Using recombinant kinases expressed in E. coli followed by mass spectrometry, the approach accurately recapitulated the well-established motif preferences of human basophilic (Protein Kinase A) and acidophilic (Casein Kinase II) kinases. These motifs, derived for PKA and CK II using only bacterial sequence data, were then further validated by utilizing them in conjunction with the scan-x software program to computationally predict known human phosphorylation sites with high confidence.The identification of protein kinase targets remains a significant bottleneck for our understanding of signal transduction in normal and diseased cellular states. Kinases recognize their substrates in part through sequence motifs on substrate proteins, which, to date, have most effectively been elucidated using combinatorial peptide library approaches. Here, we present and demonstrate the ProPeL method for easy and accurate discovery of kinase specificity motifs through the use of native bacterial proteomes that serve as  Protein phosphorylation provides one of the primary means of transducing cellular signals, and as such has been utilized by a majority of organisms that span all domains of life Many variables contribute to the likelihood of a kinase targeting a given protein in the cell including i) temporal expression of the kinase and substrate, ii) subcellular localization of the kinase and substrate, iii) physical interactions between the kinase, substrate and often other proteins, and iv) the existence of sequence specificity determinants  on the substrate protein. Given that kinase specificity motifs can vary widely . Importantly, the ProPeL methodology represents the first use of a live bacterium  acting as an in vivo peptide library for thousands of simultaneous phosphorylation reactions carried out by an exogenous kinase.Here we address the aforementioned deficiencies of both the combinatorial and “proteome-based” approaches to kinase specificity motif determination by presenting a novel and simple strategy that we call ProPeL  can phosphorylate the native E. coli proteome in a manner that is consistent with the sequence specificity of the kinase. Typically, such modifications to the host proteome would go unmeasured, however, in our approach they serve as a convenient readout of the kinase motif. In order to detect these phosphorylation sites, the bacteria are lysed, proteins are digested using trypsin, phosphopeptides are enriched using SCX/IMAC E. coli peptides are not natural substrates for the expressed kinase, phosphorylation motifs are statistically identified using the motif-xE. coli selected as a background database to account for the proteomic environment within which the reaction occurs.Briefly, a kinase of interest is cloned and expressed in E. coli, i) lacks any eukaryotic-like serine/threonine kinases, ii) has only two kinases with known serine/threonine activity, and iii) has very low levels of endogenous serine and threonine phosphorylation Importantly, the ProPeL strategy is made possible by the fact that E. coli expressing PKA, and 467 phosphorylation sites in E. coli expressing CK II. By comparison, negative controls (untransformed E. coli and E. coli expressing empty plasmid) led to the identification of only 23 endogenous phosphorylation sites, consistent with the known low background phosphorylation levels in E. coliE. coli phosphorylation As a proof of principle, we applied the ProPeL approach to two human kinases, Protein Kinase A (PKA) and Casein Kinase II (CK II), both of which have well-defined motifs p-value of 10−55, the hydrophobic cluster (I/L/M/V/F) at the +1 position, albeit smaller, still has a highly significant p-value of 10−10. Motif deconvolution using the motif-x algorithm E. coli phosphorylation sites . Similarly, scoring 348 known human CK II substrates with the CK II pLogo, and an equivalent number of random human phosphorylatable residues with the same CK II pLogo, also yielded a highly statistically significant difference in average scan-x score . These results both demonstrate that the pLogos obtained via the ProPeL methodology can be used to accurately discern the difference between a random serine or threonine residue and a true PKA or CK II phosphorylation site, and in turn that the pLogos are a strong representation of known PKA and CK II specificities.To quantitatively assess the accuracy of the motifs obtained through the ProPeL method, we used a previously devised scoring strategy, the scan-x to identify potential PKA and CK II native kinase targets in the human proteome using these same pLogos . Within just the top 20 predictions , and 4 proteins had associations with PKA either directly or through protein family members. In the case of CK II . In addition, the highest scoring candidate CK II substrate in the human proteome  has also been shown to be phosphorylated at our predicted site (Ser 312) by 27 independent tandem mass spectrometry studies p-value of <10−17. Given the limited present knowledge of the phosphorylation state of proteins, it is also striking that 80% (16/20) of the top 20 predicted CK II phosphorylation sites were previously shown to be phosphorylated ; most, in dozens of independent experiments. The remaining 4 of the top 20 predicted CK II phosphorylation sites had no prior experimental evidence of phosphorylation. However, these 4 predictions are all contained within tryptic peptides that are longer than 35 amino acids, and are thus also unlikely to be detected using standard high-throughput tandem mass spectrometry workflows.We then used e pLogos  and 3. Idictions , eight sof CK II , the 3rdscan-x as an efficient tool to predict kinase substrates within their native proteomes.The