Cartilage had no radiographic, magnetic-resonance-imaging or arthroscopic evidence for degeneration. RNA was subjected to Affymetrix microarrays followed by validation of selected transcripts by microfluidic digital polymerase-chain-reaction. The underlying biological processes were explored computationally. Transcriptome-wide gene expression was probed for association with known OA genetic risk-alleles assembled from published literature and for comparison with gene transcripts differentially expressed between healthy and OA cartilage from other studies.We generated a list of 27,641 gene transcripts in healthy cartilage. Several gene transcripts representing numerous biological processes were correlated with age and BMI and differentially expressed by sex. Based on disease-specific Ingenuity Pathways Analysis, gene transcripts associated with aging were enriched for bone/cartilage disease while the gene expression profile associated with BMI was enriched for growth-plate calcification and OA. When segregated by genetic risk-alleles, two clusters of study patients emerged, one cluster containing transcripts predicted by risk studies. When segregated by OA-associated gene transcripts, three clusters of study patients emerged, one of which is remarkably similar to gene expression pattern in OA.Our study provides a list of gene transcripts in healthy-appearing cartilage. Preliminary analysis into groupings based on OA risk-alleles and OA-associated gene transcripts reveals a subset of patients expressing OA transcripts. Prospective studies in larger cohorts are needed to assess whether these patterns are predictive for OA. Articular cartilage is a specialized connective tissue of diarthrodial joints. Several lines of evidence suggest that age , body-maStudying healthy cartilage from humans is challenging but not impossible. Cadaver knees are a significant source of tissue but often lack adequate information regarding the presence or absence of concomitant joint injuries and OA. Cartilage from patients undergoing knee amputation due to chondrosarcoma is another source but generally comes from a younger population and may have been subjected to chemotherapy drugs or radiation . Non-fibAging elevates the risk of cartilage degeneration by suppression of proteoglycan synthesis, augmented collagen cross-linking and loss of tensile strength , and incObesity is an important risk factor for OA development and progression and is associated with alterations in joint biomechanics and inflammatory environment , 26. AdiWhile aging, obesity and female sex are associated with changes in cartilage metabolism and OA , 34, 35,Lastly, it has been reported that approximately 50% of patients with meniscus injury will go on to develop OA within 10–15 years . TherefoAll procedures were approved by the Institutional Review Board (Human Research Protection Office) of Washington University School of Medicine (St. Louis). Patients diagnosed with a symptomatic tear in the posterior horn of the medial meniscus and undergoing arthroscopic surgery furnished a written informed consent as approved by the Human Research Protection Office of study institution. All patients in the study had radiographs and an MRI of the knee prior to surgery. To be considered for the study, the patients could not have any evidence for OA, chondrosis, bone marrow lesions/edema, and ligamentous injury greater than Grade I medial collateral ligament strain. Patients of any age, BMI and from both sexes were recruited.2 ± 3.9 (mean ± standard deviation), with 5 females  and 7 males . None of the patients were smokers or had diabetes.The patient cohort was relatively homogeneous . The med™ biopsy [A fragment (160–235 mg) of healthy cartilage was taken from the non-weight bearing site of the medial intercondylar notch with the use of a sharp curette, in a technique similar to a Carticel™ biopsy . The tisTissues were refrigerated for 24 hours followed by storage at 80°C until RNA isolation . The tis2 transformed before downstream analysis by Partek Genomic Suite v6.4 . Statistical significant raw P values were adjusted for multiple comparisons. The effect of all factors  was assessed at a significance level set at unadjusted P value of <0.05. Pearson correlation coefficients  were calculated using ANCOVA for age and BMI while fold change differences were calculated between males and females. Age and BMI were used a continuous variables while sex was treated as a categorical variable. The distribution of males and females across age was similar. Mean age for female participants was 50.6 years (range = 31–65 years) while that of male participants was 48.1 years (range = 40–53 years) and there was no significant difference between the mean age of females and males (P = 0.702). However, BMI of female participants (24.3 kg/m2) was significantly (P = 0.048) lower than that of male participants (28.7 kg/m2). Therefore, to circumvent the influence of any one variable on the differentially correlated or differentially expressed gene transcripts, we added all three variables  in the model.Five microgram of biotinylated cDNA  was prepared through NuGEN Ovation Pico WTA Amplification System. This system provides a fast and simple method for preparing amplified cDNA for gene expression analysis. Amplification is initiated at the 3’ end as well as randomly throughout the transcriptome in the sample, making the Ovation Pico WTA system ideal for amplification of partially degraded and compromised RNA samples from tissue. The amplified product of the Ovation Pico WTA system is optimized for gene expression analysis applications on microarrays. Following fragmentation with NuGEN Encore Biotin Module, cDNA was hybridized into the GeneChip Hybridization Oven 640 for 18 hours at 45°C on Affymetrix Gene 2.0 ST Array . The microarrays were washed and stained in the Affymetrix Fluidics Station 450 and scanned using the Affymetrix GeneChip 7G 3000 Scanner. The microarray data were analyzed with the Affymetrix GeneChip Command Console and processed with the use of Command Console  and the raw (.CEL) files generated were analyzed using Expression Console software with Affymetrix default RMA Gene analysis settings . Probe summarization , quality control analysis, and probe annotation were performed per recommended guidelines . Data were quantile normalized and logWe created a list of all detectable gene transcripts in order of their relative intensity values to provide baseline gene expression abundance in healthy articular cartilage.https://portal.genego.com). Then “one click” gene ontology was applied to obtain highly enriched and statistically significant biological processes and molecular functions. For the sake of simplicity we focused only on the biological processes, information on molecular functions can be found by plugging the gene list into GeneGo MetaCore web portal and by clicking “gene ontology molecular functions” tab.To facilitate the interpretation of differentially expressed transcripts, we used gene ontology analysis. A list of differentially expressed gene transcripts was uploaded on the GeneGo MetaCore web portal v6.22  software program was used to determine the functional pathways and human disease-associated pathways represented by the identified genes, which were associated with age , BMI S2, and sexA list of susceptibility alleles was assembled from the literature  and exprhttp://www.lifetechnologies.com and then by selecting “best coverage” and “human” under “narrow your results” tab: SNAI2 (Hs00950344_m1), PSMD5 AS1 (Hs00404560_m1), CYP7A1 (Hs00167982_m1), GDF11 (Hs00195156_m1), S100A8 (Hs00374264_g1), SFRP2 (Hs00293258_m1), ACAN (Hs00153936_m1), CHAD (Hs00154382_m1), and COL2A1 (Hs00264051_m1). These genes were chosen for three reasons: (1) those that were positively correlated with age (SNA12) and BMI (CYP7A1) and expressed at higher levels in females ; (2) those that were negatively correlated with age (PSMD5AS1) and BMI (GDF11) and expressed at lower levels in females (SFRP2) and (3) those that were neither correlated with age or BMI nor were differentially expressed by sex .To confirm microarray data, we measured the expression pattern of 9 genes via microfluidic digital PCR 96.96 Dynamic Arrays  using RNA from all 12 study samples that were used for microarrays. The selection of these genes was mainly based on their differential expression by age, BMI and sex in this study. The probe sequences can be found by locating the gene ID provided for each gene below by visiting ™ Reverse Transcription PCR System. The cycling program consisted of 10 minutes at 95°C followed by 14 cycles of 95°C for 15 seconds and then 60°C for 60 seconds. Data were analyzed using Fluidigm Reverse Transcription PCR Analysis software. Analysis was performed using 2−ΔCt approach with TATA box binding protein  as internal reference gene. The data were analyzed by SPSS software . Pearson correlation coefficients were calculated for age and BMI. The differences between the means were measured for males and females using student’s t-test and reported as mean ± standard error of the mean.The cDNAs were subjected to specific target amplification using TaqMan PreAmp Mastermix  and High Capacity cDNA Reverse Transcription Kit . The sample and assay mixtures were prepared using manufacturers’ guidelines and were loaded on the NanoFlex 4 Integrated Fluidic Circuit Controller for equal distribution followed by insertion into the BioMarkOut of 53,617 transcripts available on the microarray chip, the number of significantly differentially expressed transcripts was 1979 (3.69%) for BMI, 1889 (3.52%) for sex and 1862 (3.47%) for age. Interestingly, the number of transcripts for each category was not substantially different from each other. Among these, 1363 transcripts were exclusively associated with age, 1284 with BMI and 1216 with sex. BMI and sex had overall more common transcripts (367 transcripts) than BMI and age (193 transcripts) or age and sex (171 transcripts). 135 transcripts were common to age, BMI and sex . The numAfter removal of duplicate, non-annotated, and uncharacterized transcripts, 592 gene transcripts were differentially expressed exclusively by age. Among these, 267 gene transcripts were positively correlated with age and 325 gene transcripts were negatively correlated with age . A list After removal of duplicate, non-annotated, and uncharacterized transcripts, 509 gene transcripts were differentially expressed exclusively by BMI. Among these, 317 gene transcripts were positively correlated with BMI and 192 gene transcripts were negatively correlated with BMI . A list After removal of duplicate, non-annotated, and uncharacterized transcripts, 484 transcripts were differentially expressed by sex. To identify transcripts with the highest significant difference, the analysis was restricted to transcripts showing fold change of ≥1.5 and P<0.05. By these criteria, the number of differentially regulated transcripts was reduced to 13% (63 of 484 transcripts). More transcripts were lower in expression in females (317 transcripts) than those that were higher in expression (167 transcripts). Several gene transcripts were common between any two comparisons of patient factors. For instance, there were 193 transcripts common to age and BMI, 367 to BMI and sex and 171 to age and sex. There were 135 gene transcripts common to all three factors . After removal of duplicate, non-annotated, and uncharacterized gene transcripts, 55 gene transcripts were common to all three categories .The biological processes common to all three patient factors  were cellular hypertonic response, smooth muscle contraction, positive regulation of wound healing and production of interleukin-10 and interleukin-12 .As mentioned above, gene transcripts exclusively associated with age, BMI and sex were respectively 592, 509, and 484 which is about 8–10 fold higher than the gene transcripts common to all three factors (55). These results indicate that age, BMI and sex have differential impact on the expression profile of gene transcripts in macroscopically normal articular cartilage.2 transformed relative signal intensity values is presented in A list of 27,641 gene transcripts with their logIt is known that approximately 50% of patients with meniscus tears go on to develop OA . We quesRecent GWAS studies along with several adequately powered