nerve fibers or blood vessels per volume of periosteum (mm/mm3). Data from at least three slices per knee joint were recorded and averaged. CD68+ macrophages were quantified in both layers of the periosteum from z-series images of each field by using Imaris Pro-Software v. 6.0 . The density of CD68+ macrophages in the periosteum was reported as number of CD68+ macrophages per volume of periosteum.Density of nerve fibers, PECAM+, NF200+) that has an interlacing and/or whirling morphology, a size that is at least 10 μm thick by 70 μm long  [To determine whether changes were present in the density of nerve fibers or the formation of neuroma-like structures, frozen sections were examined at low magnification with an epifluorescent microscope. Areas that were examined included the lower half of the femur and the upper half of the tibia and included the mineralized bone, bone marrow, cartilaginous knee joint, synovium, periosteum, and knee-joint ligaments. In general, areas that contained sprouting of nerve fibers and neuroma-like structures were remarkably different from nerve fibers in the normal bone and joint in terms of morphology and density. For example, a neuroma-like structure was defined as a disordered mass of axons (CGRPl axons) -46, and l axons) ,47-49.t test was used to compare the behavioral measures of spontaneous pain (guarding and flinching) and dynamic weight bearing. The Mann-Whitney Rank Sum Test was used to compare the stimulus-evoked pain (limb use). One-way ANOVA followed by the Dunnett post hoc test was used for the immunohistochemical measures between the experimental groups. Significance level was set at P < 0.05. In all cases, the investigator responsible for behavioral testing, plotting, measuring, and counting was blind to the experimental situation of each animal.A Pain-related behaviors were evaluated in CFA-injected animals as compared with saline-treated animals. Assessment of arthritic joint pain-related behaviors, including spontaneous guarding and flinching, and limb-use analysis of the hindlimb was performed on day 28 after initial intraarticular CFA injection, and dynamic weight-bearing analysis was performed on day 23 after initial intraarticular CFA injection Figure . At day Repeated injections of CFA resulted in significant edema within the knee joint at day 28 after initial injection. In all CFA-injected mice, the inflammation was restricted to the ipsilateral joint with no evidence of inflammation present in the contralateral hindlimb.+ monocytes/macrophages into the synovial membrane  and immunonohistochemistry. At day 28 after initial injection, the repeated intraarticular injection of vehicle did not induce a visible inflammatory response in the synovium Figure  or majore Figure  and tisse Figure  of vehicy Figure .3 in vehicle-treated knees to 0.1454 ± 0.0186 mm3 on Day 28 in CFA-treated knees  as compared with vehicle-treated (365 ± 63 CD68+ profiles) or naïve mice (301 ± 42 CD68+ profiles).Quantitative image analysis revealed an increase in the volume of the synovial membrane during arthritis from 0.0044 ± 0.0003 mms Figure . A signis Figure . The CD6s Figure . When th+ , and GAP43+  revealed that CFA-induced knee arthritis resulted in a significant increase in the density of CGRP+ fibers as compared with mice treated with vehicle . Likewise, the density of NF200+ sensory nerve fibers in the femoral periosteum was significantly increased in CFA-treated mice as compared with vehicle-treated . Similarly, the density of postganglionic TH+ sympathetic nerve fibers increased significantly in the femoral periosteum of mice treated with CFA as compared with vehicle-treated mice . Finally, the density of nerve fibers undergoing sprouting (GAP43+) also increased significantly in the femoral periosteum of mice that received intraarticular injections of CFA as compared with mice injected with vehicle . The density of nerve fibers in the periosteum of vehicle-treated mice was not significantly different when compared with femoral periosteum of naïve mice (data not shown).Quantitative analysis of the density of nerve fibers  and of the nociceptive neurons of the CNS   but not changes in the density per unit areas of these inflammatory cells. Although the lack of change in macrophage/monocyte density in relation to the difference in total numbers of these cells between the groups is unknown, it is possible that that age-related loss in the macrophage infiltration and function may explain this finding [In addition to the reorganization of nerve fibers in the knee joints of the geriatric mice that received injections of CFA, we found a significant increase in the total number of CD68 finding .In general, neuroplasticity is thought to decline with age. For example, a previous study showed that the rate of axonal regeneration as determined by axonal transport of radiolabeled proteins after sciatic nerve injury is slower in 28-month-old rats as compared with 2- and 10-month old rats . In 24-mIn contrast to these studies, our results indicate that sensory and sympathetic nerve fibers that innervate the knee joints of geriatric mice not only retained the capacity to undergo a robust and inappropriate sprouting but they also formed neuroma-like structures after inflammation or injury. One potential explanation as to why the neuronal sprouting of sensory and sympathetic nerve fibers is so robust in the aged knee joint is that an age-related maintenance occurs in the synthesis and release of growth factors from skeletal tissues. In support of this hypothesis, the expression of different growth factors, including bone morphogenetic