Apis mellifera) contribute substantially to the worldwide economy and ecosystem health as pollinators. Pollen is essential to the bee’s diet, providing protein, lipids, and micronutrients. The dramatic shifts in physiology, anatomy, and behavior that accompany normal worker development are highly plastic and recent work demonstrates that development, particularly the transition from nurse to foraging roles, is greatly impacted by diet. However, the role that diet plays in the developmental transition of newly eclosed bees to nurse workers is poorly understood. To further understand honey bee nutrition and the role of diet in nurse development, we used a high-throughput screen of the transcriptome of 3 day and 8 day old worker bees fed either honey and stored pollen (rich diet) or honey alone (poor diet) within the hive. We employed a three factor  analysis of the transcriptome to determine whether diet affected nurse worker physiology and whether poor diet altered the developmental processes normally associated with aging.Honey bees (A. mellifera genome, 150 transcripts exhibited differential expression due to poor diet at 3d of age compared with 17,226 transcripts that differed due to poor diet at 8d of age, and poor diet caused more frequent down-regulation of gene expression in younger bees compared to older bees. In addition, the age-related physiological changes that accompanied early adult development differed due to the diet these young adult bees were fed. More frequent down-regulation of gene expression was observed in developing bees fed a poor diet compared to those fed an adequate diet. Functional analyses also suggest that the physiological and developmental processes occurring in well-fed bees are vastly different than those occurring in pollen deprived bees. Our data support the hypothesis that poor diet causes normal age-related development to go awry.Substantial changes in gene expression occurred due to starvation. Diet-induced changes in gene transcription occurring in younger bees were largely a subset of those occurring in older bees, but certain signatures of starvation were only evident 8 day old workers. Of the 18,542 annotated transcripts in the Poor nutrition has major consequences for the expression of genes underlying the physiology and age-related development of nurse worker bees. More work is certainly needed to fully understand the consequences of starvation and the complex biology of nutrition and development in this system, but the genes identified in the present study provide a starting point for understanding the consequences of poor diet and for mitigating the economic costs of colony starvation. Apis mellifera) contributes substantially to the worldwide economy and ecosystem health by pollinating a wide variety of plants [The European honey bee  for studying the biological signatures of dietary stress in nurse worker honey bees. Using mRNA-Seq in whole abdomens (including the digestive tract), Alaux et al.  found th [et al.  used oli [et al. ,19 and sA. mellifera abdomen exclusive of the digestive tract. We hypothesized that (1) starvation and aging would greatly impact gene expression and physiology, (2) that starvation would impact young bees differently than older bees, and that (3) early adult development (aging) would be affected by diet. We also aimed to provide testable predictions for research on development and nutrition in A. mellifera and develop targets for improving the productivity of bee colonies under nutritional stress. Here, we report that starvation greatly affects the expression of genes related to nurse physiology and development and that the processes that occur as adults develop into nurse bees are altered by sub-optimal diet.We aimed to further understand the transcriptional changes associated with starvation during early adult development. To this end, we assayed 3 day old and 8 day old bees kept in normally functioning hives and caged over only honey (poor diet) or honey and bee bread (rich diet) and allowed trophallaxis with the rest of their colony. Whole transcriptomes from abdominal carcasses  were analyzed to focus on changes in the 1,4=160.22, p=0.0002), host colony , age , diet by colony , age by diet , age by colony , and the three-way age by diet by colony interaction . Post-hoc analyses showed significant differences between 3d old bees fed the rich versus poor diet, 8d old bees fed the rich versus poor diet, and 3d versus 8d bees fed the rich diet, but no difference between 3d and 8d old bees fed only honey  had reduced hypopharyngeal glands, a classic signature of starvation. Hypopharyngeal gland (HG) size was measured in twelve classes of bees (2 ages × 2 diets × 3 host colonies). For each of these twelve treatment combinations, HGs were dissected from approximately 5 bees and 10 randomly selected acini were measured for each gland. Hypopharyngeal gland (HG) size was significantly affected by all factors tested in the model – diet  and the interaction between age and diet  were significant but the effect of age  was not  and the interaction between age and diet  were significant but the effect of age  was not  and diet  were significant, but the effect of the age × diet interaction  was not .The HG sizes of bees that were used to evaluate the predictive power of the mRNA-Seq results with qPCR (but that were not part of the mRNA sequencing experiment), were also measured. Only 8d old bees were used in this phase of the experiment, yielding 6 different classes of bees (2 diets × 3 colonies). For each of these 6 different treatment combinations, the HGs of 5 bees were dissected and 10 randomly selected acini were measured for each gland. The HGs from 8d old bees fed pollen were significantly larger than those from 8d old bees fed only honey  mapping to 12,340 transcripts  was differentially expressed , approximately 149.4 million paired end reads passed the initial quality control filters and approximately 103 million paired end reads mapped to the We investigated whether starvation impacted gene expression by first testing whether transcription was affected by the main effect of diet . The expression of 24 exons mapping to 13 transcripts was significantly affected by the main effect of diet and all were up-regulated in bees fed pollen compared to those that were fed a poor diet  or old (8d old) bees were analyzed separately to determine whether the effects of starvation varied with the age of the bee. Starvation caused more frequent down-regulation of transcripts in younger bees  compared to older bees  were differentially expressed due to the main effect of age Table . 187 71. The traX21=8.33, p=0.0039; Table X21=22.59, p<0.0001; Additional file Transcripts that were up- or down-regulated with age were investigated in pollen deprived and well-fed bees separately to determine whether the signatures of normal age-associated development differed in underfed versus well-fed bees. Transcript expression decreased with age more frequently in bees fed a poor diet  compared to bees fed a rich diet , encoding a hypothetical protein , and the expression of glucocerebrosidase was significantly lower  included those with potential roles in development and muscle and motor function. These results agree with those of Ament et al., who alsdicer and argonaute that mediate immunity towards viruses that are sometimes prevalent in failing colonies [Many more transcripts changed due to starvation in older bees  compared to younger bees (173 transcripts). Of the transcripts where expression changed due to poor diet, there was more frequent down-regulation of gene expression in younger bees (78.5%) compared to older bees (55.9%). Transcriptional changes occurring due to starvation at 3d were largely a subset of those occurring at 8d. Many more diet-related differences were observed only in bees deprived of pollen for 8d, but it is unclear whether the genes differentially expressed exclusively at 8d are so due to the longer period of starvation or because specific processes that are turned on only at later ages are also impacted by diet. The expression of MRJPs, immune genes, and genes encoding detoxification enzymes declined due to poor diet exclusively at later ages. This group of down-regulated transcripts included several members of the CYP4 and CYP6 cytochrome P450 families and one Delta GST, which have roles in insecticide detoxification , chitin-colonies ,25. One Aging was associated with the overall decline of processes such as development, transcription, and mRNA splicing, and this decline was exacerbated in pollen deprived bees. Age-related transcriptional decline was more frequent in underfed bees Tables . Bees feet al. [Further support for the hypothesis that aging is dysregulated in pollen-deprived bees becomes evident from comparing our results to examples presented in the literature, such as juvenile hormone esterase , vitellogenin, and hexamerin 70a. Nurses have highest levels of JHE in order to prevent the switch to foraging , as highet al.  find thaThe expression of relatively few genes changed with respect to the main effect of age, yet there was measurable overlap in the patterns of expression when we compared the genes that were up- or down-regulated with age in the poor versus rich diets  10 frame colonies with approximately 18,000 adults, 1 full frame face of brood, and 3.5 full frame faces of food stores. To collect newly emerged workers, sealed brood frames from approximately five colonies different from the host colonies used in the experiment were placed in a temperature-controlled room (approximately 34°C) and all adults that emerged in an 18 hour period were collected. These callow bees (≤ 18 hours of age) were marked using a small dot of non-toxic paint on their thorax and were added to the three host colonies, confined to each of two diets (a poor diet consisting of only stored honey or a rich diet of stored honey and bee bread) at a density of approximately one bee per cm2 (~40-60 bees per cage) using wire cages pushed into drawn comb over either diet. Bees were confined for 3 or 8 days in July and August of 2011. To sum, each of the three host colonies contained two dietary treatments, and the bees were sampled after being confined to that treatment for either 3 or 8 days, yielding three replicate (host) colonies for each diet (poor versus rich) by age (3d versus 8d) combination. Sampled bees were flash frozen in liquid nitrogen and maintained at -80°C until further processing.Three host colonies of 2) was measured using the Leica Applications Suite v.3.8.0 software. Measurements were analyzed using a mixed model repeated measures ANOVA testing for the effect of diet, colony, age, the two-way interactions between colony and diet, age and diet, age and colony, and the three-way interaction between age, colony, and diet. Each acinus was a repeated measure taken on the same bee and a compound symmetry covariance structure was employed to model the correlation among measures taken on the same bee. A post-hoc analysis was performed using the Tukey HSD test to determine whether there were significant differences in acini area between diets (rich versus poor diet) for each age (3d and 8d old) and between ages for each diet.Hypopharyngeal glands (HGs) of bees typically grow in preparation for nursing duties and this growth is impacted by the amount and source of dietary protein ,41. Our Abdominal carcasses were dissected from three bees per colony per treatment by removing their head and wings before making a dorsal incision through their abdominal cuticle, exposing the abdominal organs and forming a cup structure where RNAlater (Life Technologies) was added. After removing the