candidate gene studies have yielded a number of risk-alleles for OA. This allele number is now sufficiently large to allow conclusions regarding the nature of genetic susceptibility. Functional studies have revealed that effects on gene expression are likely to be one of the main mechanisms by which susceptibility to OA manifests in patients . We geneDDIT4) showed down-regulation instead of up-regulation. Of the published 9 down-regulated gene transcripts, 5 were down-regulated in Cluster 3. These clusters are likely to have varying susceptibility to developing OA, with the greatest predilection for OA in the patients from Cluster 3.Using 62 highly differentially expressed gene transcripts between healthy and OA cartilage  from the published dataset , we founSNAI2, which was positively correlated with age by microarray , was also found to exhibit similar pattern by digital PCR . Similarly, PSMD5AS1 was negatively correlated with age by microarray  and was also negatively correlated when determined by digital PCR . For BMI, CYP7A1, and GDF11 were respectively positively and negatively correlated by microarray  and showed same trend by digital PCR . S100A8 was highly expressed in females  while SFRP2 was highly expressed in males  and this pattern was consistent when measured by digital PCR as their respective fold change differences were respectively 2.85 fold (P = 0.074) and −2.60 fold (P = 0.040). In addition, we tested three cartilage genes  none of which were significantly different by age, BMI, and sex when analyzed by microarray or by digital PCR. Their correlation coefficients are provided on respective graphs  cartilage  procured from knees with meniscus tears undergoing partial meniscectomy. While some segregation for age, BMI and sex differences is seen, one of the primary roles of this dataset will be to provide near healthy cartilage transcriptome data for comparison with other cohorts on the spectrum of healthy to OA cartilage. We also noted that age, BMI and sex were differentially associated with the gene expression profile in this cartilage. Only a handful of gene transcripts and biological processes were common to all three factors .2) or sex . Interestingly, two of these patients (P4-005A and P4-009A) were also in Cluster 2 of the GWAS genes showing more similarity with expression of the OA risk-alleles. One gene in particular was associated with both, ASPN, encoding the Asporin protein which binds to TGF-β [ASPN were associated with OA, with increased expression from some variants. In our Cluster 3, all three patients demonstrated high levels of ASPN transcripts. As specific variants have been shown to result in higher gene expression, it would be interesting to determine the allelic composition of our twelve patients.Approximately 50% of patients with meniscus tears go on to develop OA . We questo TGF-β . In GWASAlthough association between these distinct expression patterns of OA risk-alleles or OA-associated gene transcripts and OA initiation warrants further investigation, our preliminary analysis suggests that a small subset of patients expresses an OA gene expression profile. While the clustering from both analyses  is informative in providing a clue for variation in individual’s response to injury, confirmation can only come from a follow-up study on patients to identify those who develop OA.Age appears to be an important factor relating to molecular changes in cartilage. The primary biological processes affected with aging included intracellular signal transduction, fat cell differentiation, cell-cell communication, and necroptotic process. The intracellular signal transduction pathway, involved in production of cytokines as well as other inflammatory compounds and enzymes, is commonly affected by several mechanisms in chondrocytes. For instance, it has been shown that nitric oxide can induce this pathway along with inflammatory gene activation. In addition, both receptors of TNFα are involved in signal transduction after being activated by TNFα during processes occurring in cartilage loss and OA , 45. SimThe biological processes negatively correlated with age were primarily immune response and leukocyte activation. It is known that the aging immune system becomes less capable of resolving inflammation. Therefore, a negative correlation of immune response with aging cartilage hints towards decreased capability to suppress inflammation in the joint. This is compounded by a decrease in leukocyte activation in cartilage, which is regulated by IL-8 through p38 mitogen activated protein kinase signaling  and trigGDF11, is negatively associated with BMI. Since this factor is a negative regulator of chondrogenesis [GDF11 was recently identified as the circulating factor that reverses age-related cardiac hypertrophy [The main biological processes positively correlated with BMI were related to regulation of memory T cell activation. It has been reported that after synovial inflammation is initiated, memory T cell activation leads to progression of inflammation in rheumatoid synovium . This suogenesis , our finertrophy .Our study also showed some influence of patients’ sex on transcriptome changes in cartilage. The gene transcripts representing processes such as detection of chemical stimulus involved in sensory perception of taste, L-ascorbic acid transport, peptidyl cysteine oxidation, aminoglycan metabolic process, and chaperone mediated protein folding were elevated in females. Their biological significance both in the context of cartilage and OA is not completely understood. However, the functional classifications repressed in females were represented by cell and tissue development and cell differentiation. Interestingly, chondrogenic differentiation of stem cells is influenced by sex as studies have demonstrated that muscle derived stem cells from females display less chondrogenic differentiation and lower quality cartilage healing than males . TherefoOur study has certain limitations. A potential limitation of the study is the use of cartilage from the notch. While the sample is not from the direct weight-bearing portion of the knee, it does come from a portion of the notch that can develop degenerative changes and OA. In addition, these findings would benefit from confirmation by immunohistochemistry as well as from an independent cohort.Taken together, these findings may identify pathways that put older and heavier patients, as well as females, at greater risk for knee OA after partial meniscectomy. While it is known that patients with previous knee surgery undergo total knee arthroplasty at a younger age than patients without previous knee surgery , the exaWhile transcriptome profiling in conjunction with OA risk-alleles and OA-related gene transcripts suggests a molecular “pre-OA” phenotype in a subset of patients, the ability to predict a population at risk for can only be assessed by future radiographic and clinical outcomes. Furthermore, validation and generalization of the findings from the current study necessitate confirmation in a distinct, larger cohort of patients.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.


Respiratory infections are important causes of morbidity and mortality in reptiles; however, the causative agents are only infrequently identified.Python regius). Eight of 12 snakes had evidence of bacterial pneumonia. High-throughput sequencing of total extracted nucleic acids from lung, esophagus and spleen revealed a novel nidovirus. PCR indicated the presence of viral RNA in lung, trachea, esophagus, liver, and spleen. In situ hybridization confirmed the presence of intracellular, intracytoplasmic viral nucleic acids in the lungs of infected snakes. Phylogenetic analysis based on a 1,136 amino acid segment of the polyprotein suggests that this virus may represent a new species in the subfamily Torovirinae.Pneumonia, tracheitis and esophagitis were reported in a collection of ball pythons (This report of a novel nidovirus in ball pythons may provide insight into the pathogenesis of respiratory disease in this species and enhances our knowledge of the diversity of nidoviruses. Nidovirales is a large order of positive sense, single-stranded RNA (ssRNA) viruses that consists of the many genera and species in the families Coronaviridae, Arteriviridae, Roniviridae and Mesoniviridae. Although the genomes of nidoviruses vary in length, ranging from 13 to 32 kilobases (kb), the organization of the genomes are similar across the entire order [Nidovirales[re order . The 5′ ovirales.Nidoviruses infect a broad range of hosts including humans and other mammals, birds, fish, insects and crustaceans –11. AlthPython regius) in upstate New York with pneumonia, tracheitis and esophagitis. The snakes were found dead between July 2011 and September 2013.Here we report the discovery of a novel nidovirus in a collection of ball pythons  or Gram’s stain for histologic examination. Eleven tissues, including lung (n = 4), liver (n = 1), spleen (n = 3) and esophagus (n = 3) from the 4 snakes were collected and stored at −20°C.Gross and histologic findings in all four snakes were primarily restricted to the respiratory and upper gastrointestinal tracts Table . HematoxTotal nucleic acids were extracted from snake samples  using the EasyMag  platform; Samples from snakes 1 and 2 were depleted of ribosomal RNA  and treated with DNAse I . cDNA synthesis was performed using SuperScript II first-strand synthesis supermix (Invitrogen). Viral discovery was performed using broadly reactive consensus PCR assays targeting common respiratory viruses of animals, including paramyxoviruses –21, reovNidovirales subfamily Torovirinae, including Breda virus, White bream virus, and Fathead minnow virus. Assembly of all Torovirinae-like reads generated a 3,408 nt contig with 33% amino acid homology to the replicase polyprotein 1ab of Fathead minnow virus. The presence of this 3,408 nt sequence in both snakes was confirmed by PCR using primers shown in Table Nucleotide sequence analysis (BLASTn) of processed reads was uninformative; however, amino acid analysis (BLASTx) revealed multiple reads with amino acid homology of <50% to the polyprotein region of the Nidovirales sequences from GenBank using ClustalW. The best-fit model of amino acid substitution, the Whelan and Goldman (WAG) matrix, was selected using the maximum likelihood method implemented in MEGA version 5.2 [Torovirinae subfamily (Figure Torovirinae.In situ hybridization to a 934 nt fragment of the genomic polyprotein 1ab region was used to assess viral infection and distribution in the lung tissue. Positive cytoplasmic staining, consistent with the presence of viral nucleic acid, was confirmed in the cytoplasm of pulmonary cells, presumably epithelial cells (Figure For phylogenetic analysis, the 3,408 nt sequence was translated with Se-Al v2.0a11, and a 1,136 amino acid fragment was aligned against all sion 5.2 . A BootsRespiratory disease can be an important cause of morbidity and mortality in both wild and captive reptiles. In captivity, reptiles, and particularly snakes, are frequently maintained in collections with a high population density in relatively small spaces. As such, disease transmission within collections can occur rapidly, and early detection and diagnosis is critical in controlling disease spread.Aeromonas sp., Pseudomonas sp., Serratia sp., no evidence of bacterial infection was found in 4 snakes. In contrast, all snakes with epithelial hyperplasia in the trachea, lung and esophagus and mononuclear inflammatory infiltrates had viral signal by PCR and ISH. In concert, these data suggest a role for this nidovirus in the pathogenesis of respiratory disease. However, unequivocal implication will require experimental infection studies.Although 8 of 12 snakes with disease in the collection showed gram-negative rods by Gram stain and follow up culture in 4 revealed the presence of Torovirinae subfamily distinct from the Torovirus and recently characterized Banifivirus genera [The identification of this novel nidovirus expands our understanding of nidoviral diversity and provides insight into the pathogenesis of respiratory disease in snakes. Phylogenetic analysis indicated that the virus belongs to a novel genus within the s genera . Due to s genera , 18, it The data set supporting the results of this article is included within the article.