protein-2, bone morphogenetic protein-7, insulin-like growth factor-1, leptin, tumor necrosis factor-α, and transforming growth factor-β are maintained and/or increased in the intervertebral discs in aged rabbits and humans -85. Addi+, TH+, and NF200+ nerve fibers in the tumor-bearing bone, irrespective of whether the tumor synthesizes NGF [A key unknown is the factor(s) driving the sprouting and neuroma formation of sensory nerve fibers and ultimately the pain in the painful inflamed joint. Previous reports exhaustively demonstrated that in both the developing and adult animal, nerve growth factor can induce marked sprouting of sensory and sympathetic nerve fibers  and sympathetic (TH+) nerve fibers. Interestingly, these sprouting nerve fibers were present at a higher density and had unique morphology that is never observed in the normal nonpainful geriatric knee joint. Although robust sprouting and neuroma formation are clearly present at 28 days after CFA injection, it remains to be determined when this nerve sprouting and neuroma formation first begins.In this study, CFA-induced inflammation of the geriatric knee joint resulted in significant pain, robust nerve sprouting, and formation of neuroma-like structures in the synovium and periosteum by both sensory (CGRPAs sprouting and reorganization of sensory and sympathetic nerve fibers has been observed in other chronic pain states in both animals and humans ,46,57-71CFA: complete Freund adjuvant; CGRP: calcitonin gene-related peptide; CNS: central nervous system; DAPI: 4',6-diamidino-2-phenyl-indole; GAP43: growth-associated protein-43; NSAIDs: nonsteroidal antiinflammatory drugs; NF200: neurofilament 200 kDa; PBS: phosphate-buffered saline; SEM: standard error of the mean; TH: tyrosine hydroxylase; TNF-α: tumor necrosis factor-alpha.None of the authors of this study claims a conflict of interest.JMJA and PWM participated in the conception and design of the experiments, in the analysis and interpretation of the data, and in the writing of the manuscript. Both authors approved the final version of the manuscript to be published.


The aim of this research was to evaluate the economic outcomes of radiotherapy (RT), temozolomide (TMZ) and nitrosourea (NT) strategies for glioblastoma patients with different prognostic factors.A Markov model was developed to track monthly patient transitions. Transition probabilities and utilities were derived primarily from published reports. Costs were estimated from the perspective of the Chinese healthcare system. The survival data with different prognostic factors were simulated using Weibull survival models. Costs over a 5-year period and quality-adjusted life years (QALYs) were estimated. Probabilistic sensitivity and one-way analyses were performed. The baseline analysis in the overall cohort showed that the TMZ strategy increased the cost and QALY relative to the RT strategy by $25,328.4 and 0.29, respectively; and the TMZ strategy increased the cost and QALY relative to the NT strategy by $23,906.5 and 0.25, respectively. Therefore, the incremental cost effectiveness ratio (ICER) per additional QALY of the TMZ strategy, relative to the RT strategy and the NT strategy, amounts to $87,940.6 and $94,968.3, respectively. Subgroups with more favorable prognostic factors achieved more health benefits with improved ICERs. Probabilistic sensitivity analyses confirmed that the TMZ strategy was not cost-effective. In general, the results were most sensitive to the cost of TMZ, which indicates that better outcomes could be achieved by decreasing the cost of TMZ.In health resource-limited settings, TMZ is not a cost-effective option for glioblastoma patients. Selecting patients with more favorable prognostic factors increases the likelihood of cost-effectiveness. Glioblastoma (GBM) is the most common and most aggressive malignant brain tumor, and it is associated with poor prognoses 6-methylguanine-DNA methyltransferase (MGMT) methylated, gained more survival benefits Temozolomide (TMZ) is a new orally administered systemic alkylating agent that crosses the blood-brain barrier (BBB) to exert antitumor activity In this economic study, we investigated the 5-year economic outcomes of three first-line strategies for newly diagnosed patients with GBM in both overall and subgroup cohorts based on Chinese clinical practice and recommendations: radiotherapy alone, nitrosourea agents plus radiotherapy and TMZ plus radiotherapy. The follow-up times of most clinical trials have not focused on the 5-year course of the disease despite the previously mentioned paucity of head-to-head comparisons of different strategies. Thus, mathematical modeling techniques must be used to provide information for decision-making. The perspective of the Chinese healthcare system was adopted to assist in determining the direct economic value of the three different first-line strategies in newly diagnosed GMB and to compare the strategies with different willingness-to-pay thresholds per quality-adjusted life-year (QALY) gains. The analysis excluded indirect societal costs .Using the R software package , a state-transition (Markov) model for newly diagnosed GBM was developed to track the 5-year disease course. We used this model to measure and compare the 5-year direct medical costs and health outcomes for the different first-line strategies for newly diagnosed GBM. The analysis was conducted from the perspective of the Chinese healthcare system. In the model, future costs and health outcomes were not discounted because most survival outcomes among GBM patients are shorter than 2 years.Patients with different