digestive tract, the remaining tissue  was separated from the thorax and this abdominal carcass was preserved at -80°C for subsequent RNA extraction. Three bees per host colony were pooled and total RNA was extracted using TriReagent (Invitrogen) according to the manufacturer’s specifications. Total RNA integrity was confirmed using Agilent’s 2100 Bioanalyzer.Twelve mRNA sequencing (mRNA-Seq) libraries (2 ages × 2 diets × 3 replicate (host) colonies) were prepared using Illumina’s TruSeq RNA Sample Preparation Kit according to the manufacturer’s protocols. Briefly, poly-A containing mRNA was purified using poly-T oligo-attached magnetic beads and was subsequently fragmented and primed for first-strand cDNA synthesis with random hexamer primers. After degrading the RNA template used for first-strand cDNA synthesis, synthesis of the second cDNA strand followed, yielding a double-stranded cDNA molecule. Adapters were ligated to both ends of the double-stranded cDNA molecules and adapter-ligated cDNAs were enriched using 15 cycles of PCR using adapter-specific primers. The libraries were validated according to the manufacturer’s protocol and the approximately 350 bp fragments were isolated from a 6% Tris Base -Boric Acid-EDTA (TBE) PAGE gel and run through the Illumina sequencer for 2 × 100 cycles at a starting concentration of 12 pM per library.A. mellifera RNA sequences listed in GenBank using the Burrows-Wheeler Aligner . Using a genome  with Topa genome  version 5 genome . First, 5 genome  and a Be5 genome  at a 5% Apis mellifera version 4.5 genome annotation [Statistical analyses of differential expression yielded a corrected significance value for each exon that mapped to each mRNA transcript in the notation . If ≥2 eDrosophila melanogaster genes orthologous to the differentially expressed A. mellifera transcripts were identified by searching for reciprocal best BLAST hits between A. mellifera mRNA sequences and D. melanogaster proteins. A FlyBase gene ID (FBgn) [A. mellifera transcripts where a D. melanogaster orthologue was identified. GeneCoDis3 [D. melanogaster genes orthologous to all A. mellifera RNAs was used to determine significant enrichment of BP GO terms and annotation clusters using a chi-square (X2) test corrected for multiple testing [The significantly differentially expressed transcripts (containing ≥2 exons) were subjected to further characterization. D (FBgn)  was assineCoDis3 -52 was tneCoDis3 -52. In aneCoDis3 -52. A re testing . SignifiA. mellifera gene expression and physiology, annotated transcripts that increased or decreased due to starvation or as individuals aged from 3d to 8d old were compared and biological processes and annotation clusters corresponding to differentially expressed orthologues were analyzed for significant enrichment. To address the hypothesis that starvation affects younger and older workers differently, comparisons were made between annotated transcripts and orthologues that were affected by diet at either 3d or 8d of age. Venn Diagrams were constructed using the annotated transcripts that were differentially expressed due to diet at 3d and those that were differentially expressed due to diet at 8d. To address the last hypothesis that development and aging are affected by diet, comparisons were made between annotated transcripts and orthologues that changed with age for bees fed either a rich diet or those fed a poor diet. Venn Diagrams were constructed using the annotated transcripts that were differentially expressed due to age for the rich diet and those that were differentially expressed due to age in bees deprived of pollen.The annotated transcripts (containing ≥2 exons) and orthologues that exhibited differential expression were compared. To address the hypothesis that diet and early adult development affect N=3) were used. These bees were not used in the previous mRNA-seq experiment, but the colonies they came from shared similar population sizes, age distributions, and distribution of food stores to the three colonies used in the mRNA-seq experiments and to each other. Dietary manipulations, fat body dissections, and RNA isolations were as described above except RNA integrity was not assayed. To test that the dietary manipulations were successful, HGs were dissected and acini were measured in the manner described above. RNA was isolated from three pooled fat bodies per colony for each age and for each dietary treatment, yielding a total of six samples (3 host colonies x 2 diets). Isolated RNA was subjected to a DNase treatment (Ambion) and the lack of genomic DNA contamination was verified by performing a PCR on the RNA with A. mellifera actin  values from three technical replicates were averaged for each target gene and two standards: A. mellifera actin (AB023025.1) and GAPDH , cdc42 (XM_394608), E75 NM_001080110), GTP-binding protein 10 (XM_396976), vitellogenin (NM_001011578), glucocerebrosidase transcript variant 1  terms that showed higher expression in starved bees compared to bees fed pollen in both Ament et al.  and Alau [et al. .Click here for fileet al. [et al. [Biological process gene ontology (GO) terms that showed reduced expression in starved bees compared to bees fed pollen in both Ament et al.  and Alau [et al. .Click here for fileet al. [et al. [The core set of biological process impacted by diet in previous studies. Biological process gene ontology (GO) terms that showed similar expression patterns in starved bees compared to bees fed pollen as determined by Ament et al.  and Alau [et al. .Click here for file2), is presented for bees raised for either 3 days (3d) or 8 days (8d) and fed a diet of honey alone (H) or honey and pollen (HP) for colonies A, B, and C. Error bars represent standard error for the mean acinus size for the five individuals tested for each diet x age combination. Significant results of a post-hoc Tukey-Kramer test on the mean acinus sizes for each diet by age combination are presented.Hypopharyngeal gland (HG) sizes of well-fed and nurse workers deprived of pollen, separated by host colony. HG size, as measured by HG acinus size , the exon annotation in NCBI’s GNOMON annotation , the D. melanogaster orthologue matching the transcript , the accession number and description for the mRNA at that genomic location (columns G and H), and whether the full model yielded a significant result for the main effect of diet (column I) or age (column K) after a correction for multiple tests. Significance of pairwise contrasts between diets at 3 days (column M) and 8 days (column O), and between young and old bees fed a poor diet (column Q) or a rich diet containing beebread (column S) are presented. Where the effect was significant, the direction and magnitude of the expression change between diets (column J), between ages (column L), between diets in only young bees (column N), between diets in older bees (column P), between ages in bees fed a poor diet (column R) and between ages in bees fed a rich diet (column T) are presented and were calculated using the expression estimates in columns U through AF. Columns U through AF include the fitted expression estimates for each exon and for each host colony x age x diet combination following a generalized linear model analysis of the raw expression values  the genome accession number (column A) and start and end positions (columns B and C) where that differentially expressed exon is located on the text and ,55).Click here for fileExon-based analysis of the number of genes changing with respect to diet or age.Click here for fileComparisons of the magnitude of age-associated (A) up- or (B) down-regulation in bees fed or deprived of pollen. For each exon that was significantly affected by age in bees fed a poor diet (deprived of pollen) or a rich diet (containing pollen), the average expression among host colonies was calculated for each age by diet combination. The magnitude of age-related change was then calculated by dividing the values obtained in young bees by the values obtained in old bees separately for each diet. This yielded estimates of up-regulation with age  and down-regulation with age  for bees fed each type of diet. Means of these values across exons are presented along with the standard error around this mean.Click here for fileet al. [et al. [Biological process gene ontology (GO) terms that were up-regulated in starved bees compared to bees fed pollen in the present study, Ament et al. , and Ala [et al. .Click here for fileet al. [et al. [Biological process gene ontology (GO) terms with reduced expression in starved bees compared to bees fed pollen in the present study, Ament et al. , and Ala [et al. .Click here for fileList of rt-PCR primers used in this study.Click here for file


Outcrossing determines the level of introgression but little is known about the relationships of outcrossing rates, genetic diversity, and differentiation particularly in a weedy taxon that coexists with its conspecific crop.Introgression plays an important role in evolution of plant species Oryza sativa f. spontanea) populations from China were analyzed using microsatellite (SSR) fingerprints to study outcrossing rate and its relationship with genetic variability and differentiation. To estimate outcrossing, six highly polymorphic SSR loci were used to analyze >5500 progeny from 216 weedy rice families, applying a mixed mating model; to estimate genetic diversity and differentiation, 22 SSR loci were analyzed based on 301 weedy individuals. Additionally, four weed-crop shared SSR loci were used to estimate the influence of introgression from rice cultivars on weedy rice differentiation. Outcrossing rates varied significantly (0.4∼11.7%) among weedy rice populations showing relatively high overall Nei's genetic diversity (0.635). The observed heterozygosity was significantly correlated with outcrossing rates among populations  although no obvious correlation between outcrossing rates and genetic diversity parameters was observed. Allelic introgression from rice cultivars to their coexisting weedy rice was detected. Weedy rice populations demonstrated considerable genetic differentiation that was correlated with their spatial distribution , and possibly also influenced by the introgression from rice cultivars.Eleven weedy rice (Outcrossing rates can significantly affect heterozygosity of populations, which may shape the evolutionary potential of weedy rice. Introgression from the conspecific crop rice can influence the genetic differentiation and possibly evolution of its coexisting weedy rice populations. Introgression, as defined in its broad sense “the transfer of genes between genetically distinguishable populations” vice versa. A recent study on modeling of rice gene flow indicated that the outcrossing ability of a pollen-recipient is an important determinant for the level of gene flow The extent of introgression between species (or populations) relies essentially on their outcrossing ability, which is usually determined by the genetic nature of a plant species Oryza sativa f. spontanea; Poaceae) provides an ideal material for studying such relationships, because outcrossing rate, genetic diversity, and differentiation of weedy populations can be estimated relatively easily.Theoretical and empirical studies have indicated evident association between outcrossing and genetic diversity/differentiation of populations Weedy rice can be defined as any spontaneously and strongly shattering rice that occurs in cultivated rice fields. It is distributed all over the world, where rice is grown. Weedy rice can be discriminated from wild rice species by its habitats because weedy rice only occurs in or nearby rice fields and the wild species