Pollinating insect populations, essential for maintaining wild plant diversity and agricultural productivity, rely on (semi)natural habitats. An increasing human population is encroaching upon and deteriorating pollinator habitats. Thus the population persistence of pollinating insects and their associated ecosystem services may depend upon on man-made novel habitats; however, their importance for ecosystem services is barely understood. We tested if man-made infrastructure (railway embankments) in an agricultural landscape establishes novel habitats that support large populations of pollinators  when compared to typical habitats for these insects, i.e., semi-natural grasslands. We also identified key environmental factors affecting the species richness and abundance of pollinators on embankments. Species richness and abundance of bees and butterflies were higher for railway embankments than for grasslands. The occurrence of bare (non-vegetated) ground on embankments positively affected bee species richness and abundance, but negatively affected butterfly populations. Species richness and abundance of butterflies positively depended on species richness of native plants on embankments, whereas bee species richness was positively affected by species richness of non-native flowering plants. The density of shrubs on embankments negatively affected the number of bee species and their abundance. Bee and hoverfly species richness were positively related to wood cover in a landscape surrounding embankments. This is the first study showing that railway embankments constitute valuable habitat for the conservation of pollinators in farmland. Specific conservation strategies involving embankments should focus on preventing habitat deterioration due to encroachment of dense shrubs and maintaining grassland vegetation with patches of bare ground. Pollinators play key roles in the ecosystem services essential for maintaining wild plant diversity Great effort has been applied to the development of protection plans in order to sustain the current level of ecosystem services provided by pollinators A supplementary or alternative solution for the above-mentioned methods is to take advantage of the unrecognized benefits of man-made habitats for pollinator diversity and abundance In the European Union as well as in the United States the overall length of railway lines amounts to more than 200 000 km Permission to access private lands, on which some of the sites were located, was obtained from landowners. Species surveys were conducted according to Polish law.The study was conducted along railway lines in the agricultural region of Kraków, Poland . All theA 200 m transect was established at each strip for pollinator surveys To compare the number of pollinator species and their abundance on embankments with those inhabiting grasslands we established a further 19 transects on extensively managed grassland or recently abandoned grassland  and length (m) were measured in the middle and at both ends of transects and then the mean was used in analyses. Plant species richness and plant height (cm) were measured in six circular plots of 1 m diameter (0.79 m2) established at each transect with a distance of 40 m between the plots. Because number of plant species and plant cover were highly, positively correlated (as indicated our preliminary study), thus to avoid multicollinearity problems, we decided to noticed only plant species richness. Plant surveys were done twice during the study in May and in July.The following environmental variables potentially affecting pollinators' food base and nesting resources were determined for embankments: bare ground cover, grassland cover, human settlement cover, species richness of native flowering plants, species richness of non-native flowering plants, length of railway lines, angle of slopes, length of slopes, shrub cover, vegetation height, water reservoir cover and woodland cover . Bare grSr = −0.644 and sr = 0.657 respectively; we used criterion of correlations Sr>|0.600|). To avoid multicollinearity we expressed vegetation height and grassland cover as residuals. Before analysis we checked if there was any spatial autocorrelation in the data by calculating Moran's statistics on correlograms  to examine the proportion of transects at which a given species was recorded for embankments and grasslands. We used the redundancy analysis (RDA) to find how species composition was related to the habitat type (embankments and reference grasslands) as well as to environmental variables measured on embankments. We applied the RDA analysis with forward selection of variables on the basis of their permutational p-values and on AIC criterion. The Wilcoxon test, χ2 tests, Fisher test and RDA analysis were conducted separately for pollinator groups in R software We used the Wilcoxon rank sum test to compare the number of pollinator species and their abundances among embankments and reference grasslands as well as the abundance of particular species in these two habitats. We applied χ1,42 = 1.47, p = 0.01) and 7.3% of butterflies  variation in species composition. The difference in composition of species was not significant for hoverflies . Altogether, 46 bee (out of 100), 13 butterfly (out of 66) and 20 hoverfly (out of 46) species were unique for embankments . There were 36 species of bees, 49 species of butterfly and 16 species of hoverfly that occurred on both embankments and grasslands .The model selection based on Akaike's criterion showed that one model explained bee species richness on embankments ,3. The m1,23 = 1.54, p = 0.02) which explained 2.18% of the variance in butterfly composition.Model selection identified two models describing butterfly species richness on embankments ,3. The m3,21 = 2.23, p = 0.005).Model selection identified seven models describing hoverfly species richness on embankments ,3. TheseBombus lapidarius and B. terrestris . The low explained variance indicate that abundance of most pollinator species similarly responded to the environmental variables. There are two groups of limiting factors for insect pollinator populations, i.e. those related to nesting requirements (for bees) and those related to foraging requirements  by the presence of embankments and their proper management. Railway embankments may thus be a text-book example of man-made alterations in the environment that alleviate conflicts between the demands of civilization and conservation of wildlife and biodiversity.Figure S1Moran's I correlograms for species richness of bees (a), butterflies (b) and hoverflies (c). Points represent Moran's I values. Envelopes of 95% confidences intervals are shown in dark-grey, envelopes of maximu Moran's I are shown in light-grey. None of spatial autocorrelations were significant after using Bonferroni correction.(DOC)Click here for additional data file.Figure S2Moran's I correlograms for abundance of bees (a), butterflies (b) and hoverflies (c). Points represent Moran's I values. Envelopes of 95% confidences intervals are shown in dark-grey, envelopes of maximum Moran's I are shown in light-grey. None of spatial autocorrelations was significant after using Bonferroni correction.(DOC)Click here for additional data file.Table S1List of all wild bee (a), butterfly (b) and hoverfly (c) species recorded within transects on embankments and grasslands. Abundance is the mean number of individuals per transect in which a given species was recorded. Occurrence is the number of transects with a given species. The total number of sites for embankments and grasslands was 25 and 19, respectively. A Wilcoxon rank sum test was used for abundance analysis. Fisher exact (F. exact) tests were used when frequencies were lower than 10, otherwise χ2 were used for occurrence analysis.(DOC)Click here for additional data file.