prognostic factors have significantly different survival times. Because no detailed clinical information was available, simulation methods were used to generate the survival rates of patients with combinations of different risk factors. In the current analysis, the prognostic factors that were included were age , methylation status of the MGMT promoter (methylated or unmethylated), surgery type  and treatment strategy . The Markov model developed for the study was used to analyze the cost-effective outcome for each risk subgroup cohort.Although new therapies for GBM have been evaluated in clinical trials, the most commonly used first-line strategies (after neurosurgery) are radiotherapy alone, radiotherapy plus nitrosourea, radiotherapy plus Gliadel Wafer and radiotherapy plus TMZ Parameter inputs for the model were: transition probabilities ; event proportions (which govern the ratios of events); direct medical costs ; and health state utilities . These data were derived from published studies or from local health systems.Cost-effectiveness ratios were measured to evaluate the outcomes of the different strategies. The main health outcomes were presented with respect to quality-adjusted life-years (QALY). Cost were converted into US dollar .The results are presented as an incremental cost-effectiveness ratio (ICER).A Markov decision model was used to evaluate the 5-year clinical and economic outcomes associated with GBM and its treatment. The cost-effectiveness model for glioma consists of three mutually exclusive health states: disease-free, disease progression and death . In the A hypothetical cohort that was clinically similar to the GBM patients in the EORTC-NCIC trial was entered in the model. The hypothetical patients were aged 18–70 years and had newly diagnosed and histologically proven GBM. The patients had a WHO performance status of 0–2 and adequate hematologic, renal, and hepatic functions. Following biopsy or surgical resection, they would receive one of three competing treatment strategies to manage their newly diagnosed GBM (first-line therapy): 1) radiotherapy (RT strategy); 2) radiotherapy and TMZ (TMZ strategy); or 3) radiotherapy and nitrosoureas (NT strategy). When the disease progressed, the patients were treated with a second-line therapy  or with the best supportive care (BSC). The second-line chemotherapy strategy was assumed to involve the PCV  regime Transition parameters and proportions were derived from randomized clinical trials or meta-analyses whenever possible. Two-parameter Weibull survival models were fitted to the data extracted from the Kaplan–Meier survival PFS and OS curves using R statistical software. Estimated scale and shape parameters and their standard errors (SEs) are listed in RT (EORTC-NCIC). The HR values between the RT and RT+NT treatment arms were derived from studies reported by Stewart et al. PFS and OS survival data for RT and RT+TMZ were derived from the clinical trials PFS data were absent for some subgroup cohorts. We assumed that the ratios of the hazard rate between OS and PFS in the overall cohort were equal to those of the subgroup cohorts at any time. Therefore, the absent Weibull parameters for PFS in some subgroup cohorts could be estimated by the following three steps: first, with the Weibull hazard function, we calculated the ratios of the hazard rate between OS and PFS at each cycle in the overall cohort; second, the hazard rates for PFS in the subgroup cohorts were calculated using the ratios ; finally, the hazard rates at each cycle were fitted with the Weibull hazard function to estimate the shape and scale parameters.The costs of each strategy  were est2 per day during radiotherapy (concomitant chemotherapy), 4 weeks off, and then six cycles of 150–200 mg/m2 for 5 days every 28 days were administered (adjuvant chemotherapy); 3) Nitrosoureas: nimustine (ACNU) is a type of nitrosourea 2, which was assumed to be administered intravenously once every 6 weeks until the tumor progressed. The PCV regime in the second-line treatment was administered at 8-week intervals: lomustine 110 mg/m2 taken orally on day 1, procarbazine 60 mg/m2 taken orally on days 8 to 21, and vincristine 1.4 mg/m2  administered intravenously on days 8 and 29 2The treatment costs were estimated according to the following schedules: 1) Radiotherapy: using a linear accelerator, a total dose of 60 Gy was delivered as the focal radiotherapy once daily at 2 Gy per fraction, 5 days/week; 2) TMZ: 75 mg/mThe EORTC-NCIC study showed that hematologic toxicity, gastrointestinal toxicity and infection were the main SAEs in the RT+TMZ combined strategy. Our model incorporated these treatment-related SAEs. SAE (grade≥3) management strategies were estimated based on patient records in local hospitals . We assuThe utility values for the various discrete health states were obtained from a previously published report All parameters, including rates, costs and utilities, were entered into the model with a statistical distribution: lognormal distributions were assigned to all input costs; beta distributions were assigned to the utilities, probabilities and proportions; and bivariate normal distributions were assigned to all Weibull parameters. Using these distributions, a probabilistic sensitivity analysis (PSA) based on a Monte-Carlo simulation  was performed to evaluate the impact of uncertainty across all of the parameters simultaneously. Following WHO recommendations, we used 3× per capita GDP of China /QALY and 3× per capita GDP of Shanghai City /QALY as the threshold values The base-case model compared the clinical outcomes to