in natural habitats. Weedy rice is characterized by its strong seed shattering and dormancy, in addition to a red pericarp, which serves as a key to distinguish weedy rice from cultivated rice. Groups of weedy rice from various parts of the world may have separate evolutionary origins Weedy rice flowers almost simultaneously in many rice planting regions with its cultivated counterpart occurring in the same fields, and spontaneous hybridization (or gene flow) between cultivated rice and weedy rice is frequently reported Using microsatellite (SSR) fingerprints, we estimated outcrossing rates, genetic diversity, and differentiation of 11 weedy rice populations collected from NE and E China. In addition, we also examined the possible allelic introgression from cultivated rice grown in the same fields. The primary objectives of this study were to (1) determine outcrossing rates and genetic diversity of the 11 weedy rice populations, and the correlation between outcrossing rates and genetic diversity; (2) estimate genetic differentiation among the weedy rice populations across a geographical range; and (3) detect the possible allelic introgression from cultivated rice at different locations that may have affected the differentiation of weedy rice.2. The estimated densities of weedy rice populations varied between 2∼60 plants per 100 m2  , and ther 100 m2 . In HeilJaponica rice cultivars (pure lines) coexisting in the same fields were also sampled from each of the 11 sites, making up a total of 106 rice plants from the sampled sites. One to three panicles from each cultivar and weedy plant were collected and placed separately in a paper seed-bag. Panicles were air dried under natural conditions.To meet our objectives, three independent sets of materials were included in this study. To estimate outcrossing rates, 16∼20 weedy rice plants (as family) were collected from each population, with a spatial distance of >2 m between sampled plants. About 20∼25 mature seeds (as progeny) from each plant were randomly collected, and a total of 400∼600 seeds from each population were sampled. Consequently, a total of 5527 progeny (seeds) from the 11 weedy rice populations were included for analyses . To estiSeeds of weedy rice and cultivated rice were germinated in the laboratory of Fudan University at the room temperature (25±3°C) about four months after the samples were collected to release potential seed dormancy. The total genomic DNA was extracted from 10-day-old fresh seedlings following a modified cetyltrimethyl ammonium bromide (CTAB) protocol http://www.gramene.org) were randomly selected and used for analyzing weedy rice populations and rice cultivars. To estimate outcrossing rates and crop specific alleles that were defined as the alleles absent in families (mother plants) but present in progeny (seeds), six highly polymorphic SSR loci were selected, as recommended by Jarne and David SSR primer pairs designed based on published genome sequence of cultivated rice (Polymerase chain reactions (PCR) were carried out in a volume of 20 µL containing 1× buffer, 1 mM each of dATP, dCTP, dGTP and dTTP, 10 mM of SSR primer, 50 ng of genomic DNA and 0.6 unit of Taq polymerase Given the co-dominant nature of SSR markers, the amplified DNA fragments of a given locus were scored as genotype data for each weedy rice individual. For outcrossing analyses, genotype data of progeny was recorded as homozygous genotypes  or heterozygous genotypes  and formatted according to the MLTR requirement to be used for outcrossing rates estimation under MLTR model mt); (2) single-locus outcrossing rate (st); (3) the proportion of biparental inbreeding to the total outcrossing rate [(mt-st)/mt] Pa) as estimated by inverse of multilocus paternity correlation Subs) estimated by the differences between correlation of pollen genotypes calculated by multilocus and single-locus The mating system of weedy rice populations was calculated from 5527 progeny using a multilocus mixed mating model f) was calculated to estimate whether the observed outcrossing rates of weedy rice populations based on SSR markers and MLTR model deviated from theoretically expected outcrossing rates under inbreeding equilibrium (IE). Inbreeding coefficient (f) was obtained as follows: (1) calculating the most possible maternal genotypes observed in each weedy rice population based on the MLTR model; (2) calculating inbreeding coefficient (Mf) of maternal plants in each population based on maternal genotypes by the formula: Mf  = 1 - oH/eH, where oH and eH indicated the observed heterozygosity and expected heterozygosity, respectively; (3) calculating the expected inbreeding coefficient (eqf) in each population by the formula: eqf  =  (1 - mt)/(1+mt) t-test of paired samples was performed to detect the significant differences between the observed (Mf) and expected (eqf) coefficients.The parameter of inbreeding coefficient (isF) A); (3) the percentage of polymorphic loci (P); (4) Nei's genetic diversity (GD) oH). Private alleles of each weedy rice population were analyzed using the GDA  software ver. 1.1 Genetic diversity was estimated from 301 plants of 11 weedy rice populations based on 22 SSR loci using the MSA  software ver. 4.05 A, GD, and oH) were analyzed using the Pearson's correlation method packaged in the SPSS software ver. 12.0 (http://www.spss.com/). The correlation was illustrated by a linear model (y = a+ bx) using the STATISTICA ver. 6.0 .Correlations between obtained outcrossing rates from mating estimation and genetic diversity parameters  estimating frequency of alleles from paternal and maternal plants by MLTR software; (2) determining paternal specific alleles by comparing alleles from both parents, alleles that only appear in pollen donors were defined as paternal specific allele; (3) determining the paternal specific alleles that could only come from the crop by examining alleles in cultivated rice coexisting in the same fields, as crop specific alleles.Nei's unbiased genetic distances of 11 weedy rice populations (301 plants) and rice varieties (106 plants) that coexisted in the same fields were calculated together based on four shared SSR primer pairs  that highly represented genetic diversity and structure of the crop and weed, using the MSA software ver. 4.05. An UPGMA tree of weedy and cultivated rice was constructed based on the cluster analysis of Nei's genetic distances, using the software NTSYS ver. 2.02 mt), and 2.9% by single-locus outcrossing rate (st), indicating in general very low frequency of outcrossing in weedy rice populations, which correspond to the self-pollinating nature of weedy rice. A relatively high level of variation in outcrossing rates was found among populations (mt ranging from 0.4∼11.7% and st from 0.3∼8.6%). The lowest multilocus outcrossing rate was found in the JL-4 population and the highest in the HLJ-4 population  and expected inbreeding coefficient  between Mf and eqf, suggesting in general that the outcrossing rates detected in this study were reliable (Average outcrossing rates of the 11 weedy rice populations were 4.1% as estimated by multilocus outcrossing rate (pulation . To examreliable . The inbreliable . A low lreliable .Pa), indicating the average number of genetically distinct pollen donors (father plants) involved in hybridization, was nearly four plants in weedy rice populations, with the highest number of five plants and the lowest of one plant (Average effective paternity (ne plant . In addine plant .isF) estimated from 22 SSR primer pairs of 301 plants was as high as 0.987 (GD) of the 11 weedy rice populations was 0.635, with the highest value in the HLJ-4 population (0.453) and the lowest in the JL-2 population (0.145) (oH) varied considerably among the 11 weedy rice populations, with the highest value of oH = 0.023 in the HJL-4 and JL-1 populations, and the lowest value as oH = 0.000 in JL-4, JS-2, and JS-3 populations  . The obsulations .mt, r2 = 0.783; P<0.001; for st, r2 = 0.895; P<0.001) between outcrossing rates and observed heterozygosity in the 11 weedy rice populations. The correlation between multilocus outcrossing rates and observed heterozygosity could be well described by a linear model y = 0.1937x+0.0003  between the genetic distances and spatial distances of weedy rice populations, indicating that genetic differentiation among weedy rice populations was significantly correlated with their geographical distribution. The possible flow of SSR alleles from cultivated rice to coexisting weedy rice populations was examined by analyzing the paternity in weedy rice seedlings (progeny), given that a weedy rice mother plant (family) was always surrounded by cultivated rice plants . When the 11 coexisting rice cultivars were included in the cluster analysis, the structure of weedy rice populations indicated by the UPGMA tree was slightly altered . In genet-test of paired samples demonstrated that the observed inbreeding coefficients (Mf) were in significant accordance with the expected inbreeding coefficients (eqf) in all 11 weedy rice populations. This statistic showed strong reliability of the estimated outcrossing rates of the self-pollination weedy rice based on the SSR fingerprints and MLTR modeling in this study. Therefore, the detected variation in outcrossing rates among weedy rice populations may well reflect the true nature of outcrossing in weedy rice, with a low possibility of errors from the experimental procedures.Results from the present study based on >200 families  and >5500 progeny (seeds) indicated a low to moderate level of outcrossing rates (0.4∼11.7%) of weedy rice populations, although with considerable variation. The Outcrossing rate is usually determined by the genetic ability to outcross (which is a relatively stable character for a particular plant population or species) and the environmental conditions under which the outcrossing events occur In addition to variation in outcrossing rates, we also found that the proportion of biparental inbreeding  to the total outcrossing was considerably variable among weedy rice populations (ranging from ca.11∼65%). Usually, biparental inbreeding is associated with the fine-scale spatial genetic structure, in which the neighboring individuals are genetically identical or extremely close oH) in the corresponding weedy rice populations, although no such a correlation between outcrossing rates and Nei's genetic diversity or average number of alleles per locus was observed. In other words, outcrossing can considerably promote heterozygosity in weedy rice populations. Wright et al. Our results, based on the fingerprinting of 22 SSR loci from another independent set of weedy rice samples with >300 individuals from the 11 populations, demonstrated a considerable among-population variation in genetic diversity, as estimated by the average number of alleles per locus, the percentage of polymorphic loci, and Nei's genetic diversity. Interestingly, these results showed a significantly positive correlation between outcrossing rates and observed heterozygosity  although the detected geographical effect might be associated with differences between north (Heilongjiang and Jilin) and south (Jiangsu) regions. Genetic differentiation of weedy rice populations has been reported by many authors et al. Our results from the cluster analysis based on the coefficients of genetic distances showed evident differentiation among the 11 weedy rice populations. The Mantel test further indicated a significant correlation between the genetic and spatial distances of weedy rice populations across a geographical region (isF) Japonica rice fields from NE and E China, where the farming conditions for rice are very similar