Canis lupus L.) from 14 territories in Scandinavia in relation to roads. We used resource and step selection functions, including >12000 field-checked GPS-positions and 315 kill sites. Wolves displayed ambivalent responses to roads depending on the spatial scale, road type, time of day, behavioral state, and reproductive status. At the site scale (approximately 0.1 km2), they selected for roads when traveling, nearly doubling their travel speed. Breeding wolves moved the fastest. At the patch scale (10 km2), house density rather than road density was a significant negative predictor of wolf patch selection. At the home range scale (approximately 1000 km2), breeding wolves increased gravel road use with increasing road availability, although at a lower rate than expected. Wolves have adapted to use roads for ease of travel, but at the same time developed a cryptic behavior to avoid human encounters. This behavioral plasticity may have been important in allowing the successful recovery of wolf populations in industrialized countries. However, we emphasize the role of roads as a potential cause of increased human-caused mortality.Throughout their recent recovery in several industrialized countries, large carnivores have had to cope with a changed landscape dominated by human infrastructure. Population growth depends on the ability of individuals to adapt to these changes by making use of new habitat features and at the same time to avoid increased risks of mortality associated with human infrastructure. We analyzed the summer movements of 19 GPS-collared resident wolves ( We studied how wolves in Scandinavia respond to roads built to ease human travel but degrading habitat quality for many wildlife species. Wolves responded with ambivalence: They both selected and avoided roads, all depending on the spatial and temporal scale and their behavioral status. To understand the multi-scale effects of human infrastructure on animal behavior is important with regard to the recent come-back of many wildlife species to now industrialized countries. Roads are man-made habitat features that are hardly comparable to any natural habitat: they are linear, have an open canopy, a hard surface, and often have parallel open-canopy strips with ground-cover vegetation on both sides. Connected with other roads, they form a network causing fragmentation of natural habitats. Roads are among the most recent of man-made habitat alterations, having spread dramatically during the past century following the development of motor vehicles . ReviewsThe wolf is a pack-living, highly mobile species that defends large pack territories by frequent scent-marking . Roads mOn the other hand, roads have been shown to increase mortality of wolves directly due to traffic accidents and indirectly by increasing access for hunters and poachers . FragmenIn summary, roads pose a trade-off for wolves between human-induced negative effects  and the positive effects resulting from increased ease of travel, efficient scent-marking, and access to prey. Our study examines ambivalent responses toward roads at different spatial and temporal scales from single wolf steps up to the landscape level for wolves in Scandinavia. After the functional extinction of the Scandinavian wolves in the 1960s , a coupl2), we examined how road type, time of day, and wolf behavioral state affect road use and the distance wolves stay from roads (Johnson’s fourth order). We predicted that wolves 1) prefer to use gravel roads and avoid main roads; 2) use roads more often and are closer to roads during night than daytime; and 3) use roads mainly while traveling and avoid being close to roads while handling prey and resting. Thirdly, we examined whether the wolves’ selection of patches within the home range  was dependent on road density. We expected wolves to prefer areas of low road density in order to avoid human disturbance, and we expected breeding wolves to show a stronger avoidance than nonbreeders. Finally, at the home range scale , we tested whether road use by wolves was a function of road density. We predicted a functional response due to human disturbance, that is wolves living in territories with high road densities use roads relatively less frequently than wolves in territories with low road densities. Our study focused on the summer period because 1) all gravel roads are available for wolves and people as opposed to winter when only some parts of gravel roads are snow-ploughed, and 2) breeding wolves may be more constrained during this period when movements are restricted to denning or rendezvous sites.Habitat selection is a process acting at different spatial scales. According to 2). The wolf territories were primarily covered by boreal coniferous forest (mean ± SE: 81.7±1.3% for n = 14 territories) dominated by Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies L.), with some deciduous species, of which birch (Betula pubescens Ehrh.) and aspen (Populus tremula L.) were most abundant. Mire was the second most frequent land cover type within the wolf territories (10.5±1.2%), followed by water (4.6±0.8%), agricultural fields (1.6±0.6%), open areas , and built-up areas (0.2%). The density of main roads within territories averaged 0.19±0.02 km/km2, and the maximum distance to main roads ranged from 3.72 to 14.88 km  are the most important prey of wolves in Scandinavia, with a population density of approximately 1–2 moose/km2 in summer. For Scandinavian wolf packs, moose represent more than 95% of the food biomass in summer (Capreolus capreolus L.), semi-domestic and wild reindeer (Rangifer tarandus L.), red deer (Cervus elaphus L.), and domestic sheep (Ovis aries L.). Smaller prey are also available for the wolf, including beaver (Castor fiber L.), badger (Meles meles L.), mountain and European hares , capercaillie , and black grouse (Lyrurus tetrix L.) (Moose (n summer . Other utrix L.) .As part of the Scandinavian Wolf Research Project (SKANDULV), the data for this study were collected on 19 adult scent-marking wolves resident in 14 territories between 2002 and 2010 . The wolOur original data sets consisted of 13188 hourly and 18910 half-hourly GPS positions in total. GPS success that is the percentage of successful positioning attempts per data set, averaged 81% (range 29–99%). We restricted our analysis of travel speed to half-hourly positions only and used positions at hourly intervals for all other analyses. In order to detect prey remains, we created 100 m buffers around all positions . We dissAll cluster positions and most of the single positions, that is >12000 positions in total, were visited in the field a few days after the wolf was present and searched for prey remains with the help of dogs . This reAs the wolf population was cross-border, we joined digital maps of Norway and Sweden. Vector data of roads and houses were derived from the Norwegian  and Swedish (1:100000) national maps. We categorized roads as either gravel roads (“Enskilda vägar” in Sweden and “Privatvei” in Norway) or main roads . In general, main roads were paved. Due to the large size of the study area , we wereLand cover data were derived from satellite-based maps that had been classified and approved by national authorities in both countries. In Norway, the SatVeg raster data (Source: Norwegian Environment Agency) had a pixel size of 30 m. The Swedish Corine Land Cover map with a pixel size of 25 m was provided by the National Land Survey of Sweden. For this study, we simplified the original habitat classes (24 in Norway and 59 in Sweden) into four land cover categories: Forest, Water, Mire, and Open. The last class included built-up areas, agricultural fields, and other terrestrial, nonforested areas.P > 0.05) variables and interactions in a stepwise backward procedure. For the final model, we reported the marginal and conditional R2 for LMMs as proposed by We calculated wolf travel speed from individual steps with a maximum time length of 35 min and minimum step length of 200 m, resulting in a total of 3188 steps from 25 data sets. We categorized start and end positions of each step as being either on or off of a road, within a threshold of 30 m. The distance of 30 m corresponds to the inaccuracy of GPS positions . In thisFor the site level habitat selection study , travel-to-cluster, cluster-to-travel and cluster-to-cluster steps . We exclThe response variable of the step selection models was a binary term with 1 for the used wolf locations, that is the end points of the real steps, and 0 for the end points of the random steps. To match the random steps to the corresponding real step, we applied conditional logistic regression (R 3.0.0) following the approach chosen by We estimated SSFs for all steps compiled, and separately for all combinations of time of day and behavioral state, that is at the end point of the real step the wolf was either handling prey (Kill), resting at a bed cluster (Rest), or traveling  . We ran r < 0.60) predictors and subsequently eliminated nonsignificant (P > 0.05) predictors in a stepwise backward procedure. We cross-validated the final models by excluding one wolf territory at the time, estimating model coefficients for the retained territories, and using these coefficients to predict the SSF for all real and random steps of the excluded territories. The predicted SSFs were ranked within each stratum of 11 paired steps with ranks from 1 to 11. From each stratum, we randomly sampled one random step. We then used a paired Wilcoxon signed rank test to validate whether the ranks of the real steps were higher than the ranks of the random steps. If P > 0.05, model fit was regarded as insufficient and the model was rejected.With the second set of SSF-models, we tested whether distance to human infrastructure  and land cover type predicted the choice of where wolves ended a step. Here, we included a quadratic term for distance to infrastructure to test for selection of intermediate distances. After checking predictor variables for collinearity with a pairwise Pearson rank correlation, we started with a full model that included all uncorrelated  within each home range to describe habitat availability.To test whether the wolves selected patches with low road densities within their home ranges , the density of houses (km−2), and the percentage availability of the different land cover types. These variables were the fixed factors in mixed effects logistic regression models (GLMM), and we included the data set-ID nested within territory as a random factor. The response was a binary term of 1 for used and 0 for available patches. We started with a full model including all noncorrelated (Pearson’s r < 0.6) predictor variables in linear and quadratic form and used stepwise backward selection to exclude nonsignificant variables (P > 0.05). We did this modeling procedure separately for breeding and nonbreeding wolves in combination with the three different behaviors .For each used and random patch, we derived the density of gravel and main roads  of nonbreeders 818±138 km2; breeders 377±39 km2; t = 3.062, P = 0.009).We explored the relationship between road availability and use at the home range scale  in a stepwise backward procedure.For gravel road availability, we created 30 m buffers along all gravel roads and calculated the proportion of the land area covered by the buffered roads within individual home ranges. Gravel road use was