the results from clinical phase 3 trials. The median OS time from the trials, in addition to models for the overall cohort, and subgroups differentiated by age, MGMT methylation status and surgery status, are shown in The base case cost-effectiveness results  were bas2. This estimate was determined using a range from 1.61 to 1.83 m2. The body surface area was thus determined to be the second most substantial effect factor, leading to a change in the net health benefit from −2.15 to −2.53 QALYs. The other important effect factors of the model were the utility of the PFS in TMZ adjuvant chemotherapy and the HR for the TMZ strategy versus the RT strategy. Other factors, such as the costs of managing SAEs and incidences of SAEs, had little impact (not shown in the tornado diagram).The one-way sensitivity analyses reveal that some model parameters had a substantial impact on the net health benefit of the TMZ strategy compared to the RT strategy. The most influential parameter presented in the tornado graphs  was the The PSA comprising 1,000 simulations measured the probabilities of meeting the ICER thresholds of $11,034 and $38,376 per additional QALY for the TMZ strategy compared to RT and NT strategies for GBM patients. The results are presented in The cost-effectiveness acceptability curves (CEACs) show the preferred first-line strategies for GBM in the overall cohort and the 8 subgroups when a range of cost-per-QALY thresholds is taken into account. The CEAC plot shows that when the threshold was $11,034, the likelihood of achieving cost-effectiveness with the RT strategy might be higher than the likelihood of achieving cost-effectiveness with the TMZ and NT strategies in the overall cohort and in 7 subgroups. In the subgroup with MGMT methylated patients, the NT strategy achieved a similar probability of cost-effectiveness as the RT strategy. When the threshold was $38,376, the NT strategy achieved the maximum likelihood of cost-effectiveness in the subgroups with MGMT methylated, complete resection, partial resection, biopsy only, age<50 years and age 50∼60 years .Since TMZ was introduced as a first-line treatment in newly diagnosed GBM, survival rates and quality of life have both improved. However, the widespread use of TMZ has resulted in a dramatic increase in healthcare costs. An economic evaluation of the recommended TMZ strategy as a first-line therapy in a health resource–limited setting can help policy-makers, physicians and patients to make the proper decisions. Using decision-analytic modeling techniques, we estimated the cost-effectiveness of three first-line GBM strategies in an overall cohort and in 8 subgroups with different prognostic factors from the perspective of the Chinese healthcare system over a 5-year period.Our results suggest that the TMZ strategy as a first-line treatment for GBM may possess significant advantages in relation to health benefits. However, the gap between the costs of TMZ and payment capacity in a health resource-limited setting may be too great to allow the TMZ strategy to be recognized as the most appropriate approach. In the overall cohort, the TMZ strategy (when compared to the RT and NT strategies) revealed benefits that were achieved at an incremental cost per QALY of $ 87,940.6 and $ 94,968.3, respectively. These costs are far greater than the societal willingness-to-pay thresholds for each additional quality-adjusted life-years gained . These higher ratios are largely attributable to the high cost and the relatively limited survival benefits associated with TMZ. The ICERs identified in other health economic analyses from relatively health resource-rich regions ranged from €37,000 per life-year gained to €42,840 per QALY gained for the TMZ strategy compared with the RT strategy The one-way sensitivity analysis shows that the results of the model were driven by certain key parameters (particularly the cost of TMZ). As Several limitations of the current analysis must be considered. First, the estimation of the Weibull parameters of PFS for the subgroups of age and surgery type was an inevitable limitation. The Weibull parameters of PFS determined the survival rates of the disease-free patients. However, the results of the comparison of the median PFS times between the model and the trial in the MGMT subgroups indicates that the estimation method used in this study minimized this bias. Second, because few head-to-head trials have been conducted for the three first-line strategies for GBM, an indirect comparison was used in this study. Similar patient characteristics for the three strategies were assumed in our indirect comparison, and the results of the indirect comparison were imputed into the analytical model. When there is no direct comparison trial, indirect comparisons using well-recognized methods have been accepted by many researchers around the world. When direct comparison data are available, this analysis could be updated. Third, we did not fully explore other therapeutic strategies for first- and second-line treatments of GBM, especially targeted therapies such as bevacizumab In conclusion, in the Chinese healthcare setting , the addition of TMZ to radiotherapy in patients with GBM would not be a cost-effective approach compared to radiotherapy alone or radiotherapy plus nitrosourea agents. However, better economic outcomes are likely to occur when subgroups with more favorable prognostic factors receive TMZ. Decreasing the price of TMZ might be one potential way to counter the restrictive Chinese reimbursement policies.Table S1Baseline Clinical Data.(DOCX)Click here for additional data file.