in the irrigated eco-system. Normally, rice was cultivated once a year or rotated with wheat or oil rape at the following seasons, but rice varieties used across the region can vary greatly. Given the frequent gene flow from cultivated rice to weedy rice Commonly, the causes of genetic differentiation among plant populations across a given geographical range can be associated with factors such as immigration/migration and environmental heterogeneity among populations As a conspecific weed of cultivated rice, individuals of weedy rice are commonly surrounded by densely populated crop in the field. Recurrent gene flow from a particular rice variety or different varieties grown at different years in the same fields can considerably shape the genetic composition of weedy rice populations, and enhance genetic differentiation among weedy rice populations by incorporating crop alleles from different varieties 2) of weedy rice plants that were surrounded by cultivated rice plants in the fields. More importantly, comparison between the paternal-specific alleles found in weedy rice and alleles detected in cultivated rice from the same fields demonstrated some paternal-specific alleles that were also detected in rice varieties. This finding confirms the allelic introgression from diverse rice cultivars to weedy rice, supporting our hypothesis of introgression from rice crop that may influence genetic structure and differentiation of weedy rice populations. In fact, farmers frequently change rice varieties between years for cultivation among regions, where considerable genetic diversity of rice varieties has been detected Data from outcrossing rate estimation in this study revealed some paternal-specific SSR alleles (paternity) that was not detected in the genotypes of maternal plants (weedy rice families). This finding suggests that the pollen donors of many outcrossing (or hybridization) events were most likely from cultivated rice, given the relatively low density (∼10 plants/100 mThe above findings have implications for understanding adaptive evolution of weedy rice in human-influenced agro-ecosystems. Weedy rice always occurs sympatrically with cultivated rice, and genes from cultivated rice can be easily incorporated into the gene pool of weedy rice through recurrent crop-to-weed gene flow and introgression. This progress may promote the persistence and better adaptation of weedy rice to the human-influenced habitats via gene flow and introgression although other factors such as selection and habitat heterogeneity may also play their roles. The knowledge of biological bases and the relationships among outcrossing, genetic diversity, and differentiation in weedy populations is essential for addressing adaptive evolution of weedy rice, in addition to assessing environmental biosafety impacts caused by crop-to-weed transgene flow from GM rice.In summary, results from this study demonstrate a considerable variation in outcrossing rates and genetic diversity among weedy rice populations from a given geographical range. The correlation between outcrossing rates and observed heterozygosity provides evidence that a high level of outcrossing may influence genetic variability and potential evolution of weedy rice populations. This might be true for other self-pollinating plant species. Our results also show considerable genetic differentiation among weedy rice populations across a given geographical range. Noticeably, the genetic structure and differentiation of weedy rice populations could be affected by cultivated rice


However, the requirement for KLB in the propagation of FGF21 action in living animals has yet to be evaluated. To answer this question, we tested FGF21 in mice with total body ablation of KLB (KLBKO) and found no detectable activity. Firstly, we demonstrate that the disruption of KLB entirely abrogates acute FGF21 signaling in adipose tissue. We go on to show that this signaling defect translates to the absence of FGF21 mediated metabolic improvements in DIO mice. Indeed, KLBKO mice are totally refractory to FGF21-induced normalization of glucose homeostasis, attenuation of dyslipidemia, elevation of energy expenditure and weight loss. The lack of FGF21-driven effects was further substantiated at the transcriptional level with no FGF21 target gene signature detectable in adipose tissue and liver of KLBKO animals. Taken together our data show that KLB is a vital component of the FGF21 in vivo signaling machinery and is critically required for FGF21 action at the whole organism level.FGF21 has gradually become a focal point in metabolic research given its intriguing and complex biology and relevance to drug discovery. Despite the large amount of accumulated data, there remains a dearth of understanding of FGF21 physiology at the molecular/whole organism level. The scaffold protein βklotho (KLB) has previously been demonstrated  The identification of FGF21 as a metabolic regulator has triggered extensive study in part due to the therapeutic potential of FGF21 to treat metabolic disease in vitroin vivo has recently become apparent in vitroin vivoStrong evidence exists that FGF21 binds KLB and that this interaction is a necessary step in FGF21 receptor complex activation in vivo.In the present study we sought to clarify the extent to which KLB is required for the function of FGF21 in animals. To achieve this goal we generated mice with whole body deletion of KLB (KLBKO) and subjected them to the acute and chronic treatment with FGF21. The outcomes of this investigation are indicative that FGF21 cannot induce acute signaling, act on its target genes or produce integrated metabolic effects in KLB-deficient animals. Taken together our data show that KLB is a necessary co-factor for FGF21 action CAACTATAGCAGCTGAGACTGACG and ACAAATCTTCAGTCGGTGGTG). Prior to study mice were crossed onto the C57BL/6 background for at least 10 generations.KLB mutant mice (KLBKO) were generated utilizing the FLP/FRT recombination system by Taconic-Artemis . Exons 1–4 of the mouse KLB gene were flanked with FRT sites. After administration of hormones, superovulated BALB/c females were mated with BALB/c males. Blastocysts were then isolated from the uterus at day 3.5. A flat tip, piezo actuated microinjection-pipette was used to inject targeted C57BL/6NTac ES cells into each blastocyst. After recovery, injected blastocysts were transferred to each uterine horn 2.5 days post coitus of pseudopregnant NMRI females. Chimerism was measured in chimeras (G0) by coat color contribution of ES cells to the BALB/c host (black/white). Highly chimeric mice were bred to C57BL/6-Tg (CAG-Flpe) females to excise the targeted region. Genotype was assessed using PCR with the following primer sequences  facility with 12 h/12 h light/dark cycle. Animal protocols in this study were approved by the Eli Lilly and Co. Animal Use and Care Committee (Protocol No. 12225).Male WT and KLBKO mice (n = 4 per time point) were maintained on a standard chow diet  prior to treatment. To determine acute signaling responses mice were administered 1 mg/kg of recombinant human FGF21 and then sacrificed at specified time points.Male WT and KLBKO mice (n = 5 per group) were maintained on a calorie-rich diet consisting of 40% fat, 39% carbohydrate, and 21% protein caloric content  and had free access to food and water before randomization by weight. Mice were administered FGF21 for a period of 14 days via continuous infusion using osmotic mini-pumps  at dose of 1 mg/kg/day. Prior sacrifice glucose levels were determined using Precision G Blood Glucose Testing System . Following sacrifice tissues were rapidly dissected and flash frozen in liquid nitrogen.Blood samples were collected on ice temperature prior to storage of plasma at −80°C. Serum metabolites were measured by small-scale enzymatic assays for glucose, β-hydroxybutyrate, triglycerides, and cholesterol (Stanbio Laboratory). Insulin , leptin  and Total Adiponectin (BioVendor Inc.) were measured by specific ELISA.RNA was isolated from tissues using TRIzol reagent  or by homogenization of frozen samples in Lysing Matrix D shaker tubes  and was reverse transcribed into cDNA using a High-Capacity cDNA Reverse Transcription Kit . Reactions were performed in triplicate on an ABI Prism 7900HT (PE Applied Biosystems) and were normalized to PPIA mRNA.Immunoblotting of WAT and liver extracts from WT and KLBKO mice was performed using an anti-KLB (R&D Systems), and β-actin as a loading control (Sigma).Data are presented as mean±SEM. Statistical analysis was performed using one-way ANOVA, followed by Dunnett’s multiple comparisons test where appropriate. Differences were considered significant when P = <0.05.We first investigated levels of KLB transcript in WT and KLBKO mice in tissues which have been suggested to have detectable KLB expression, namely, WAT, BAT, liver, pancreas, hypothalamus, and ileum , and prein vivoFollowing administration of exogenous FGF21 we examined acute signaling in WAT, a tissue we and others have recently shown to be the primary mediator for the metabolic actions of FGF21 When maintained on either chow or HFD for a period of 8 weeks the KLBKO mice were indistinguishable from WT counterparts with regards to food intake, energy expenditure (EE) and fed serum glucose indicative that at baseline the impact of FGF21/FGFR1/KLB axis on energy homeostasis is masked. These findings are reminiscent of the phenotype observed in the FGF21KO Given the absence of acute FGF21 signaling we went on to examine the metabolic effects of chronic FGF21 administration. Consistent with the established anti-obesity action of FGF21 in vivo mechanism of FGF21 action. Confirming this hypothesis we saw an increase in serum adiponectin  but consistent with Yang et al (2012). We suggest that the differences between our study and Tomiyama et al (2010) may be due to the quality/purity of protein utilized in these experiments. Indeed, Tomiyama et al (2010) employed non-purified supernatants from FGF21 cDNA transfected cells while the test article used in this study and others At the signaling level we show that removal of KLB abrogates the ability of FGF21 to activate WAT in a manner found in WT animals  indicatiCoupled with the signaling response there was a profound FGF21-induced shift in energy homeostasis but only in WT animals. Furthermore, FGF21 mediated effects on weight loss and glucose/lipid control in WT mice were all absent in the KLBKO animals. Thus, lack of WAT engagement at the signaling level due to KLB ablation translates to a complete inability of FGF21 to affect metabolic endpoints  A–E. Intin vivoOf note, the metabolic effects of FGF21 in WT mice were largely reflected at the transcriptional level with significant induction of “browning” related genes in WAT such as UCP1 and PGC1α , a phenoin vivoIn the liver cholesterol 7α-hydroxylase (CYP7A1), a rate-limiting enzyme responsible for conversion of cholesterol to bile acids, plays a key role in regulating lipid and bile salt homeostasis and a key target gene for FGF19 action SCD1 , which wet al (2012) describe severely attenuated signaling in KLBKO mice following FGF21 treatment similar in magnitude to the effect we communicate in this report. However, to study the role of KLB in the majority of their FGF21 metabolic studies a cross of KLBKO mice to FGF21 transgenic (FGF21Tg) mice was utilized et al’s (2012) results may not be directly comparable to our more discrete pharmacologic studies. Nevertheless, in the whole body KLBKO/FGF21Tg mice deletion of KLB significantly impairs the efficacy of FGF21 In parallel with our work, an independent group has recently communicated the metabolic phenotype of both whole body and adipose specific KLB null mice in vivo. Loss of KLB Furthermore, Ding and colleagues (2012) also conditionally deleted KLB from adipose. Analogous to our recent report on FGFR1 adipose null mice in vivo action and present with the new developmental opportunities to design novel therapeutics that target FGF21 pathway.Taken as a whole our data span the gamut of FGF21 biology from acute signaling to the integrated metabolic effects at the whole body level and demonstrate the ultimate need of KLB in supporting the action of FGF21 in living animals. Coupled with our earlier data on adipose specific deletion of FGFR1