the proportion of hourly positions per data set within 30 m of the closest gravel road. We ran linear regression models of the proportion of wolf positions on gravel roads, with proportion of gravel road area as the main predictor. If wolves used gravel roads in proportion to their density, we would expect a linear relationship with slope = 1 and intercept = 0. In addition we added the following covariates into the full model: Home range size, median Julian date of the study period, and sex of wolves. We eliminated nonsignificant variables  = 1.54 km/h]. On roads, wolves traveled on average 1.8 times faster at a speed of 3.84 km/h . Speed was intermediate if wolves traveled partly on roads . The final LMM of travel speed included the variable Road in interaction with Reproduction, and Time of day , but there was no such preference or avoidance of main roads (P = 0.097). The separate SSFs for different times of day and behavior revealed that wolves preferred to use roads of either type for traveling, but not for other behaviors. They were 3.5 and 5.2 times more likely to travel on gravel roads during day and night, respectively  ended on gravel roads and 30 (1.0%) on main roads. Taking into account road availability in the SSF-models, wolves were 3.1 times more likely eβ,  to end avely eβ, , than ofFor each combination of time of day and behavior, the distance to the closest gravel road and/or main road was a significant predictor of the SSF . HoweverWhile resting during day time, wolves preferred intermediate distances to gravel roads, and they were 1.4 times more likely to bed at distances of 1–1.5 km from the closest gravel road as compared to directly at the road . They prWhile traveling during day time, wolves preferred areas far from main roads  and at iβ) if the gravel road density was increased by 1 km/km2  of the land area of the home ranges (n = 33). Road use varied highly among nonbreeding wolves (n = 12), with on average 4.4±2.5% (range 0.7–14.8%) of hourly positions on gravel roads  was less variable (mean = 3.3±0.6%) and positively related to road availability . The slope was significantly lower than 1  (Bison bison L.)   and bisoison L.)  traveledTerritory maintenance by scent-marking, which allows efficient communication toward intruders, is another plausible explanation for the extensive use of roads by wolves. Wolves regularly scent-mark along roads , and havAlthough at the site scale Scandinavian wolves displayed an overall preference for roads during summer, this preference was dependent on the road type, with main roads being less attractive than gravel roads. These findings support results from other studies that showed that wolves decreased use of roads and paths or increased their distance from these features with increasing size of roads or increasing rate of human use . Time ofResource selection is a function of the behavioral state of an animal . AlthougMinimizing the probability of encountering humans has been identified as an important factor shaping habitat selection of wolves within the home range, and road density has been used as a proxy for this . HoweverFinally, we expected gravel road use by wolves to vary with gravel road density across home ranges. This functional response of wolves to roads was only supported for breeding wolves in Scandinavia. Although road use by breeders was positively correlated with gravel road density, the functional response was less than proportional to gravel road availability, that is breeding wolves used gravel roads less frequently than expected as gravel road densities increased. In a study of two wolf packs tracked on snow in Alberta, Canada, Ursus arctos L., Ursus americanus Pallas) habitat by limiting the mortality risk of hunting and poaching , Swedish University of Agricultural Sciences, the Swedish Association for Hunting and Wildlife Management, the Swedish Carnivore Association, several county municipalities, and Hedmark University College.This SKANDULV study was supported by the


Survivors of critical illness are at increased risk of fractures. This may be due to increased osteoclast formation during critical illness, leading to trabecular bone loss. Such bone loss has also been observed in Paget’s disease, and has been related to deficient autophagy. Deficient autophagy has also been documented in vital organs and skeletal muscle of critically ill patients. The objective of this study was to investigate whether deficient autophagy can be linked to critical illness-induced bone loss.in vitro and their precursor cells isolated from peripheral blood of critically ill patients and from matched healthy volunteers were analysed for the expression of autophagy genes , and proteins ) and for autophagy and epigenetic signalling factors via PCR arrays and were treated with the autophagy inducer rapamycin. The effect of rapamycin was also investigated at the tissue level in an in vivo rabbit model of critical illness.Osteoclasts grown in vitro from the blood precursor cells isolated from critically ill patients, which accumulated p62, and displayed reduced expression of Atg5, Atg7, and LC3–II compared to healthy controls, suggesting deficient autophagy, whilst addition of rapamycin reduced osteoclast formation. PCR arrays revealed a down-regulation of histone methyltransferases coupled with an up-regulation of negative regulators of autophagy. Critically ill rabbits displayed a reduction in trabecular and cortical bone, which was rescued with rapamycin.Many more osteoclasts formed in vitro and reduced the amount of bone loss in critically ill rabbits in vivo. These findings may help to develop novel therapeutic targets to prevent critical illness-induced bone loss.Deficient autophagy in osteoclasts and their blood precursor cells at least partially explained aberrant osteoclast formation during critical illness and was linked to global histone hypomethylation. Treatment with the autophagy activator Rapamycin reduced patient osteoclast formation  After 6 weeks, the tibiae were processed to paraffin and sections of 5 μm thickness were cut and stained for H&E, and the amount of trabecular bone quantified by ImageJ® software  [t test was used for the comparison of normally distributed data (presented as mean ± SD), and the Mann–Whitney U test for data that were not normally distributed . P values less than or equal to 0.05 were considered statistically significant. Statistical significance is indicated on all graphs as follows: *: p < 0.05, **: p < 0.01, ***: p < 0.001.The data were processed using the Statistical software package StatView 5.0.1 (SAS Institute Inc.). Student’s (Day 0 of culture) from critically ill patients displayed lower levels of Atg5 protein than those from healthy controls. Levels of LC3–II were unchanged, and an accumulation of p62 protein was observed, suggesting autophagy was already supressed in these non-cultured precursor cells. Additionally, in PBMCs which had been differentiated in culture for 14 days, protein levels of both Atg5 and LC3–II were decreased when compared to cultured cells from healthy controls. In addition, p62 protein levels were increased, suggesting an accumulation in p62  and histone H3 methylated Lys27 (H3K27) was lower than in those from healthy controls, whilst levels of unmethylated histone H3 were unchanged Fig. . The autIn order to examine the direct effect of an autophagy inducer in critically ill rabbits, trabecular, and cortical area were analysed in sick rabbits treated with rapamycin or saline. After 3 days, the number of trabeculae was not significantly lower in critically ill rabbits compared to controls (15.3 % (p = 0.20); Fig. To investigate the effect of rapamycin on osteoblast differentiation the gene expression of RUNX2, OSX, and COL1A1 were measured in the trabecular bone of critically ill rabbits treated with rapamycin or saline and in healthy control rabbits. The expression of these 3 markers was significantly lower in the critically ill rabbits than in healthy controls (RUNX2: 54 % (p ≤ 0.01), OSX: 64.3 % (p ≤ 0.01) and COL1A1: 57.9 % (p ≤ 0.01) Fig. . AdminisOsteoclast differentiation in trabecular bone was assessed through the gene expression of markers of 2 alternative signalling pathways attributing to osteoclastogenesis, RANKL/RANK/OPG, and CD16, as we have previously shown that CD16 signalling is essential for osteoclastogenesis during critical illness . No chanin vitro, where PBMC osteoclast precursors in the blood of critically ill patients displayed markers of supressed autophagy, which was maintained upon differentiation into mature osteoclasts. Furthermore, treatment with the autophagy inducer rapamycin resulted in an inhibition of pathological osteoclast formation from patient PBMCs. Interestingly, global histone methylation was reduced in patient PBMCs and osteoclasts, and this was associated with an increase in the expression of autophagy-inhibiting genes such as BCL2L1. It is known that female survivors of critical illness display increased fracture risk up to 8 years following ICU discharge [In the current study we have investigated the role of autophagy in critical illness-induced bone loss, and the potential role of epigenetic changes. The cellular control of autophagy in human critical illness was studied ischarge , 4. In tin vitro phenomenon. However, the observation of an accumulation in p62 protein in patient osteoclasts reinforces the hypothesis that the autophagy process is deficient during critical illness.We have previously shown that autophagy is insufficiently activated in liver, kidney, and skeletal muscle of critically ill rabbits , 8. AddiInduction of autophagy with the mTOR inhibitor rapamycin significantly reduced aberrant osteoclast formation in patient PBMC cultures, but had no effect on healthy control PBMCs, supporting the hypothesis that deficient autophagy plays a key role in the extreme osteoclast formation observed in critical illness.in vitro. The expression of key developmental genes is often regulated by H3K9 and H3K27 hyper- or hypomethylation, and in mature osteoclasts, H3K27 at the promoter region of NFATc1 gene is markedly hypomethylated [in vitro  raised levels of exogenously expressed HTT, reduced cell viability, and increased the number of cells bearing mutant HTT aggregates [The epigenetic regulation of autophagy has recently been highlighted as a key factor in cell survival. In particular, methyltransferases such as G9a have been shown to coordinate the transcriptional activation of key regulators of autophagosome formation by remodeling the chromatin landscape . In the thylated . In ordethylated , and blogregates . Importagregates . These gin vitro and improved bone phenotype observed in critically ill rabbits treated with rapamycin is due to the immunosuppressive effects of rapamycin rather than its autophagy-inducing properties. In addition, we cannot exclude the possibility that the bone-sparing effects observed with rapamycin are not due to direct effects on osteoclasts and osteoblasts in the trabecular bone, but are secondary to alterations in the immune system, inflammation, or angiogenesis. Consequently, further work is required to identify therapies that induce autophagy but do not cause immunosuppressant side effects in critically ill patients.We have previously shown that rabbits made critically ill for one week displayed bone loss and displayed signs of insufficiently activated autophagy in other organs and tissues , 21. In in vitro and in an in vivo rabbit model of critical illness, reduced the amount of bone loss. These findings may help to develop novel therapeutic targets to prevent critical illness-induced bone loss.The present study has confirmed that PBMCs from critically ill patients are predisposed to form an increased number of mature osteoclasts compared to PBMCs from healthy controls. This increase in osteoclast formation is associated with deficient autophagy, which may be mediated by global histone modifications. Treatment with the autophagy inducer rapamycin reversed these findings