The function of microRNAs (miRNAs) during alcoholic liver disease (ALD) has recently become of great interest in biological research. Studies have shown that ALD associated miRNAs play a crucial role in the regulation of liver-inflammatory agents such as tumour necrosis factor-alpha (TNF-α), one of the key inflammatory agents responsible for liver fibrosis (liver scarring) and the critical contributor of alcoholic liver disease. Lipopolysaccharide (LPS), a component of the cell wall of gram-negative bacteria, is responsible for TNF-α release by Kupffer cells. miRNAs are the critical mediators of LPS signalling in Kupffer cells, hepatocytes and hepatic stellate cells. Certain miRNAs, in particular miR-155 and miR-21, show a positive correlation in up-regulation of LPS signalling when they are exposed to ethanol. ALD is related to enhanced gut permeability that allows the levels of LPS to increase, leads to increased secretion of TNF-α by the Kupffer cells and subsequently promotes alcoholic liver injury through specific miRNAs. Meanwhile, two of the most frequently dysregulated miRNAs in steatohepatitis, miR-122 and miR-34a are the critical mediators in ethanol/LPS activated survival signalling during ALD. In this review, we summarize recent findings regarding the experimental and clinical aspects of functions of specific microRNAs, focusing mainly on inflammation and cell survival after ethanol/LPS treatment, and advances on the role of circulating miRNAs in human alcoholic disorders. IntroductionLPSTLR4TNFαKupffer cells and increase in TNFα secretionmicroRNAsmiRNA and epigeneticsmiR-155 and ethanol exposuremiR-155 and TNFαmiR-155 and NF-κBmiRNA and endotoxin altered permeabilitymicroRNA and hepatic cell survivalCirculating miRNAs as stable blood-based markers for ALDsConclusionAlcoholic liver disease presents a global health concern One control point regulating inflammatory response and cellular apoptosis in ALD involves microRNA targeting of critical inflammation/apoptosis signalling proteins. MicroRNAs are short functional RNAs that cause reduced expression of their target genes through post-transcriptional mechanisms. In general, this targeting involves imperfect base-pairing between the microRNA and the cognate mRNA target, resulting in altered protein production Lipopolysaccharide exposure is initiated by the liver's detoxification process upon exposure to gram-negative bacterial cell wall Toll-like receptors were first discovered in Drosophila and later as corresponding human homologs There is ample evidence for increased inflammatory cascade activation in ALD. Toll-like receptor 4 is expressed in all cell types of the liver; thus, gut-derived endotoxin can modulate the function of all liver cells in ALD Some feedback mechanisms exist to limit LPS mediated toxic effects. Several soluble decoy receptors, such as soluble TLR4, and splice variants of signal-transduction proteins, including MyD88-s, IRAK-M and TAG, are the key regulators Tumour necrosis factor-alpha is an inflammatory agent in the liver which has been found to be a key contributor to alcoholic liver disease in vivo and in vitroInactivation of Kupffer cells with gadolinium chloride or clodronate injection can ameliorate alcohol- induced liver disease Kupffer cells are resident macrophages in the liver that become activated upon recognition of an LPS signal both microRNAs are segments of RNA, which serve as epigenetic regulators. The first discovery and observation of miRNA's function occurred in 1993, and over the subsequent 10 years, 300+ miRNA sequences have been discovered. Today, ∼17,000 miRNAs have been described in 142 species. Of these, around 1000 are present in the human genome Epigenetics, in its essence, defines the alternation of gene expression without disruption of DNA sequences. Epigenetic regulation is altered and manipulated by several factors, including DNA methylation, modifications of histones and RNA silencing by non-protein-coding RNAs (ncRNAs) Altered DNA methylation occurs after alcohol consumption during initial periods of alcohol abuse. Global hypomethylation of DNA in liver after long-term ethanol exposure has been reported miRNA is also a modulator of epigenetics in the liver at a post-transcriptional level. It is responsible for gene expression regulation. They are responsible for the hindering of several translational elements to include: initiation, elongation, degradation, and degradation of target mRNA in vitro. Since miR-155 plays such a crucial role in the stimulation of LPS-induced TNF-α production, this study stated that the inhibition of miR-155 prevents alcohol-induced sensitization to LPS. Inversely, it was found that the up-regulation of miR-155 increased Kupffer cell sensitivity to LPS signalling. Experimentation on alcohol-fed mice showed that alcohol exposure up-regulates miR-155 via NF-κB, thus linking NF-κB with miR-155.miRNA is a known regulator of Kupffer cell response to LPS. Three miRNAs in particular, miR-155, miR-125b and