Despite the initial promise of myoblast transfer therapy to restore dystrophin in Duchenne muscular dystrophy patients, clinical efficacy has been limited, primarily by poor cell survival post-transplantation. Murine muscle derived stem cells (MDSCs) isolated from slowly adhering cells (SACs) via the preplate technique, induce greater muscle regeneration than murine myoblasts, primarily due to improved post-transplantation survival, which is conferred by their increased stress resistance capacity. Aldehyde dehydrogenase (ALDH) represents a family of enzymes with important morphogenic as well as oxidative damage mitigating roles and has been found to be a marker of stem cells in both normal and malignant tissue. In this study, we hypothesized that elevated ALDH levels could identify murine and human muscle derived cell (hMDC) progenitors, endowed with enhanced stress resistance and muscle regeneration capacity.hi subpopulations isolated by fluorescence activate cell sorting demonstrated increased proliferation and myogenic differentiation capacities compared to their ALDHlo counterparts when cultivated in oxidative and inflammatory stress media conditions. This behavior correlated with increased intracellular levels of reduced glutathione and superoxide dismutase. ALDHhi murine myoblasts were observed to exhibit an increased muscle regenerative potential compared to ALDHlo myoblasts, undergo multipotent differentiation (osteogenic and chondrogenic), and were found predominately in the SAC fraction, characteristics that are also observed in murine MDSCs. Likewise, human ALDHhi hMDCs demonstrated superior muscle regenerative capacity compared to ALDHlo hMDCs.Skeletal muscle progenitors were isolated from murine and human skeletal muscle by a modified preplate technique and unfractionated enzymatic digestion, respectively. ALDHThe methodology of isolating myogenic cells on the basis of elevated ALDH activity yielded cells with increased stress resistance, a behavior that conferred increased regenerative capacity of dystrophic murine skeletal muscle. This result demonstrates the critical role of stress resistance in myogenic cell therapy as well as confirms the role of ALDH as a marker for rapid isolation of murine and human myogenic progenitors for cell therapy. Duchenne muscular dystrophy is a degenerative muscle disease caused by a mutation of the gene encoding dystrophin, a protein that anchors the myofiber cytoskeleton to the basal lamina, resulting in muscle fiber necrosis and progressive weakness + cells, and myo-endothelial cells Numerous myogenic progenitors have been isolated from post-natal murine and human skeletal muscle for cell therapy such as satellite cells, myoblasts, MDSCs, side-population cells, Sk-DN/Sk-34 cells, pericytes, mesangioblasts, human SMALDPreviously we demonstrated the central role that the intracellular antioxidant glutathione (GSH) plays in the increased survival and muscle regenerative capacity of MDSCs. Increased levels of GSH in MDSCs compared to myoblasts was correlated with the increased rates of survival, proliferation, and myogenic differentiation in conditions of oxidative and inflammatory stress In the current study, we sought to further validate this hypothesis through the isolation of myogenic progenitors with enhanced stress resistance using elevated expression of cytosolic aldehyde dehydrogenase (ALDH1A1) as a marker for this trait. ALDH represents a family of intracellular enzymes that regulate retinoic acid (RA) concentration, which plays an important role in embryonic myogenesis, by driving the expression of multiple myogenic regulatory factors (MRFs) in murine and human embryonic stem cells hi and ALDHlo) were isolated from cultured murine and human skeletal muscle by fluorescence activated cell sorting (FACS) as depicted in lo and ALDHhi subpopulations were isolated from preplate derived murine myoblasts and MDSCs in addition to unfractionated human muscle derived cells (hMDCs) lo and ALDHhi populations to withstand oxidative and inflammatory stress conditions, in terms of proliferation and myogenic differentiation, was examined in addition to their skeletal muscle regeneration capacity in vivo. This stress resistance capacity was then correlated with intracellular antioxidant levels, in the form of GSH and superoxide dismutase (SOD). Observations of increased ALDH activity in human muscle derived cells have been made previously by Jean et al. and Vauchez et al., who demonstrated the myogenic capacity of these subpopulations both in vitro and in vivo hi human myoblasts when exposed to H2O2 in vitro, yet found no analogous ALDHhi subpopulation in murine muscle isolates hi myoblasts in vitro, by comparing the osteogenic and chondrogenic potential of ALDHhi to that of ALDHlo myoblasts. A summary of the findings described in the present study can be found in the Subpopulations of skeletal muscle derived cells that expressed high and low levels of ALDH (ALDHhi and isolated from heterogeneous populations of murine and human muscle derived cells.Cells with elevated ALDH levels become fluorescent when exposed to boron-dipyrromethene (BODIPY) labeled amino acetaldehyde  and can be isolated using fluorescence activated cell sorting (FACS). The non-polar BODIPY-aminoacetaldehyde diffuses freely into the cytoplasm and is converted by cytosolic ALDH1A1 to the negatively charged BODIPY-aminoacetate, which accumulates in the cytoplasm and causes the cell to fluoresce with an emission peak at 513 nm hi cell subpopulations were isolated from rapidly adhering preplate myoblasts, slowly adhering MDSCs as well as unfractionated hMDCs hi and ALDHlo subpopulations of murine myoblasts. Dead cells were excluded from FACS isolations by detection of nuclear propidium iodide staining (hi and ALDHlo cell isolations were determined by ALDH fluorescence extremes of DEAB inhibited controls (and low side scatter) of each population as illustrated in lo and ALDHhi myoblasts represented roughly 1–5% of the total myoblast population (ALDHstaining . The gatpulation .hi cells compared to ALDHlo cells immediately following FACS isolation (data not shown). That is, the mechanical stress of the cell segregation process of flow cytometry typically had a more deleterious effect on the ALDHlo cell viability compared to the ALDHhi cells.We observed an elevated median Aldefluor fluorescence in murine MDSCs compared to myoblasts, despite the absence of a difference in autofluorescence in their untreated controls (data not shown). In addition, MDSCs appear to have a more homogenous elevated ALDH activity than myoblasts, indicating less heterogeneity in ALDH expression. The red overlay in lo and ALDHhi cells isolated from dissociated murine skeletal muscle  has been observed in previous studies despite the consistent observation of enrichment in the MDSC population in successive preplate cycles beyond PP2 To verify this trend of increased ALDH activity in slowly adhering MDSCs, the preplate technique was performed using ALDHl muscle . A trendlo and ALDHhi subpopulations of murine myoblasts were isolated by FACS as depicted in Myoblasts isolated from rapidly adhering cells (RACs) using the preplate technique have been previously characterized as a heterogeneous population in various states of quiescence, activation, and differentiation lo and ALDHhi subpopulations from the RACs, we quantified the oxidative (hydrogen peroxide or H2O2) and inflammatory (TNF-α) stress resistance of these subpopulations during proliferation and myogenic differentiation Following FACS isolation of ALDHlo and ALDHhi subpopulations of murine myoblasts in terms of proliferation and myogenic differentiation. The quantification of proliferation rates of ALDHlo and ALDHhi myoblasts was performed using live cell imaging microscopy in conditions of oxidative and inflammatory stress. This study showed a significant proliferation advantage by the ALDHhi cells compared to ALDHlo cells (lo and ALDHhi myoblasts was induced by a 4d exposure to low serum (2% horse serum) differentiation medium (DM) conditions in the presence of oxidative and inflammatory stress  expressing myofibers  prior to exposure to hydrogen peroxide to determine whether the proliferation rate could be elevated to that of ALDHhi cells hi myoblasts were treated with a GSH sequestrator, DEM, prior to oxidative stress exposure in order to alter their intracellular antioxidant levels To further elucidate the role of intracellular antioxidant levels in the stress resistance capacity of ALDH sorted myoblasts, we altered the cells antioxidant levels prior to oxidative stress challenge. ALDHlo murine myoblasts compared to ALDHhi myoblasts seen in lo cells were increased with XJB (500 nM) prior to oxidative stress exposure, the proliferation rate was significantly increased, to a level statistically equivalent to that observed in the ALDHhi myoblasts (hi myoblasts were decreased by treatment with DEM (50 µM), the proliferation rate was significantly decreased to that observed in the ALDHlo myoblasts , a potent inhibitor of ALDH, we observed that DEAB impaired ALDHhi myoblast proliferation in the absence of oxidative stress (hi cells in oxidative stress conditions (250 µM H2O2) except at a single time point, 24 hrs   . This betaining)  suggestiyoblasts . Howevercoalesce . Even afyoblasts .hi myoblasts (lo myoblasts following BMP-4 stimulation (hi myoblasts (Osteogenic differentiation of murine myoblasts was induced via bone morphogenic protein-4 (BMP-4) stimulation. The unsorted myoblast population increased their alkaline phosphatase expression, an early marker of osteogenic differentiation, in response to BMP-4 stimulation , which wyoblasts . Howevermulation . As was yoblasts .lo myoblasts experienced some form of growth retardation in chondrogenic media compared to ALDHhi myoblasts to the extent that ALDHlo myoblasts did not form a condensed pellet. Furthermore increased RA production by the ALDHhi cell would not be expected to increase the chondrogenic differentiation capacity of these cells. The inability of ALDHlo myoblasts to survive and proliferate in chondrogenic media impaired their capacity for chondrogenic differentiation, as measured by pellet size and amount of GAG production. Consequently, we believe that the increased proliferation potential of ALDHhi myoblasts may not only improve the myogenic differentiation but also facilitate increased chondrogenic and osteogenic differentiation capacities.While the differences in osteogenic differentiation in ALDH sorted myoblasts may either be attributed to increased osteogenic capacity or increased media stress resistance, the case of chondrogenic differentiation capacity suggests the latter mechanism. It seems clear that ALDHlo and ALDHhi subpopulations of murine MDSCs were isolated by FACS in the same manner as murine myoblasts as depicted in lo and ALDHhi MDSCs is illustrated in Given the heterogeneity of ALDH activity and stress resistance in myoblasts, we hypothesized that such heterogeneity may not be observed in MDSC populations since we have previously