These beneficial effects of taVNS also exist in pinealectomized rats, which otherwise would show overt and continuous hyperglycemia, hyperinsulinemia, and high HbAlc levels. We concluded that multiple taVNS sessions are antidiabetic in T2D through triggering of tidal secretion of melatonin. This finding may have potential importance in developing new approaches to the treatment of T2D, which is highly prevalent, incurable with any current approaches, and very costly to the world.Melatonin plays a protective role in type 2 diabetes (T2D) through regulation of glucose metabolism. Whether transcutaneous vagus nerve stimulation (taVNS) is antidiabetic and whether a modulated melatonin production is involved in the antidiabetic mechanism of taVNS is unknown. In this study, once daily 30min noninvasive taVNS was administered in Zucker diabetic fatty  and Zucker lean  littermates under anesthesia for 5 consecutive weeks. The acute and chronic influences of taVNS on the secretion of melatonin were studied as well as the effects of taVNS on blood glucose metabolism. We found that naïve ZDF rats develop hyperglycemia naturally with age. Each taVNS session would trigger a tidal secretion of melatonin both during and after the taVNS procedure and induce an acute two-phase glycemic change, a steep increase followed by a gradual decrease. Once daily taVNS sessions eventually reduced the glucose concentration to a normal level in seven days and effectively maintained the normal glycemic and plasma glycosylated hemoglobin (HbA Type 2 diabetes (T2D) is a metabolic disorder characterized by high blood sugar levels due to increased insulin resistance, in which the body’s cells have lost the ability to respond adequately to relatively normal levels of insulin . T2D is Melatonin (N-acetyl-5-methoxytryptamine) is the hormone that functions as the mediator of photoperiodic information to the central nervous system in vertebrates and allows central circadian regulation of numerous physiological homeostatic mechanisms –6. It waAcupuncture has been shown to decrease blood glucose level in T2D  and is bIn our previous studies, taVNS ameliorated depression , a condiad libitum except on the days when the rats be fasted for 6 hours and a blood glucose level test will be taken. The body weight was recorded weekly and average daily food intake calculated in a whole for each group during the experiment. Rats entered the experimental procedure at 8wk of age, divided into ZDF and ZL groups according to the rat’s size, appearance, and blood glucose level. The ZDF or ZL rats were further divided into subgroups randomly. The investigators were not blinded to the group allocation during the experiment because treatment procedures, for example the taVNS or melatonin injection, must be kept in one group, and the tests used in this study are all objective. For this study, only male ZDF rats of the same age were used to avoid a possible confounding effect from gender and age differences on the levels of endogenous melatonin, glucagon, insulin, and other possible hormones. The experimental protocol was approved by the Institutional Animal Care and Use Committee in China Academy of Chinese Medical Sciences. Principles of laboratory animal care were followed.Male Zucker diabetic fatty  rats and Zucker lean  littermates were purchased at 5wk old from Vital River Laboratories International Inc. . Animals were housed under controlled temperature (21°C±2°C), relative humidity (50%±10%) and artificial light . Littermates from the same or foster mother were housed in one large cage with distilled water and a standard rat diet pellets available All the time points recorded in this study are in accordance with the taVNS occurrences, i.e. the first taVNS session occurs on day 1, the seventh day recorded as W1, and so on. For taVNS, under 2% isoflurane inhalation anesthesia, two oppositely charged magnetic electrodes (+/-) were placed over the auricular concha region, inside and outside respectively, of each ear. Saline was applied between an electrode and the skin to improve electric conductivity. A session of 30min transcutaneous electrical stimulation at frequencies of 2/15 Hz  and an intensity of 2mA was applied via an electrical stimulator . The procedures were given in the afternoon after a blood glucose concentration test and a blood sample collection at designated time points. Auricular margin was used as the sham acupoint. The electroacupuncture condition at auricular margin was same as that at taVNS except the stimulate location .By using Ascensia Breeze Blood Glucose Monitoring System , the glucose concentration was tested from tail tip-prick blood samples. To determine the acute effects of taVNS on glucose concentration, blood glucose was tested for the first 5 consecutive days, 6 times per day: immediately before, 15min during, immediately after, 30, 60, and 120 min after the taVNS procedure. The chronic effect of taVNS on glucose metabolism was determined by weekly measurements of glucose concentration at 0 (baseline), 1, 2, 3, 4, and 5 weeks, before daily taVNS or melatonin treatments. The test range was 0.6–33.3 mmol/L. Any concentration over the testing limit was recorded as 33.3 mmol/L for statistical purpose.For the melatonin injection group, melatonin was injected intraperitoneally once daily in the afternoon and after blood glucose test (4 P.M.) at designated time points. Melatonin was purchased from Sigma Chemical Co.  and dissolved in 5% ethanol saline (v/v) immediately before use. The injected dose was 60mg/kg body weight.lc were examined in pinealectomized rats. The pineal gland was removed from rats according to a reported method [To find out the role of taVNS in pinealectomized rats and to explore whether there are other melatonin sources, the blood concentration of glucose, melatonin, insulin, and HbAd method . Brieflylc were evaluated using enzyme linked immunosorbent assay (ELISA) kits purchased from R&D System  and analyzed by Huanya Biomedicine Technology Co. LTD . The results were read using a microplate reader  at wavelengths of 450nm.Blood samples were collected from right atrium upon final sample collection or from tail tip veins at other time points, centrifuged for 10min at 110 g in cold room and plasma was collected. All plasma samples were wrapped in foil and stored at -80°C until use. Concentrations of plasma melatonin, serotonin, glucagon, insulin, and HbABy running GraphPad InStat version 3 for Windows, raw data of body weight, food intake, glucose concentration, and ELISA results at last time point were compared using repeated measure One-way ANOVA to detect differences among treatment groups, followed by Tukey-Kramer Multiple Comparisons Test to determine sources of differences. Data were presented as mean ± standard deviation. Differences were considered to be statistically significant at the level of α = 0.05. For the acute effect of taVNS on the secretion of hormones during taVNS session and two hours immediately after it, only average of raw data and standard difference were used to produce the dotted lines with error bar for secretion-shape and secretion-duration comparing propose.The body weight change in naïve rats would be considered natural. Compared with that in naïve rats, the body weight of taVNS treated ZL rats was lower after three weeks of treatment and for ZDF rats two weeks after. For the daily melatonin administrated ZDF rats, the body weight was comparably lower than naïve ZDF rats two weeks after the beginning of injection .As compared for the whole duration of treatments, the average daily food intake for different treatment groups was not significantly different except the melatonin injection ZDF rats, which ate less food (P<0.05) as compared with naïve ZDF rats .As detected from samples taken on day 1, 3, and 5, each taVNS session would induce tidal melatonin release with multiple parabolic waves. The waves existed both during and after the taVNS session . A compaBoth of the concentrations of glucagon Fig  and 2d aAt 8 weeks of age, the glycemic level in naive ZDF rats was 19.793±7.158 mmol/L . ZDF rats develop hyperglycemia naturally with age but the procession could be broken/reversed by taVNS . AlthougThe antidiabetic effects of long term taVNS treatment is reflected on the well-controlled glucose metabolism in taVNS treated ZDF rats, as compared with that in naïve  and pineThe baseline glycemic concentration of ZL rats was 6.733±0.724 mmol/L . It decreased to a lower level at week one of the consecutive daily electric stimulation in both taVNS and auricular margin electroacupuncture (AMEA) treated rats. The taVNS showed no significant influence on the glycemic level in ZL rats thereafter .lc in naive ZDF rats was significantly higher than in ZL littermates (lc to a much higher level, taVNS or melatonin treatment maintained it at a lower level comparable with that in naive ZL rats (At an age of 12 wks, the plasma HbAtermates . While p ZL rats .The effect of long-term treatments on the secretion of melatonin, including taVNS, AMEA, as well as melatonin injection, was shown in The group of ZDF rats treated with once daily melatonin injection was used as a positive control. Results from this group were distributed in In this study we found that taVNS triggers melatonin release in a tidal manner and inhibits the progression of T2D. Physiologically, melatonin is secreted from both pineal and extrapineal sources –26. The Melatonin modulates glucose metabolism through receptor-dependent influences on glucagon and insulin secretion ,27,28. IIt has been reported that melatonin increases glucagon production from pancreatic α-cells  but inhiMelatonin is reported to inhibit insulin production through an MT2 receptor mediated cGMP signaling pathway in β-cells  with a fThe α- and β-cells may not release hormones at the same rhythm for reasons that, acute exposure of MT2 to physiological or supraphysiological concentrations of melatonin would induce a concentration- and time-dependent receptor desensitization and internalization, which would take 8–24 h to resensitize , whereasAlthough taVNS is antidiabetic in ZDF rats, it has limited efficiency in ZL control. This may be due to a hyperinsulinemia induced sympathetic hyperactivity which reduces melatonin production and increases insulin resistance in T2D rats . The taVA theoretic cascade in T2D following taVNS is shown in Our current findings may have potential importance in developing new approaches to the treatment of T2D, which is highly prevalent, incurable with any current approaches, and very costly to the world. We suggest taVNS or short half-life melatonin agonists for the treatment of the illness. One of the advantages of our finding is that taVNS clinics can be easily setup worldwide.