aforementioned results demonstrate that the motifs obtained via the ProPeL methodology can be used to scan whole proteomes in order to predict new high-confidence phosphorylation sites specific to a given kinase. Therefore, in addition to uncovering the motifs for kinases with unknown sequence specificities, by using a bacterial expression system, the ProPeL methodology can be used in conjunction with scan-x predictions, and to compare these results to those obtained using the combinatorial peptide library based Scansite predictor scan-x and Scansite PKA and CK II specific predictors  or to purify large quantities of intact and active recombinant kinases. Additionally, when compared to the most current combinatorial peptide library approaches Aside from its simplicity, the ProPeL approach has a number of other benefits over the well-established combinatorial peptide library approaches and the recent proteome-derived library approaches . Most siE. coli intracellular environment.In the present study, large portions of the ProPeL methodology were carried out independently by two different laboratories (one lab for each kinase), using slightly varying interpretations of the SCX/IMAC protocol (see Methods section for details). The fact that these independent experiments yielded hundreds of phosphorylation sites, both of which accurately recapitulated the expected motifs, provides evidence for the robustness of the approach. Additionally, the use of acidophilic (Casein Kinase II) and basophilic (Protein Kinase A) kinases from opposite sides of the kinase phylogeny suggests a potential for applicability to intermediate kinases without significant bias from the E. coli, we demonstrate here that the diversity of the E. coli proteome is more than sufficient for the discovery of protein kinase motifs, which typically contain no more than two to three major specificity-determining positions motif-x we were able to detect numerous motifs with 3 fixed positions  as a way to interrogate different sequence repertoires available for motif determination.While the human proteome is much larger than that of ositions , thus inE. coli, including kinases, may result in a range of toxicities to the bacterial host. Because of this, as with other protein expression systems, some kinases will likely require some fine-tuning of expression and culture conditions. Additionally, there exist kinases that require specific ligand binding . In this way, recombinant kinases may be expressed, purified, and activated in any appropriate system, while still realizing the benefits of the low background phosphorylation levels in the E. coli proteome.It is well established that expressing a variety of exogenous proteins in We also wish to note that while compiling this manuscript we became aware of work by the Huber lab at the University of Illinois demonstrating the use of bacterial phosphorylation to determine the specificity of several plant receptor-like kinases (RLKs). Their successful results (simultaneously under review at the time of this manuscript’s submission) further suggest that the methodology is broadly applicable and can be used to query the specificity of kinases from a wide range of species – the overwhelming majority of which remain yet unknown.Even in this post-genomic era, elucidation of cellular interactions remains a significant bottleneck for our understanding of molecular mechanisms in humans and other species. ProPeL can rapidly decode the sequence-based determinants of kinase specificity and provide experimentalists with the first line of hypothesis generation necessary to fully annotate the breadth of kinase-substrate interactions. In turn, we can better understand the role of protein phosphorylation and how it affects cell physiology in health and disease.Escherichia coli OverExpress C41(DE3) cells from Lucigen were transformed and plated on Luria-Bertani (LB) plates supplemented with 50 µg/mL carbenicillin, with an untransformed control. Colonies were inoculated in LB broth supplemented with 50 µg/mL carbenicillin and grown up overnight at 37°C with shaking at 200 rpm. Overnight cultures were diluted 10 fold into fresh media and grown under the same conditions until OD600 reached 1, at which point protein expression was induced with 1 mM Isopropyl-β-D-1-thiogalactopyranoside (IPTG) and grown overnight. Cultures were centrifuged at 3000 g for 20 minutes and cell pellets were stored at −80°C until lysis.Human his-tagged full-length PRKACA/pBEV construct was provided by Vertex Pharmaceuticals Inc Cell lysate was prepared according to Villén and Gygi Phosphopeptide enrichment was performed according to Villén and Gygi. Fifteen mg of each protein sample was reduced, alkylated and digested with trypsin. Peptides were desalted with 500 mg 3 cc tC18 SepPak Vac solid-phase extraction cartridges (Waters) and dried in a SpeedVac. Samples were fractionated by HPLC using a Resource S column  with 8 fractions collected according to Macek et al. A plasmid containing the human CSNK2A1 gene in an Invitrogen Gateway donor vector (pDONR223) was kindly provided by The Broad Institute and was transferred using the standard Gateway protocol to the pDEST17 backbone for bacterial expression (Life Technologies). Escherichia coli OverExpress C41(DE3) cells (Lucigen) were transformed and plated on Luria-Bertani (LB) plates supplemented with 100 µg/mL ampicillin, with the empty vector pUC19 (New England Biolabs) serving as a control. Colonies were inoculated in LB broth supplemented with 100 µg/mL ampicillin and grown up overnight at 37°C with shaking at 250 rpm. Overnight cultures were diluted 50 fold into fresh media and grown under the same conditions until OD600 reached 0.6–0.7, at which