reported that MDSCs are highly resistant to stress lo and ALDHhi subpopulations. No significant differences in proliferation  and one intermediate inflammatory stress dose . While these trends suggest that there may be a slight difference in stress resistance between ALDHhi and ALDHlo MDSCs, these trends were not consistently statistically significant.In contrast to myoblasts, MDSCs did not display a high degree of heterogeneity in stress resistance behavior when sorted into ALDHferation  or myogeferation  capacitymdx mouse gastrocnemius, both ALDHlo and ALDHhi MDSC subpopulations induced robust muscle regeneration and formation of numerous dystrophin expressing muscle fibers; however, no significant difference in the RIs of ALDHlo and ALDHhi MDSCs was observed  and inflammatory stress (TNF-α). Vauchez et al. and Jean et al. have previously isolated ALDHhi progenitors from human muscle hi cells to be absent in murine muscle, despite the isolation of ALDHhi cells from other murine tissues by others In the current study, ALDHhi cells are primarily responsible for the increased oxidative stress resistance. This conclusion is not entirely consistent with the findings of Jean et al. who observed a dramatic loss in viability of human ALDHhi myoblasts in oxidative stress when ALDH activity was inhibited by DEAB hi myoblasts were treated with DEAB. The difference in our observations however likely stems from differences in our experimental design. Jean et al. used survival as their metric of oxidative stress resistance, whereas we measured proliferation, which clearly employs different cellular pathways and may have a different sensitivity of oxidative stress. Jean et al. observed differences in survival at H2O2 concentrations of 400–650 µM, far greater than the concentration we required (250 µM) to observe differences in proliferation. We concluded that ALDH activity likely plays a role but does not represent a major determinant in the oxidative stress capacity of ALDHhi cells. However, increased reservoirs of intracellular antioxidants such as GSH and SOD in murine and human ALDHhi murine myoblasts and hMDCs appear to have a greater impact on oxidative stress resistance than ALDH activity.While ALDH has been shown to mitigate oxidative damage such as lipid peroxidation, our results indicate that increased intracellular antioxidant levels observed in ALDHhi murine myoblasts and hMDCs in the absence of H2O2 and TNF-α suggests an inherent elevated myogenic capacity compared to their ALDHlo counterparts, clearly implicating the RA producing activity of ALDH. These results are corroborated by those of Vauchez et al., who observed increased myogenic capacity of ALDHhi human myoblasts hi cells as discussed previously. This conclusion is reinforced by the observation that ALDHlo myoblasts, while capable of myogenic differentiation, lose this capacity rapidly with increasing stress doses.ALDH mediated production RA has been shown to promote myogenic differentiation but play a more nuanced or synergistic role in osteogenic and chondrogenic differentiation hi myoblasts may be expected to have increased osteogenic differentiation compared to ALDHlo myoblasts when treated with BMP-4 as we observed in our murine muscle derived cells. Vauchez et al. also observed osteogenic differentiation in their ALDHhi human cells however they did not compare this capacity to their ALDHlo cells hi cells to undergo chondrogenesis at an enhanced rate compared to ALDHlo cells. However, we observed not only increased proliferation in chondrogenic media but also increased GAG production by ALDHhi myoblasts. In this case the RA does not necessarily have a role in promoting chondrogenesis and almost certainly does not have a role in promoting proliferation in this setting. We posit that this result may simply be explained by an increased capacity of these cells to survive the chondrogenic and osteogenic media conditions, given the poor proliferation of ALDHlo myoblasts in chondrogenic media and inability to form a dense chondrogenic pellet.RA has been shown to promote osteogenesis synergistically with bone morphogenic protein hi cells in late preplate cells (slowly adhering cells), a population enriched with MDSCs which exhibit elevated oxidative stress resistance hi murine muscle derived cells  as promulgated by the Committee of Care and Use of Laboratory Animals of the Institute of Laboratory Sciences, the National Academy of Sciences, and the National Research Council. The University of Pittsburgh's Institutional Care and Use Committee (IACUC) reviewed and approved all the procedures performed in these studies (IACUC protocol #: 0902596A-2).Murine myoblasts and MDSCs were isolated from the skeletal muscle of 3-wk-old C57BL/6J female mice  as previously described using a modified preplate technique The preplate technique segregates muscle derived cell populations by how quickly they adhere to collagen-coated plates as described elsewhere 2 as described previously Myoblasts were isolated from rapidly adhering cell fractions of the early preplate cycles while MDSCs were isolated from the slowly adhering cell fraction of the late preplate cells (PP6). All cells were cultured and expanded in PM in collagen-coated flasks at 37°C and 5% CO2. PM consists of 10% horse serum (GIBCO), 10% FBS (GIBCO), 1% chick embryo extract , and 1% penicillin–streptomycin (GIBCO) in DMEM high glucose (Invitrogen). Following enzymatic dissociation, human muscle cells were cultured for three to four days in the flasks of their original seeding and were passaged one to two times as necessary prior to FACS. This protocol is illustrated in Human gastrocnemius muscle biopsies, 9 in total, were obtained  from the National Disease Research Interchange (NDRI) and the Center for Organ Recovery and Education (CORE). All biopsies were taken from patients with no history of neuromuscular/skeletal disease, sepsis, chemotherapy, drug abuse, or ventilatory support greater than 1 month. Furthermore, NDRI and CORE obtain written informed consent from the patient's family prior to harvesting any organs or tissues. Acquisition of muscle from the NDRI and CORE, and subsequent muscle stem cell isolation, was performed in accordance to the protocol reviewed and approved by the University of Pittsburgh's Institutional Review Board . Tissue was finely minced then digested for 60 min at 37°C with 2.4 U/mL dispase (GIBCO), type-I and type-IV collagenases . The digested tissue was pelleted and resuspended in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S), then passed through a 100 µm filter followed by a 70 µm filter to obtain a single cell suspension. Cells were then treated with red cell lysis buffer for 15 minutes before being cultured in PM in collagen-I coated flasks at 37°C and 5% CO6) of each population were resuspended in Aldefluor buffer, which contains an ABC transport inhibitor that prevents efflux of the Aldefluor dye, and incubated at 37°C with BAAA according to the manufacturer's instructions . Cells were washed in Aldefluor buffer and maintained in 4°C throughout the cell sorting process. ALDH activity was assessed using the FL1 channel of a BD FACSAria Cell Sorting System and FACSDiva software (version 6.1.2) . Collected cells were gated on their fluorescence intensity, which corresponds to their ALDH activity levels, as well as low side scatter (SCClo). Sorted cells were recaptured in cold (4°C) PM and immediately plated in collagen-I coated flasks and normal incubation conditions (5% CO2 at 37°C).Cultured murine and human cells were trypsinized, washed in cold PBS, and counted using a hemocytometer. Cells  and inflammatory stress  and immediately placed in a live cell imager. Cells were imaged at 30 min intervals in three 10× microscope fields per well over a 48 hr time period under normal incubation conditions (5% CO2 at 37°C).Time-lapse live cell microscopy was employed to monitor the rate of proliferation under conditions of oxidative and inflammatory stress 2 in 24-well plates for 2 d in PM or until near confluence. PM was then exchanged for differentiation media  treated to simulate oxidative stress  and inflammatory stress . The media was exchanged daily to maintain constant stress levels The capacity of ALDH sorted cell populations to differentiate into MHC expressing cells under varying conditions of oxidative and inflammatory stress was quantified in vitro using the MDI metric. Cells were plated at 1000 cells/cmThe antioxidant capacity was measured in terms of the activity of the major intracellular antioxidant molecules: GSH and SOD. Levels of GSH were measured colorimetrically  using a spectrophotometer . GSH detection is mediated by capture of all thiol mercaptans (RSH) from mechanically homogenized cells into thioethers by 4-chloro-1-methyl-7-trifluromethyl-quinolinium methylsulfate followed by the formation of a chromophoric thione in those GSH specific thioethers using NaOH. GSH levels were quantified by chromophoric thione absorbance at 400 nm. Total activity of SOD was measured using a colorimetric assay . SOD levels of chemically lysed cells  were measured using a xanthine/xanthine oxidase system in which superoxide-chromagen absorbance (490 nm) is lowered by the presence of SOD.lo and ALDHhi myoblasts were modified by a chemical antioxidant  and DEM, a pro-oxidant (Sigma-Aldrich). XJB is a membrane permeable, mitochondrial targeted nitroxide antioxidant that has proven to be cytoprotective in disease states associated with oxidative stress, such as hemorrhagic shock, and mitigates apoptotic cell death in vitro lo myoblasts were pretreated with 500 nM XJB in PM for 2 hrs prior to exposure to oxidative stress (250 µM H2O2 in PM) conditions. ALDHhi myoblasts were pretreated with 50 µM DEM for 2 hrs prior to exposure to oxidative stress (250 µM H2O2 in PM) conditions. In both cases the myoblasts were washed in PBS prior to stress exposure to insure all active antioxidants or pro-oxidants were intracellular and not in the media. The rates of proliferation and survival of these pretreated myoblasts were quantified using a live cell imager  and compared to that of non-pretreated controls in identical oxidative stress conditions.The intracellular antioxidant levels of ALDHhi myoblasts with 50 µM of DEAB in PM was used 24 hrs prior to oxidative stress exposure. A concentration of 50 µM DEAB was maintained during the oxidative stress exposure to maintain ALDH inhibition throughout the experiment. The rates of proliferation and survival were quantified using a live cell imager and ImageJ software (NIH).To examine the role of ALDH in the antioxidant capacity of ALDH sorted myoblasts a potent ALDH inhibitor, DEAB, was used. Pretreatment of ALDH5 cells injected is recorded. ALDH sorted murine cells were injected into the gastrocnemius muscles of female mdx mice , which is a murine model of Duchenne muscular dystrophy using a protocol previously described mdx mouse is a C57BL strain homozygous for a spontaneous X-linked mutation of the dystrophin gene and lacks dystrophin protein expression mdx/SCID mice  aged 6–8 weeks using a protocol previously described 6 cells per 20 µl, prior to injection. Animals were sacrificed at 10 d after injection to conform to previous human cell transplantation experiments in our lab Skeletal muscle regeneration capacity of ALDH sorted myogenic cells was quantified using the regeneration index metric as previously described Differentiation of myogenic cells into MHC expressing cells in vitro was quantified using the MDI metric described previously. Briefly, samples were blocked with 5% HS and incubated with monoclonal antibodies for MHC  followed by Cy3-conjugated anti-mouse antibodies. Nuclei