Absence of cost-effectiveness (CE) analyses limits the relevance of large-scale nutrition interventions in low-income countries. We analyzed if the effect of invitation to food supplementation early in pregnancy combined with multiple micronutrient supplements (MMS) on infant survival represented value for money compared to invitation to food supplementation at usual time in pregnancy combined with iron-folic acid.Outcome data, infant mortality (IM) rates, came from MINIMat trial . In MINIMat, women were randomized to early (E around 9 weeks of pregnancy) or usual invitation (U around 20 weeks) to food supplementation and daily doses of 30 mg, or 60 mg iron with 400 μgm of folic acid, or MMS with 15 micronutrients including 30 mg iron and 400 μgm of folic acid. In MINIMat, EMMS significantly reduced IM compared to UFe60F (U plus 60 mg iron 400 μgm Folic acid). We present incremental CE ratios for incrementing UFe60F to EMMS. Costing data came mainly from a published study.By incrementing UFe60F to EMMS, one extra IM could be averted at a cost of US$907 and US$797 for NGO run and government run CNCs, respectively, and at US$1024 for a hypothetical scenario of highest cost. These comparisons generated one extra life year (LY) saved at US$30, US$27, and US$34, respectively.Incrementing UFe60F to EMMS in pregnancy seems worthwhile from health economic and public health standpoints.ISRCTN16581394; Date of registration: Feb 16, 2009.Maternal and Infant Nutrition Interventions, Matlab;  The Bangladesh Integrated Nutrition Project (BINP) and its successor National Nutrition Program (NNP) implemented food supplementation programs for malnourished pregnant and lactating women and undernourished children less than two years of age , 2. ThesIt has been debated whether to move from the routine iron-folic acid supplementation in pregnancy to multiple micronutrients (MMS) in low- and middle-income settings . This moThese studies were conducted in populations often at risk of both macro- (food) and micronutrient deficiencies and MMS was not given in combination with food supplements. Prenatal food supplementation in the Gambia was associated with 46 % reduction in mortality during the first week of life . A CochrIn a randomized trial with the acronym MINIMat  we have shown a substantial reduction in infant mortality (IM) with early invitation to prenatal food supplementation (E - around week 9 pregnancy) combined with MMS (EMMS) compared to invitation to food supplementation at usual time (U- around week 20) in pregnancy and 60 mg iron 400 μgm folic acid (UFe60F) .Since there are questions on the affordability of large-scale food supplementation interventions and whether or not to move from the routine iron folic acid supplementation to MMS, we examined if EMMS represented value for money. We present incremental cost-effectiveness ratios (ICERs) for incrementing usual invitation to food supplementation combined with iron-folic acid to invitation to food supplementation early in pregnancy combined with multiple micronutrients for women in rural Bangladesh.Outcome data came from MINIMat trial . MINIMat was a randomized trial , conductOutcome data, IM, came from intent-to-treat analysis of MINIMat trial comparing UFe60F and EMMS arms, HR 0.38 (95 % CI: 0.18 to 0.78) . TherefoAdherence to food and micronutrients also came from MINIMat study; 60 food packets and 113 capsules in UFe60F arm, and 94 food packets and 107 capsules in EMMS arm .We assumed the effects of MINIMat intervention were accumulated from June 2002 through June 2004 and this can be represented by reductions in IM in the EMMS arm compared to the UFe60F arm.By using life expectancy (LE) at birth, 70 years in the year 2012 , we calcMINIMat intervention was distributed from September 2001 to end of 2003. The costing study was conducted from October 1999 to March 2000 [The direct cost for the intervention included food and micronutrient supplements, staff, training and meeting, administration, capital, community volunteer time, and recurrent activities. The indirect cost included the cost of participants’ time.Most cost data were available from Khan and Ahmed , while dCNCs operated for malnourished pregnant and lactating women and children under two years of age. Per day at the NGO run CNCs, there were 9.36 pregnant women, 9.71 lactating women and, and 10.29 under two-year-olds. In total 19.07 pregnant and lactating women represented 19.07 adult equivalents and, 10.29 children represented 5.15 adult equivalents equal to 24.22 adult equivalents. This was because each pregnant or lactating woman was offered four packets and each child was offered two packets of food supplement; thus, two children equals to one adult. There were 7579 (24.22*313) person days per year . Pregnant women represented 2930 (9.36*313) person days a year and used about 32 % (9.36/29.36*100) of working time at the CNC [Staff cost was from Khan and Ahmed , who derCapital costs, space for CNCs, and instruments for screening, maternal height and weight measuring scales, were available from Khan and Ahmed . From thParticipant cost was estimated at cost of a laborer when labor cost is the lowest . UNICEF For ICERs for extra IM averted we calculated the difference of costs for supplementing 1000 pregnant women in EMMS and UFe60F arms, difference of IM rates between these two arms and then divided the cost difference by the difference of IM rates. For ICERs for LY saved, the same steps were followed after multiplying the IM averted (IM rate in UFe60F – IM rate in EMMS) with discounted figures of LE at birth. For sensitivity analyses, we used the lower and upper limits of 95 % CI of HR from the intent-to-treat analyses comparing EMMS and UFe60F arms in MINIMat, and converted the resulted number of infant deaths to IM rates. The above-described steps for calculating ICERs were then followed and ICERs for extra IM averted and extra LY gained were calculated.Using the adherence levels and costs from Table The ICERs for one extra IM averted by switching from UFe60F to EMMS were US$907, and US$797, respectively, for NGO run and government run CNCs Table . This fiThe IM rates associated with the lower limit of 95 % CI of HR from the intent-to-treat analysis showed ICERs for one extra IM averted by switching UFE60F to EMMS were US$685, and US$602, respectively for NGO run and government run CNCs, and US$774 for the hypothetical highest cost scenario Table . ICERs rThe IM rates associated with the upper limit of 95 % CI of HR showed ICERs for one extra IM averted were US$2577, and US$2266, respectively for NGO run and government run CNCs; and US$2911 for the hypothetical highest cost scenario Table . Under tOur incremental CE analyses have shown by using actual delivery modes one extra IM could be averted at a cost of about US$797 to US$907 by switching from UFe60F to EMMS in pregnancy. Our analyses have also shown one extra LY can be saved at a cost of US$27 to US$30 by switching from UFe60F to EMMS. We consider the amount of expenditures to attain these gains, which is less than per capita gross domestic product (GDP) US$958 in the year 2013 in Bangladesh  represenThe effect data in the CE analysis are considered to be valid, as the trial design offered unbiased estimates of treatment effects. All pregnant women in the area were invited to participate in the study, and the pool of pregnant women under trial was probably representative of rural settings in Bangladesh, where women in general are short, and there is occasional food insecurity. Therefore the ICERs presented may be considered relevant for similar settings. The cost data were collected within the time frame and situation similar to that of the MINIMat trial and we believe it reasonably represents costs incurred by these interventions.et al. questioned the affordability and cost-effectiveness of the program [et al. questioning the US$60 million budget as erroneous and incomplete.As the overall cost of these programs is high, an evaluation by Hossain  program . By desiIn a micronutrient trial  conducteThe cost per extra IM averted estimates, US$757 to US$907, from government run and NGO run CNCs are less than per capita GDP, US$958, in Bangladesh in the year 2013 , which aA community based newborn care intervention in Bangladesh has shown that a neonatal death could be averted at a cost of US$ 2939 , which iA recent study indicated that monitoring and start-up costs might not be trivial . The cosWe presented cost per pregnant woman for NGO run and government run CNCs, and for a hypothetical highest cost scenario. Since food cost comprises the largest component cost, low food cost if delivered through government run CNCs resulted in substantially lower ICERs for extra IM averted compared to ICERs for NGO run CNCs and the hypothetical highest cost scenario. These differences are also evident in case of one extra IM averted based on effect estimates from the upper and lower limits of 95 % CI of HR comparing UFe60F and EMMS. Depending on prevalence of risk factors for IM and responsiveness of these risk factors to prenatal food and micronutrient interventions, the effect estimates as captured in 95 % CI of HR may result in substantial reductions in IM from prenatal food and micronutrient interventions and highly favorable ICERs may be observed. Since actual show-ups of enrollees were higher for government run CNCs  possiblyWe do not have data on treatment cost for the side effects of micronutrients and other intangible costs. Similarly, our analysis is based on one positive outcome, IM reduction; we did not include other tangible and intangible benefits of the intervention that may have occurred .Our analysis shows that at US$797 to US$907 and US$27 to US$30 respectively, one extra IM can be averted and one extra LY can be saved by switching from invitation to food supplementation at usual time in pregnancy and iron-folic acid supplementation to an early initiation to food supplementation combined with MMS. These figures are below per capita GDP, US$958, for the year 2013 in Bangladesh. Compared to the economic venture, the health gains can be considered high in low-income countries and in the global context. These results should aid decision-making that prioritizes pregnant women’s health. Further research is needed to understand if better infant survival can be achieved by improving diet during early pregnancy.