miR-146a, have been of interest in studies and shown to be key contributors to LPS regulation The up-regulation of TNF-α production in Kupffer cells by LPS and alcohol was determined in two individual experiments: one in which LPS alone was increased, the other in which ethanol exposure alone was increased. In both experiments the production of TNF-α by resident macrophages was increased. A tertiary experiment also proved that miR-155 is the bridge step between ethanol and LPS up-regulation. In this experiment, miR-155 induced cells produced more TNF-α than the controls with no miR-155 overexposure. Lipopolysaccharide levels were also higher though no LPS was introduced. This led to the conclusion that miR-155 is directly involved in LPS-induced TNF-α production, not ethanol Studies have linked the inhibition of miR-155 to the NF-κB inhibitor. NF-κB inhibitors have proven to mediate the up-regulation of miR-155 in Kupffer cells Another regulator of gut-endotoxin permeability, other than ethanol, is miRNA. It was discovered that miRNA increases the permeability of the intestinal lumen in a similar way to ethanol. miRNA, however, does so by affecting the Zonula occludens 1 (ZO-1) protein negatively to induce intestinal lumen permeability. The ZO-1 protein is a critical component that insures the permeable response of the intestinal lumen to endotoxins. Just like with alcohol, the permeability of the intestinal lumen leads to higher absorption of endotoxins that are later transported to the liver to be detoxified. In this process, the detoxification of these endotoxins leads to an LPS-TNF-α chain reaction that causes alcoholic liver disease. In addition to its effects on gut permeability, ethanol also up-regulates miRNA responsible for the reduction of lumen permeability Several investigations have demonstrated the regeneration and remodelling potentials of the adult cells in the liver after injury including hepatocytes and cholangiocytes and with newer studies showing the plasticity of hepatic stellate cell (HSC) and perhaps other cell types as well The key pathophysiological features of ALD are altered lipid metabolism and hepatocyte apoptosis. The results of recent studies show a significant down-regulation of miR-122, a liver-specific miRNA The development of minimally invasive tests for the detection and monitoring of ALD could greatly reduce the worldwide health burden of alcoholic liver injury. The demonstration that miRNA profiles could reveal smoke-related effects in the liver of mice exposed to ethanol established a proof-of-principle for the use of miRNAs to evaluate early steps of ALD process, which could be extended to other risk factors. However, the most useful approach would be to distinguish specific biomarkers circulating in the bloodstream before alcoholic liver injury become clinically apparent. To this end, miRNAs can potentially become useful biomarkers for human ALD. Actually, deregulated miRNAs are found in liver as well as in the blood of ALD patients. Because of their innate stability, miRNAs may be detected blood based assays for ALD. miRNAs are present in plasma in a stable form, making them feasible biomarkers for the detection of ALD and other liver disorders via TLR4. Both of these receptors have been identified as key components of alcoholic liver disease since both their deactivations help reduce progression of the disease. Upon activation of TLR4, macrophages in the liver release inflammatory agents that cause liver fibrosis. One particular macrophage of interest is the Kupffer cell, which releases TNF-α. The role miRNA takes part in this process involves the regulation of the LPS signalling to the TLR4 receptor. It is miRNA that regulates the LPS reception in Kupffer cells. It is also known that ethanol regulates miRNA (miR-122 and miR-34a) in the liver that contributes to hepatocytes/HSC survival. Therefore, as alcohol consumption increases, so does miRNA in the liver and thus the reception of LPS and TNF-α release. Although still under study, microRNAs have been shown to play a pivotal role in the development, causation and regulation of alcoholic liver disease.The role of miRNA in alcoholic liver disease is certainly a crucial one. In the past decade and in recent years its role in alcoholic liver disease has found keen interest. miRNA's interaction with alcohol plays a pivotal role in alcohol-induced liver scarring and fibrosis. The process that leads to alcoholic liver disease begins, of course, with ethanol consumption and exposure. Ethanol exposed mice have shown loss of permeability of the intestinal lumen. This permeability alternation occurs in two ways: First, with the direct influence of ethanol on the permeability of the intestinal lumen and allowing endotoxins to enter the liver. Second, ethanol induces miRNA, a known mediator of intestinal permeability, in the intestinal lumen to do the same. Once in the liver these endotoxins, produced by the decomposition of gram-negative bacteria and pathogens, follow a pathway that leads to liver inflammation. The endotoxins that are taken in by the intestinal lumen find their way to the liver, where they are detoxified. The detoxification of the endotoxins from gram-negative bacteria releases a particular kind of endotoxin called LPS, which once in the liver, attaches to TLR4, and sets in action the pathway that leads to alcoholic liver disease. Toll-like receptor 4 is not composed of two coreceptors, MD-2 and CD14, but these two proteins may be required for response of cell to LPS stimulation