were stained blue with 4, 6-diamidino-2-phenylindole . At least 200 cell nuclei were counted for each MDI measurement.All murine skeletal muscle tissue samples were frozen in 2-methylbutane cooled in liquid nitrogen, then stored at –80°C. 8 µm cryosections were fixed in 5% formalin for 2 min and blocked in 5% donkey serum for 1h. Skeletal muscle sections were stained for dystrophin (DYS) using an anti-dystrophin primary antibody  and a secondary anti-rabbit antibody  using a protocol previously described hi, ALDHlo and unsorted cells were prepared in pellet form (2.5×105 cells) or as a monolayer (1.5×104 cells/well in 12 well plates). For osteogenesis, murine myoblasts were plated on collagen-I coated 12 well plates and maintained for 4 d in osteogenic media , which included dexamethasone, glutamine, ascorbate, penicillin/streptomycin (P/S), β-glycerophosphate and bone morphogenic protein-4 (50 ng/ml). The cells were stained for alkaline phosphatase following the manufacturer's instructions . Chondrogenesis of murine myoblasts was studied by culturing the cells in a pellet culture by incubating the cell pellets for 21 d in chondrogenic media , which contains dexamethasone, ascorbate, ITS+, P/S, sodium pyruvate, proline, glutamine and TGF-beta-3 (10 ng/ml) . All pellets were fixed in 10% formalin for 24 hrs followed by dehydration, paraffin embedding, sectioning, and Alcian blue staining.ALDHt-test, where p<0.05 was considered to be statistically significant. Comparisons of multiple groups were completed using one-way ANOVA followed by Tukey post-hoc comparisons.Data are expressed as a mean with its standard deviation. Direct comparisons between two cell populations were made using an unpaired, two-tailed Student's Figure S1Isolation diagram of murine and human ALDH sorted cells. (a) Murine myoblasts and MDSCs were isolated by a modified preplate technique, as described previously. ALDHhi and ALDHlo subpopulations of these muscle derived cells were isolated by FACS for subsequent expansion in proliferation medium. (b) hMDCs were isolated by enzymatic digestion of human skeletal muscle. Following 3-4 d of culture, ALDHhi and ALDHlo subpopulations of hMDCs were isolated by FACS for subsequent expansion in proliferation medium.(TIF)Click here for additional data file.Table S1Data summary of ALDH sorted populations isolated from skeletal muscle.(TIFF)Click here for additional data file.


KRAS mutations are frequent in colorectal cancer (CRC) and have the potential to activate proliferation and inhibit cell death through effects on MAPK/ERK and PI3K/Akt signaling pathways. Because diet is one of the most important determinants of CRC incidence and progression, we studied the effects of the dietary triterpenoid ursolic acid (UA) on proliferation and cell death induction in human CRC derived KRAS mutated cell lines.Our results show that UA decreases cell proliferation and induces cell death while decreasing signaling through KRAS as indicated by a decrease in ERK and Akt phosphorylation (western blot). UA also induced cell death.TP53 mutated cells are known to be resistant to the chemotherapeutic drug 5-FU. Caspase independent apoptosis (Tunel assay), was increased 6 fold by co-incubation of UA with 5-FU. However, apoptosis was only a small percentage of the total cell death induced by UA. In order to explain these observations, we looked into effects on autophagy. Autophagy is emerging as a promising therapeutic target for drug resistant tumors. UA modulated autophagy by inducing the accumulation of LC3 II and p62 levels an effect dependent on JNK activation.In conclusion, this study shows UA’s anticancer potential as a modulator of KRAS signaling and cell death mechanisms increasing sensitivity to the chemotherapeutic drug 5-FU.


The capacity for subjects to learn to volitionally control localized brain activity using neurofeedback is actively being investigated. We aimed to investigate the ability of healthy volunteers to quickly learn to use visual feedback during real-time functional MRI (rtfMRI) to modulate brain activity within their anterior right insular cortex (RIC) localized during a blink suppression task, an approach of possible interest in the use of rtfMRI to reduce urges. The RIC region of interest (RIC-ROI) was functionally localized using a blink suppression task, and blood-oxygen level dependent (BOLD) signal changes within RIC-ROI used to create a constantly updating display fed back to the subject in the scanner. Subjects were instructed to use emotional imagery to try and increase activity within RIC-ROI during four feedback training runs (FB1–FB4). A “control” run (CNTRL) before training and a “transfer” run (XSFR) after training were performed without feedback to assess for baseline abilities and learning effects. Fourteen participants completed all neurofeedback training runs. At the group-level, increased BOLD activity was seen in the anterior RIC during all the FB runs, but a significant increase in the functionally defined RIC-ROI was only attained during FB2. In atlas-defined insular cortex ROIs, significant increases were seen bilaterally during the CNTRL, FB1, FB2, and FB4 runs. Increased activity within the insular cortices did not show lateralization. Training did, however, result in a significant increase in functional connectivity between the RIC-ROI and the medial frontal gyrus when comparing FB4 to FB1. Since neurofeedback training did not lead to an increase in BOLD signal across all feedback runs, we suggest that learning to control one’s brain activity in this fashion may require longer or repeated rtfMRI training sessions. Technological advances in computer hardware and functional MRI (fMRI) data processing software have made it possible to analyze neural activity as measured by changes in blood-oxygen level dependent (BOLD) contrast almost as quickly as images are acquired. This real-time fMRI (rtfMRI) approach allows for the displaying of measures of localized brain activity back to a subject in a scanner and investigation of their ability to learn to volitionally control their own brain activity . The useAn increasing number of rtfMRI studies have been reported suggesting that healthy subjects can learn through operant training to use neurofeedback to control the activity in a wide range of cerebral regions. These regions include the anterior cingulate cortex , right iRecently, rtfMRI-based neurofeedback has demonstrated the potential to lead to clinical effects in certain patient populations. Preliminary studies suggest neurofeedback may have benefit in patients suffering from chronic pain , tinnituA number of imaging studies from our lab and other investigators have supported the presence of abnormal limbic-motor coupling in patients with the neuropsychiatric disorder Tourette Syndrome  as well In the present study, we sought to develop and establish an rtfMRI-based neurofeedback training methodology that could be used for future investigation as a therapeutic intervention in neuropsychiatric conditions associated with disordered suppression where a role for the insular cortex has been implicated such as Tourette syndrome , obsessiGiven that the preferential recruitment of the insula during tasks involving recall and imagery of emotionally relevant events , along wWe enrolled a total of sixteen healthy volunteers, aged 29.3 ± 7.8 years . All participants had normal neurological examinations and all but one were right-handed by the Edinburgh Handedness Inventory . The stu3 isotropic resolution, TR = 10 ms, TE = 4.96 ms, flip angle = 19°).Images were acquired with a 3T scanner and 8-channel head coil  foam-padded to restrict head motion and improve subject comfort. Functional T2*-weighted images were acquired using gradient echo, echo planar imaging using the imaging acquisition parameters: matrix size = 64 × 64, field of view (FOV) = 22 cm × 22 cm, TR = 1000 ms (800 ms for first six subjects), TE = 30 ms, flip angle = 70°, bandwidth = 250 kHz. Each scan consisted of 14 or 17 slices that covered most of the brain except for the cerebellum . High-order shimming was applied to lessen the field inhomogeneities during data collection and improve the signal-to-noise ratio in areas prone to susceptibility artifacts. A high-resolution magnetization-prepared rapid gradient echo anatomical scan was acquired for each subject for superposition of functional maps upon brain anatomy and to allow for image normalization to a standardized brain space . Simultaneous electrooculography was used to ensure subjects were suppressing blinking . BOLD signal responses during the blink suppression run were then explored in all three dimensions in the vicinity of the anatomically derived position of the ROI until a position was found that visually led to the maximum amount of buildup in BOLD signal in the ROI during the blink suppression blocks . A composite map of the RIC-ROI for all subjects, created by summing RIC-ROI masks with each assigned a value of 1 and co-registered to a standard stereotactic space .Real-time fMRI data were acquired and exported in real-time to a console at the scanner running Analysis of Functional NeuroImages (AFNI) software , which aic space , revealeFigure 1B) was used to average out any unspecific activation and cancel out non-specific activation and global scanning effects. The mean BOLD signal within the specified ROIs were extracted and exported in real-time to a dedicated Linux workstation. In-house Python routines were developed to read BOLD signal changes, perform basic mathematical operations, and produce a dynamic visual display that conforms to standard block fMRI experimental design. The feedback display consisted of a red column with a height that was continuously updated after an initial baseline rest block at each TR using the following equation:A reference ROI (REF-ROI) encompassing the entire brain volume in an axial slice distant to the insular cortex ROI . During the feedback runs (“FB”), participants were shown a continuously updated feedback display and instructed to increase the red column’s height toward the goal bar by focusing their thinking on recall or imagery of emotionally relevant events – a mental task based on a previously reported demonstration of subjects to use this strategy during neurofeedback training to modulate activity within the anterior RIC . A final 30 s rest block allowed the delayed hemodynamic response from the final active block to be included in the analysis.Each scanning run began with 10 s for scanner signal stabilization and participant acclimation to the scanner environment before an initial 30 s baseline rest block. The active GO and FB blocks were alternated with rest blocks during which participants were encouraged to relax and think of the letters “A, B, C” or numbers “1, 2, 3” in order to divert their focus from the emotional mental imagery used during the active blocks of the scanning runs. Each scanning run consisted of a total of the initial baseline rest block followed by five active regulation blocks (GO or FB) separated by rest blocks of 30 s each , followed by four FB training scanning runs (FB1–FB4), and a final post-training or “transfer” GO run (XSFR). The CNTRL run was performed to evaluate the ability of participants to modulate activity within the RIC-ROI before neurofeedback training and the XSFR task was performed to evaluate if neurofeedback training led to the ability of participants to modulate brain activity in the RIC-ROI without the presence of neurofeedback. After each of the scanning runs, participants were asked to briefly describe the type of emotional imagery they used during the previous run (with or without feedback). If