point protein expression was induced with 1 mM Isopropyl-β-D-1-thiogalactopyranoside (IPTG) and grown for a further 3 hours. Cultures were pelleted by centrifugation at 3000 g and 4°C for 10 minutes. Pellets were stored at −70°C until lysis.Cell lysate was prepared according to Villén and Gygi Phosphopeptide enrichment was performed according to Villén and Gygi. Samples were reduced, alkylated and digested with trypsin. Peptides were desalted with 500 mg 3 cc tC18 SepPak Vac solid-phase extraction cartridges (Waters), snap-frozen in liquid nitrogen and lyophilized. Samples were fractionated by HPLC using a polySULFOETHYL A SCX column (PolyLC) with 12 fractions collected, snap frozen in liquid nitrogen, partially lyophilized to remove acetonitrile, and desalted using 100 mg 1 cc SepPaks (Waters). Phosphopeptide enrichment was performed with PhosSelect iron affinity gel (Sigma) and desalted with StageTips made from C18 material (3M), as described in Rappsilber et al. For each of PKA and a negative control (untransformed cells), 8 SCX fractions were collected and enriched with IMAC as described above.For each of CK II and a negative control , 12 SCX fractions were collected and enriched with IMAC as described above.Before analysis with mass spectrometry, each fraction (either from a set of 8 or a set of 12) was dried down and resuspended in 30 uL of loading Buffer A (see below). Fractions 5, 6, 7 and 8 for a given starting sample were combined, dried and then resuspended to 30 uL loading buffer. For the samples with 12 fractions, fractions 9, 10, 11 and 12 were similarly combined before mass spectrometry was performed. For each fraction (or combined fractions), 4 uL was loaded (using buffer A) onto a C18 nanocapillary column with a pulled tip that sprayed directly into the inlet of a Thermo Fisher Scientific LTQ Orbitrap XL mass spectrometer.Peptides were eluted from the column into the inlet of the mass spectrometer using an Agilent 1200 HPLC binary pump with a gradient that changed solvents from 100% to 65% Buffer A (0% to 35% Buffer B) over a 48 minute time period .During the course of the injection, the mass spectrometer was run using a TOP10 method .E. coli proteome which included decoy database entries Each fraction’s spectra were searched using SEQUEST Following SEQUEST analysis, peptides from spectra containing predicted serine and threonine phosphorylated peptides were summarized for motif analysis. In order to minimize false positives, for each of the two classes of peptide charges z = +2 and z = +3 and greater, minimum XCORR thresholds were chosen to be above the value of the highest XCORR for a decoy hit from the database. The deltaXCORR (the difference between the first and second hits to the databases) was always required to be 0.08. This corresponded to a predicted False Discovery Rate (FDR) of 0%, however it was still subject to statistical variations and may have included some small contamination of false positives.For each input sample, peptides from each fraction (or combined fractions) were identified with a predicted FDR of 0% as described, and then the peptides were combined into a single list of non-redundant peptides for each fraction. Redundant peptides occurring across fractions, but not across samples were highly specific for a particular kinase, and many peptides were identified in more than one independent spectrum. Negative controls occasionally shared peptides with positive samples and with other negative controls.E. coli proteome The final lists of peptides used for each kinases’ motif analysis consisted of phosphopeptides that were not contained in controls nor previously reported to be found in the normal http://plogo.uconn.edu) are currently in preparation.pLogo images were generated using in-house software. Specifically, pLogos depict residues proportional to the log-odds of their binomial probabilities with respect to a given background motif-x analyses for both the PKA and CK II MS/MS peptide identification results were carried out using an internal version of the motif-x web tool scan-x analyses of known and random substrates were carried out using an internal version of the scan-x software (described in detail in reference http://phosphosite.org), while random substrates were obtained by randomly choosing an equivalent number of serine/threonine 15 mers from the human proteome. A whole proteome scan was also carried out using the PKA and CK II pLogos against the entire SwissProt Human proteome containing nearly 1.17 million serine- and threonine-centered 15 mers to generate a ranked list of the highest scoring predicted substrates for those respective kinases.single human kinase were retrieved. Sites shown to be phosphorylated by PKA were called “positive PKA sites” while those known to be phosphorylated by a different kinase were called “negative PKA sites”. Similarly, sites shown to be phosphorylated by CK II were called “positive CK II sites” while all other kinase specific phosphorylation sites were called “negative CK II sites”. scan-x analyses were carried out using an internal version of the scan-x software (described in detail in reference http://scansite3.mit.edu) with either the CK II or PKA matrices selected under minimum stringency setting . To plot the ROC curves, sensitivity and specificity were calculated as previously described To obtain a “gold-standard” data set for ROC curve generation all human phosphorylation sites within the PhosphoSitePlus database which were only known to be phosphorylated by a Table 1Raw mass spectrometry phosphorylated peptide sequence results for the Protein Kinase A (PKA), Casein Kinase II (CK II), and control ProPeL experiments.(XLS)Click here for additional data file.