The usefulness of the nutritional screening tool Minimal Eating Observation and Nutrition Form - Version II (MEONF-II) relative to Nutritional Risk Screening 2002 (NRS 2002) remains untested. Here we attempted to fill this gap by testing the diagnostic performance and user-friendliness of the MEONF-II and the NRS 2002 in relation to the Mini Nutritional Assessment (MNA) among hospital inpatients.Eighty seven hospital inpatients were assessed for nutritional status with the 18-item MNA (considered as the gold standard), and screened with the NRS 2002 and the MEONF-II.The MEONF-II sensitivity (0.61), specificity (0.79), and accuracy (0.68) were acceptable. The corresponding figures for NRS 2002 were 0.37, 0.82 and 0.55, respectively. MEONF-II and NRS 2002 took five minutes each to complete. Assessors considered MEONF-II instructions and items to be easy to understand and complete (96-99%), and the items to be relevant (87%). For NRS 2002, the corresponding figures were 75-93% and 79%, respectively.The MEONF-II is an easy to use, relatively quick and sensitive screening tool to assess risk of undernutrition among hospital inpatients. With respect to user-friendliness and sensitivity the MEONF-II seems to perform better than the NRS 2002, although larger studies are needed for firm conclusions. The different scoring systems for undernutrition appear to identify overlapping but not identical patient groups. A potential limitation with the study is that the MNA was used as gold standard among patients younger than 65 years. Undernutrition is associated with poorer health, compromised ability to recover from medical conditions and increased mortality . People In Sweden, it is recommended that undernutrition risk screening should include at least the following three criteria: unintentional weight loss, eating difficulties, and low BMI . These cMEONF-II is based within an interdisciplinary nursing framework, combining descriptions of mealtime problems with classical signs of undernutrition in order to facilitate detection of problems in need of interventions ,9. In a Ninety six persons (18+ years old) receiving inpatient care at four wards  at a hospital in southern Sweden were approached. Fifteen of the patients were younger than 65 years. There were no predefined inclusion or exclusion criteria used. Data from 87 patients were analyzed. Reasons for not participating (n = 7) were aphasia (n = 1), language difficulties (n = 1), dementia (n = 1), mental illness (n = 1), and not wanting to participate (n = 3). In another two cases the forms were incomplete and had to be excluded. Informed consent was obtained. The sample has previously been used as part of a larger study regarding MEONF-II cut-off scores . The stuBackground data  were recorded and assessments were conducted according to the MNA, MEONF-II and NRS 2002.The MNA was developed for use among elderly patients ≥ 65 years) ,13. The 5 years ,The main purpose of the NRS 2002 is to identify patients who may benefit from nutritional interventions . It consFood intake includes "difficulty manipulating food on plate", "difficulty conveying food to the mouth" and "difficulty to maintain good sitting position during meals". Swallowing/mouth includes "difficulty chewing", "difficulty coping with food in mouth" and "difficulty swallowing". Energy/appetite includes "lacks energy to complete an entire meal", "poor appetite" and "eats less than 3/4 of food served" [MEONF  during one set day in November 2010.The order of the nutritional assessments in the protocol was, first MEONF-II, thereafter NRS 2002, and finally MNA. Meal-time observations (MEONF-II) were conducted during lunch or dinner. Height and weight were recorded in the morning and other observations at convenient time points during the day. The four nurses received written and oral information about the study and the included assessment methods. The information was provided to the four nurses in a single group session lasting for about one hour the day before data collection. The researchers were available to answer questions during the data collection day.User-friendliness of the three tools was evaluated by recording the time required to complete each tool and by inquiring the assessors of their perceived ease of understanding and following instructions, ease of understanding and completing items, and whether items were perceived as relevant. This was done following each patient assessment (n = 87).The diagnostic performance