the emotional imagery was of a personal nature, participants were informed they did not have to answer the question.post hoc using AFNI and the afni_proc.py processing stream. The first 10 scans of each session were excluded from data analysis to account for T1 equilibration effects and subject scanner acclimation. Functional scanning images were corrected for motion and realigned using the last scan as a reference . Images were spatially smoothed using an isotropic 8-mm FWHM Gaussian filter to accommodate individual anatomical variability. The realigned images were co-registered to the high-resolution anatomical images and subsequently transformed into Talairach space  to test for within-group differences in task-related changes in BOLD. Family-wise error (FWE) correction for multiple comparisons was performed using Monte Carlo-based simulations with the AFNI program 3dClustSim. We set overall significance at p ≤ 0.01 FWE corrected by using a voxel threshold of p ≤ 0.005 and a cluster size threshold of 113 voxels.Task-related changes in BOLD signal at the individual level were estimated at each voxel using a block-design function convolved with a standard gamma-aviate hemodynamic response function and a general linear model (GLM). Covariates derived from motion parameters were included into the GLM to take into account artifacts caused by head motion. Group-level analysis was performed using a simplified mixed-effects model  to assess treatment effect across all six training runs and across the four FB training runs across subjects, and paired t tests were used to evaluate XSFR vs. CNTRL, FB4 vs. FB1, and increases in mean percent BOLD signal above a resting baseline for each neurofeedback run for the “best performers” and “best performances,” respectively. The emotional valence of each subject’s self-reported emotional imagery was also used to test whether negative or positive emotional valence was associated with better neurofeedback performance. Details of self-reported imagery were further assessed for similar themes and grouped in order to compare the effects of specific mental strategies in their ability to lead to significant increases in RIC-ROI over baseline during the neurofeedback runs. Significance level for t test comparisons was set at a threshold of p ≤ 0.05.Exploratory ROI analyses were also performed including assessing the “best performers” and the “best performances.” The “best performers” were evaluated because it is unlikely that all participants are able to quickly learn to use rtfMRI-guided neurofeedback to modulate brain activity within a relatively short training period. The “best performances” were evaluated to assess if a particular themes in the types of emotional imagery found to be most successful in increasing the activity in RIC-ROI could be identified. The “best performers” group included those participants who had a significant increase in BOLD signal during neurofeedback blocks for at least two of the four FB runs. The “best performances” included the top third performances for each neurofeedback scanning run independent of the participant. Paired and one-sample ) single subject average image based on the Destrieux atlas in Freesurfer . Both ROIs were smoothed using an 8 mm FWHM kernel analogous to that used for the rtfMRI data analysis and then intensity filtered to limit the overall size of the ROI, approximate the structures in Talairach space, and minimize artifactual increases in statistical thresholds due to large surface areas relative to volumes . A lateralization index (LI) was calculated for each subject using a normalized difference between percent signal change extracted from the target (%RIC) and contralateral ROI (%LIC) using the equation: LI = (%RIC - %LIC)/(%RIC+%LIC), as has been applied elsewhere .The residual BOLD times series from the whole brain analysis were used for the connectivity analysis to assess whether neurofeedback training altered underlying connectivity between RIC-ROI and another brain region. For each run and each subject, the time series of the functionally localized RIC-ROI was used as seed and correlated with each voxel in the brain. Individual correlation maps were then transformed into Talairach space , and r vOne participant was unable to remain still during the neurofeedback runs and had to have her scanning terminated. Technical scanner issues forced neurofeedback scanning to be terminated shortly after starting neurofeedback training in another participant. Thus, 14 participants  completed all four FB runs were included in the final analysis. Software glitches resulted in two of the14 participants not having usable CNTRL runs and one not having a usable XSFR run such that 12 CNTRL runs and 13 XSFR runs were available for final analysis.Figure 1C).Fifteen participants completed the functional localizer blink suppression scanning and simultaneous electrooculography confirmed all participants were attempting to suppress blinking during functional localization run see . RegionsFigure 3, Table 1) revealed significantly increased BOLD activity within the region of the anterior RIC during all four training scanning runs when visual neurofeedback was provided (FB1–FB4), but not when there was no visual feedback (CNTRL and XSFR). Reported cognitive strategies employed by participants included both positive mental imagery , as well as negative imagery . Cognitive strategies that were associated with the best performances during neurofeedback training included both negative and positive mental imagery. Examples of the most successful thoughts were rather negative including thoughts of exerting an extreme effort, details of friend’s death and sadness, hunger and confinement, emotional memories, painful emotional experiences, pain in body parts; however, some of the most successful thoughts were positive including fond and hometown memories, sipping tea, and hearing a pleasant song.Group-level voxel-wise analysis (n = 14) level during the FB2 training run . Additionally, there was no significant effect for the treatment condition of neurofeedback training  = 2.39, p = 0.11). Using the atlas-defined structural anterior insular cortex ROIs, activation during neurofeedback training was significantly increased bilaterally during the CNTRL , FB1 , FB2 , and FB4 , training runs . Sub-group analysis of the “best performers” showed significant increases in RIC-ROI during the FB1 (p = 0.02) and FB2 (p = 0.002) training runs. Sub-group analysis of the “best performances” revealed significant increases during the CNTRL (p = 0.001), FB1 (p = 0.001), FB2 (p = 0.004), FB3 (p = 0.015), and FB4 (p = 0.009) training runs . No significant increases in RIC-ROI were observed in the final XSFR run for either the “best performers” or the “best performances.” Although voxel-wise imaging analysis revealed greater significant cluster sizes in the region of the right compared to left insular cortex, group-level and sub-group ROI analyses showed no lateralization in the insular cortex activations during any of neurofeedback training runs .Mean percent BOLD signal change during rtfMRI-guided neurofeedback was significantly increased in the functionally localized RIC-ROI at the group (n = 24) and negative (n = 24) valence could be ascribed to the type mental strategy employed, no significant difference (p > 0.05) was observed between imagery with positive and negative valence in leading to a greater BOLD increase within RIC-ROI . Self-reported mental imagery topics were then grouped into seven major themes with a trend toward significant increases observed for mental strategies involving bad memories and/or pain (p = 0.052) and positive thoughts of friends, family, and/or God .The emotional valence of the mental imagery used by subjects was unable to be assessed for 33 of the total of 81 usable neurofeedback runs across subjects due to insufficient detail in the self-reported summaries provided by subjects. Comparing the runs where positive . This cluster of significantly increased connectivity included a small portion of the anterior cingulate cortex. The medial frontal gyrus cluster showed a significant effect for the treatment condition  = 4.83, p = 0.010), with increasing connectivity seen across successive training runs that disappeared for the XSFR run . A significant increase in connectivity between RIC-ROI and the medial frontal gyrus was observed across the neurofeedback training runs with visual feedback , but not between the CNTRL run and XSFR run (p = 0.56). There were no significant differences in connectivity detected between the CNTRL and XSFR runs.Neurofeedback training across FB runs resulted in an increase in functional connectivity between the RIC-ROI and medial frontal gyrus  with the first training run (FB1). The medial frontal gyrus is associated with high-level executive functions including monitoring of ongoing actions and performance outcomes, as well as adjusting behavior and learning . SimilarAlterations in functional connectivity induced by rtfMRI-based neurofeedback are increasingly being reported. In one study that involved trying to train subjects to modulate activity within their supplementary motor area, decreased connectivity between the supplementary motor area and subcortical regions including the striatum and thalamus was seen . IncreasFollowing the neurofeedback training paradigm outlined in this study, participants did not demonstrate an ability to increase activity within the insular cortex when the visual neurofeedback signal was withheld. In fact, even by evaluating the best performers and the best performances separately, no significant increases were seen in the final XSFR run, which was designed to detect whether subjects learned how to modulate brain activity in the absence of active feedback. This is in contrast to some prior reports showing healthy subjects were able to retain an improved ability to modulate their brain activity immediately following neurofeedback training . The majIn addition to assessing whether study participants learned how to self-modulate brain activity in the insular cortex immediately following neurofeedback training, we tested whether participants were able to increase activity within the insular cortex before any training began. No significant increase in insular cortex activity was detected in our voxel-wise group analysis, but we did detect a significant increase bilaterally in our anatomical insular cortex ROI analysis. It is possible that some individuals may be able to activate their insular cortices through focused emotional imagery even without neurofeedback training. Indeed, the existence of this type of potentially intrinsic human ability has been recently exploited as an approach to testing for cognitive awareness in individuals in a vegetative state . While aOne limitation to our study, and a shared limitation with most other rtfMRI-based studies, is the limited amount of time during which participants are actually devoting to neurofeedback training. After setup and localization and anatomic scanning, subjects engaged in a total of four training runs, with each run consisting of a total of 2½ min devoted to active regulation training blocks. More effective neurofeedback training may take longer and/or repeated training sessions. Training to modulate brain rhythms with EEG biofeedback may take weeks to see significant effects. In a recent rtfMRI study, schizophrenia patients were trained to modulate brain activity in their bilateral anterior insula cortices using a training paradigm that consisted of three training runs per day for four days spread out over 2 weeks . DespiteThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.