Prior research has shown that adoptees have a higher rate of substance use disorders (SUDs) than nonadoptees. But these findings have not been verified with a population-based sample of adult adoptees in the United States. Also, no previous adoption study has measured the prevalence of each specific substance use disorder (SUD). We aimed to compare lifetime prevalence rates and odds ratios of SUDs in adopted and nonadopted adults.The data come from the National Epidemiologic Survey on Alcohol and Related Conditions (NESARC). The main outcome measure was the prevalence of lifetime SUDs in adopted (n = 378) and nonadopted adults (n = 42503). Adoptees and nonadoptees were compared to estimate the odds of lifetime SUDs using logistic regression analysis. Adoptees had higher prevalence rates of lifetime SUDs than nonadoptees. Overall, adoptees had a 1.87-fold increase  in the odds of any lifetime SUD compared to nonadoptees. For each SUD, adoptees had higher odds for alcohol abuse/dependence (AOR 1.84), nicotine dependence (AOR 1.78), cannabis abuse/dependence (AOR 1.77), cocaine abuse/dependence (AOR 2.54), amphetamine abuse/dependence (AOR 3.14), hallucinogen abuse/dependence (AOR 2.85), opioid abuse/dependence (AOR 2.21), and other drug abuse/dependence (AOR 2.87) compared to nonadoptees. This study also identified two adoption-specific risk factors  associated with any lifetime SUD.This study demonstrated an increased risk of lifetime SUDs in adopted adults. The findings can be useful for clinicians and policy makers to provide education, prevention, and support for adoptees and their families. The annual number of adopted children in the United States increased 15% from 118,779 in 1990 to 136,000 in 2008 Nevertheless, compared to nonadopted persons, adoptees still have higher rates of psychiatric disorders Several studies in the United States also reported higher rates of SUDs among adoptees. We previously reported that the proportion of adoptees was 14 times higher than expected in two SUD treatment programs The higher SUD rates among adoptees are associated with genetic and environmental risk factors The purpose of this study was to investigate lifetime prevalence rates and odds ratios of SUDs comparing adoptees and nonadoptees in a nationally representative sample in the United States. We hypothesized that lifetime prevalence rates of SUDs are higher in adoptees than nonadoptees. We also investigated which demographic variables are risk factors for SUD specific to adoption. To our knowledge, this is the first study investigating odds ratios for SUDs among adopted and nonadopted adults using a large population-based sample in the United States.This study was approved by the Research & Development Committee of Minneapolis VA Health Care System. Oversight by the Institutional Review Board was exempted because the current study used de-identified public data.The study data were drawn from the National Epidemiologic Survey on Alcohol and Related Conditions (NESARC) Adoption status was determined by face-to-face structured interviews with the following two questions: (1) “Did you live with at least one of your biological or birth parents at any time when you were growing up, that is before you were 18 years old?” When participants answered “no,” the following questions were asked: (2) “When you were growing up, before the age of 18, were you raised by adoptive parents?” In this study, adoptees were defined when the question #1 was answered “no” and the question #2 was answered “yes.” By this criteria, adopted individuals in this study were raised by adoptive parents, but never lived with any biological parents when they were growing up.Diagnoses of lifetime SUDs were made by the Alcohol Use Disorder and Associated Disabilities Interview Schedule–DSM-IV Version (AUDADIS-IV) Lifetime SUDs evaluated were as follows: (1) nicotine dependence; (2) alcohol abuse or dependence; (3) cannabis abuse or dependence; (4) cocaine abuse or dependence; (5) opioid (painkillers or heroin) abuse or dependence; (6) amphetamine abuse or dependence; (7) hallucinogen abuse or dependence; and (8) other drug  abuse or dependence. Although sedatives, tranquilizers, inhalants, and solvents were independently assessed, we have lumped into the category of “other drug” because of the small percentages of these drug disorders.Participants were classified into two groups based on adoption status (adopted and nonadopted). Prevalence rates of demographic