of the MEONF-II and NRS 2002 was assessed by calculating the sensitivity, specificity, positive and negative predictive values  and accuracy ,19, usinTime to complete the three screening tools was analyzed by Friedman's two-way analysis of variance by ranks followed by post-hoc Wilcoxon signed ranks tests. Other user-friendliness data, and undernutrition risk according to the three tools were analyzed using Cochran's Q test followed by post-hoc analyses (McNemar). P-values were considered significant if < 0.05 . AnalyseDemographical data are presented in Table The proportion of people classified as at risk for undernutrition according to NRS 2002 (29%) was significantly lower than found using MEONF-II  and MNA . There was no significant difference (p = 0.021) in proportions found using the MEONF-II and MNA following Bonferroni correction (p = 0.063)  of the MEONF-II was 61% Table . For theA positive MEONF-II result, indicating risk for undernutrition, was associated with a PPV of 82%; that is, a 82% probability that the individual really was undernourished (according to the 18-item MNA). A negative MEONF-II result was associated with a NPV of 57%; that is, a 57% probability that the individual really was not undernourished. For the NRS 2002, PPV and NPV were 76% and 47%, respectively. The exact proportions of agreement (accuracies) according to the various methods were 68% for MEONF-II and 55% for NRS 2002 in relation to the 18-item MNA Table .Overlapping 95% CIs between the NRS 2002 and MEONF-II suggested no statistically significant differences in sensitivity, specificity, PPV and NPV between these tools.Analyses of data only from those ≥65 years old (n = 72) yielded similar results for both the MEONF-II  and NRS 2002 .The median time required to conduct assessments according to the NRS 2002 and MEONF-II was 5 minutes each; in contrast, the 18-item MNA took twice as long to complete Table . AssessoThis study provides support for the validity and user-friendliness of the MEONF-II and the NRS 2002. Perceived user-friendliness of the MEONF-II was somewhat better than that of the NRS 2002. It was indicated, although not significantly, that the MEONF-II had better sensitivity than the NRS 2002 in comparison to the MNA.The MNA has commonly been used as the comparator, or gold standard nutritional screening tool  .The MEONF-II is a screening tool designed to detect risk of undernutrition, not only those with manifest undernutrition. As such, it is reasonable for sensitivity to be given priority at the cost of specificity since over-identification is preferable to under-identification, given that positive screening results are followed by in-depth assessment ,22. In tThe MEONF-II demonstrated good user-friendliness in terms of time to complete, ease of understanding of items, as well as ease of completion. In these respects, our observations suggest that MEONF-II compares favourably to the NRS 2002. One reason for this may be that it helps nurses identifying problems and intervene directly, either themselves or by involving other professionals. It should be noticed that time consumption was low when using MEONF-II, despite the fact that this assessment was done before NRS 2002 and MNA. It could otherwise be expected that the time needed would be lower for tools used as second and third since several items are shared between the tools. One should, however, be careful in the interpretation of these findings since it was only four nurses that rated user-friendliness and there may be a learning curve for each of these nurses, affecting rating of user-friendliness and time needed for completing forms. Anyhow, user-friendliness is of fundamental importance for successful clinical implementation of nutritional screening tools. As most screenings are carried out by nurses, their perspective in this respect must be taken into account.The MEONF-II is an easy to use, relatively quick and sensitive screening tool to assess risk of undernutrition among hospital inpatients. High sensitivity is of primary concern in nutritional screening. With respect to user- friendliness and sensitivity, the MEONF-II appears to perform well compared to the NRS 2002, although larger studies are needed for firm conclusions. The different scoring systems for undernutrition appear to identify overlapping but not identical patient groups. However, the appropriateness of using the MNA as gold standard among patients younger than 65 years can be questioned.The authors declare that they have no competing interests.All authors participated in the design of the study, read and approved the final manuscript. AW informed the data collectors, performed the statistical analysis, and drafted the manuscript. EN coordinated and supervised the data collectors. PH helped to draft the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6955/10/24/prepubMEONF-II . The file contains the nutritional screening tool Minimal Eating Observation and Nutrition Form - Version II (MEONF-II).Click here for file