Elevated fasting levels of branched chain amino acids  in venous blood are associated with a variety of metabolic impairments, including increased risk of type 2 diabetes (T2D). Fasting BCAA levels are influenced by non-dietary factors. However, it is unknown whether fasting BCAAs can be altered through manipulation of dietary intake alone.To test whether a specific dietary intervention, using differences in BCAA intake, alters fasting BCAA levels independent of other factors.Five healthy male volunteers underwent 4 days of a low and 4 days of a high BCAA content dietary intervention . All food and supplements were provided. Fasting BCAAs were measured from venous blood samples by mass spectrometry at baseline and after each intervention.p<0.0001 for all). Fasting valine was significantly lower (p=0.02) and fasting isoleucine and leucine were numerically lower following the low BCAA content vs. the high BCAA content diet levels. The inter-individual response to the dietary interventions was variable and not explained by adherence.Diets were isocaloric; contained equal percentages of calories from carbohydrate, fats, and protein; and differed from each other in BCAA content (1.5±0.1 vs. 14.0±0.6 g for valine; 4.5±0.9 g vs. 13.8±0.5 g for isoleucine; 2.1±0.2 g vs. 27.1±1.0 g for leucine; Short-term dietary manipulation of BCAA intake led to modest changes in fasting levels of BCAAs. The approach from our pilot study can be expanded to test the metabolic implications of dietary BCAA manipulation. Elevated fasting levels of branched chain amino acids  measured in venous blood have been associated with metabolic risk factors for type 2 diabetes (T2D), including obesity, insulin resistance, and elevated fasting glucose –5. AmongIn animals, dietary manipulation of BCAAs can alter glycemic metabolism. In obese mice, nutritional interventions that increase all three BCAAs in the diet led to increased fasting levels of all three BCAAs and promoted insulin resistance . A seconNo human study to date has examined the effect of dietary manipulation of BCAA intake on fasting levels of BCAAs. One group has demonstrated that intravenous infusion of amino acids (containing BCAAs and non-BCAAs) acutely worsened insulin sensitivity . Yet, th2, change of weight by more than five pounds during the month prior to enrollment, use of nicotine-containing products during the study, use of drugs of abuse, conditions causing intestinal malabsorption, and inability to adhere to a prespecified meal plan, including standardized meals, abstinence from alcohol, and limitation to one caffeinated beverage per day. An initial exclusion threshold of BMI greater than 25 kg/m2 was changed to 28 kg/m2 to facilitate recruitment.We recruited and enrolled five healthy, adult men, age 20–40 years, into this single-center, single-blind, crossover nutritional intervention study. This cohort size was selected to provide 75% power to detect a difference in fasting BCAAs at alpha=0.05, assuming an effect of diet as large or larger than that observed in prior animal studies . EnrollmThe protocol was approved by the local institutional review board, and informed consent was obtained from all study participants. The trial was registered on Clinical Trials.gov (NCT02110602) prior to enrollment. Participants were recruited through local advertising between March and July 2014, and all were compensated for their study involvement.Participants attended five visits at the Massachusetts General Hospital Clinical Research Center (CRC); participants arrived to each study visit after having fasted for at least 8 h. At Visit 1, weight and fasting blood glucose were measured from a venous sample and a full medical history was obtained. A research nutritionist interviewed each participant, calculated daily caloric requirements using the Mifflin St. Joer equation, and cross-checked the output with 25–30 kilocalories per kilogram of participant body weight. Activity factors were based on the results of the Paffenbarger questionnaire. This information was used to construct the study diets with adequate calories personalized to each participant. Between Visits 1 and 2 (an approximately 2-week period), participants consumed their native diet and completed a 4-day food log, which was used to estimate nutrient content of the native diet. Food logs were not used to estimate caloric requirements for the study diets.Measurement of BCAAs below). After these measurements were completed, participants were provided with 4 days of meals and supplements for the first study period.At Visit 2, the 4-day food log was reviewed with assistance from a research nutritionist. Weight and fasting blood work for measurement of venous glucose, insulin, and BCAAs was performed just prior to starting the first study diet. Glucose was measured with a hexokinase assay ; insulin was measured using a radio-immunoassay ; and BCAAs were measured using an established platform  varied for each participant, the supplement levels in each nutritional arm were adjusted to achieve the target percentage of total protein from BCAAs at each meal.BCAAs are found in most protein-containing foods; therefore, the food components of the study diet interventions were generally low in protein. To achieve a low BCAA content intervention with an amount of protein typical for an American diet, participants consumed maple syrup urine disease (MSUD) Express  with each prepared meal. This BCAA-free amino acid supplement allowed for the food to contain a low amount of protein from the BCAAs for the low BCAA content diet but to have equal calories from protein as compared with the high BCAA content diet. The low BCAA content diet was intended to provide approximately 1% of protein calories from valine and isoleucine and 2% of protein calories from leucine, mirroring the 1:2:1 ratio for valine:leucine:isoleucine observed in a typical American diet. To achieve a high BCAA content intervention with an amount of protein typical for an American diet, participants consumed L-isoleucine, L-leucine, and L-valine supplements  with each prepared meal. The high BCAA content diet was intended to provide approximately 14% of protein calories from valine and isoleucine and 28% of protein calories from leucine, again mirroring the 1:2:1 ratio for valine:leucine:isoleucine observed in a typical American diet.Fasting BCAAs were analyzed on a validated platform for amino acid measurement ; the plaLiquid chromatography tandem mass spectrometer (LC-MS/MS) peak areas were integrated and relative quantitative information was used in these analyses. The signal-to-noise ratio (S/N) of the metabolite peak area was greater than 30 in every sample , and therefore exceeded thresholds typically used to define limits of detection (S/N>3) and quantitation (S/N>10). All LC-MS analyses were performed using a 4000 QTrap triple quadrupole mass spectrometer  coupled to either an 1100 or 1200 Series pump  or an HTS PAL autosampler  equipped with a column heater.Internal standard peak areas were monitored for quality control, and individual samples with peak areas differing from the group by more than two standard deviations were re-analyzed. MultiQuant software  was used for automated peak integration, and metabolite peaks were manually reviewed for quality of integration and compared against a known standard to confirm identity.Analyses were conducted using GraphPad Prism .t-tests were used to compare the effects of study diets. A threshold of alpha<0.05 was the threshold for statistical significance.The 4-day dietary intake data were analyzed using Nutrition Data System for Research software ; University of Minnesota, Minneapolis, MN). Pronutra  was used to analyze the nutrient content of the study diets. All fasting BCAA measurements were log transformed to approximate a normal distribution. Mean and standard error were provided for all quantitative variables, and paired 2. All participated in regular exercise at baseline and consumed at least two meals prior to starting the study.Five participants were enrolled into the study and all completed both controlled diet interventions . All volNutritional characteristics of the native (baseline) diet and study diets are shown in p=0.006), leucine (p=0.005), and isoleucine (p=0.007), and the high BCAA diet had higher amounts of valine (p<0.0001), leucine (p<0.0001), and isoleucine (p<0.0001). Consequently, the amount of each BCAA was different in the low and high BCAA diet .By design, the low BCAA and high BCAA diets differed from the native diet and from each other in BCAA content . As compp=0.52 and p=0.79, respectively) or high BCAA content intervention . Weight at baseline was 81.4±5.2 kg and was slightly lower when compared with the weight following either the low BCAA intervention  or high BCAA intervention ; weight was not different at the end of the low and high BCAA content interventions .During the low BCAA diet, participants consumed 90% of the study meals and supplements and consumed less than one non-study meal. During the high BCAA diet, participants consumed 97% of the study meals and supplements and consumed less than one non-study meal. Participants adhered to not more than one coffee or tea per day and did not consume alcohol during both diets. Each participant maintained baseline levels of exercise during both study periods. Metabolic parameters remain unchanged during study diets. Fasting glucose and insulin at baseline were 89±3.02 mg/dL and 6.3±1.2 µIU/mL, respectively, and there was no change in either fasting glucose or insulin as a result of the low (p=0.02) and numerically lower for leucine (p=0.16) and isoleucine (p=0.38). Thus, a consistent pattern of modest differences in fasting BCAA levels was observed between the two study diets.The fasting BCAA levels for each participant, separately, are shown in Subjects 1, 3, and 4 had the largest differences in fasting valine, leucine, and isoleucine following the low and high BCAA diets, and these differences followed the expected pattern of higher levels following the high BCAA diet as compared with the low BCAA diet. Subjects 2 and 5 had smaller differences in the amino acids following the low and high BCAA interventions, and these differences did not always follow the expected pattern.There were no adverse events during the study interventions. The participants rated the quality of the dietary interventions relatively high . For the low and high BCAA interventions, respectively, taste of food was rated (mean [lowest–highest]) 8.8 –10 and 9In this pilot study, we tested whether a specific dietary intervention, using differences in BCAA intake, alters fasting BCAA levels independent of other factors. Participants tolerated the diets, rated them favorably on qualitative measures, and adhered closely to the prescribed interventions. Fasting valine was lower and fasting leucine and isoleucine trended lower after a low BCAA content diet as compared with after a high BCAA content diet. Group trends were for fasting valine, leucine, and isoleucine to be lower after the low BCAA diet and higher after the high BCAA diet as compared with levels on a native diet, but these effects were not consistent across subjects.While the nutritional intervention in this study had modest effect, our findings suggest that interindividual differences, not characterized in this study, may have a more important role than dietary intake in determining fasting levels of BCAAs. This result is consistent with larger observational studies that have found no association between BCAA intake, as assessed by dietary recall, and fasting BCAA levels . Genome-Fasting levels of BCAAs are strongly influenced by protein degradation  and uptaThese results indicate that application of only a low or high BCAA content nutritional intervention in the short term without physical activity restriction is unlikely to result in a consistent decrease or increase, respectively, in circulating BCAAs from a native diet across individuals. Furthermore, because participants indicated they could adhere to either diet for less than 8 weeks, this approach is unlikely to be effective for prevention or treatment of chronic metabolic conditions.However, our pilot study showed that modest differences in fasting BCAAs can be achieved in groups of individuals by comparing levels after a low BCAA diet and a high BCAA diet. The randomized, crossover design of this study increases the likelihood that any observed group differences were related to the intervention itself. While a statistical separation in fasting valine levels on a low versus high BCAA diet was achieved in this study with five individuals, larger studies using our interventions could achieve this separation for fasting leucine and isoleucine as well. Assuming a similar effect size as observed in this study, we would have 85% power to detect a significant difference in fasting leucine and isoleucine levels with 16 or 47 subjects, respectively. A study of such size could use the nutritional intervention from this study to explore mechanisms linking fasting BCAAs to altered metabolism.The nutritional intervention platform developed here may be applied to other amino acids and nutrient metabolites. Our approach is flexible, incorporates food and specific supplements, and, because we used a metabolite profiling platform that can quantify amino acid and non-amino acid metabolites simultaneously, permits measurement of a broad range of dietary hypotheses. Whether fasting levels of other amino acids or nutrient metabolites with possible metabolic effects can be more easily modulated through dietary intake should be tested in different experiments. Thus, our platform offers a structured method of testing the influence of controlled dietary manipulation on fasting levels of specific nutrient metabolites and their implication on different conditions.Strengths of this study include a design of a novel controlled dietary intervention, crossover application in random order to the low and high BCAA diets so that each subject served as his own control, high adherence to study diet, and measurement of BCAA levels in the fasted state before and after each intervention. Still, we acknowledge limitations to our study. We studied healthy subjects and for only 4 days on each intervention. This pilot study provided data to demonstrate that our method could be applied to other populations. It is possible that dietary manipulation of BCAA would be more effective in obese or insulin resistant individuals, who are known to have dysregulation of BCAA metabolism , 3, or wIn conclusion, we constructed a novel nutritional intervention to test whether short-term intake of a controlled diet could alter circulating levels of BCAAs in this pilot study. The intervention was more effective at providing a modest difference in BCAA levels following the low and high BCAA diets than in consistently lowering or raising circulating BCAAs from the native diet. Future studies can utilize the interventional construct developed here, along with physical activity restriction, to test the effect of BCAA or other amino acid manipulation under different experimental settings.