variables and lifetime SUDs were obtained using cross-tabulations. Comparison of the two groups was performed for demographic variables to estimate odds ratios (OR) and 95% confidence intervals using a series of logistic regression. The variables (1) age, (2) race, (3) education, (4) personal income, and (5) marital status were coded as indicator variables using (1) ages of 45 or older, (2) white, (3) education of 0–11 years, (4) personal income of $0–19999, and (5) married/cohabitation as the reference category.For main outcome variables , we conducted logistic regression to examine the effects of adoption status on lifetime SUDs. First, unadjusted odds ratios (OR) and 95% confidence intervals were estimated. Second, adjusted odds ratios (AOR) and 95% confidence intervals were estimated while adjusting for covariates . The covariates were selected based on the known correlates of adoption or of SUD.To identify adoption-specific risk factors for any lifetime SUD, a series of logistic regression analyses were conducted using the entire sample. Interaction effects between adoption status and other predictor variables  were examined in logistic models to determine if any of these variables modified the effect of adoption status on SUD.Of the 43093 adults in the NESARC, 42881 participants (99.5%) were included for data analysis after excluding 212 individuals who did not know their adoption status. The rate of adoptees (n = 378) in this study sample (n = 42881) was 0.88%.Adoptees also had higher odds of both diagnostic categories of abuse and dependence compared to nonadoptees as follows: alcohol abuse , alcohol dependence , any illicit drug abuse , and any illicit drug dependence .Our findings from the population-based NESARC data showed that adoptees have higher rates of lifetime SUDs than nonadoptees. These findings are consistent with two large European studies Importantly, adoptees’ odds ratios were high for both abuse and dependence . The odds ratios were slightly higher for dependence than abuse, but these differences were not significant on the 95% CI. This findings applied to both alcohol abuse/dependence as well as drug abuse/dependence. From a public health perspective, these data indicate that adoptees have a wide range of SUD including abuse and dependence.These adoption studies convey both theoretical and practical implications. On a theoretical level, they indicate a linkage between being adopted and having a propensity to SUD years later in adulthood. The most likely linkage is a genetic one Our study identified two risk factors increasing the prevalence of lifetime SUD specific to adoption status. The first and largest risk factor was Hispanic race-ethnicity . Factors in the Hispanic adoptees that might account for this greater vulnerability to SUD include a higher prevalence of foreign-born adoptees with more adverse intrauterine factors  and/or environmental factors in the family-of-adoption . Or conversely, Hispanic nonadoptees may possess protective factors against SUD, such as higher levels of parental monitoring against SUD The second risk factor was single marital status . However, we have no previous studies pointing to a higher single marital status as observed in this study. Other remaining demographic characteristics  failed to show statistical differences. Although male gender was a risk factor for SUD in population studies Several limitations should be considered in interpreting our findings. First, the NESARC data did not provide information regarding the status of international or domestic adoption Adoptees had higher odds for lifetime SUDs than nonadoptees in this study using NESARC data. Despite the advantages of adoptees’ higher educational levels probably due to being raised by higher educated, higher income adopting parents, adoptees are still at higher risk to lifetime SUD. Awareness of adopted persons and their adoptive parents to this risk may help in primary prevention  and in secondary prevention . The findings can also be useful for clinicians and policy makers to provide education, prevention, and support for adoptees and their families.


The APC tumor suppressor gene is frequently mutated in human colorectal cancer, with nonsense mutations accounting for 30% of all mutations in this gene. Reintroduction of the WT APC gene into cancer cells generally reduces tumorigenicity or induces apoptosis. In this study, we explored the possibility of using drugs to induce premature termination codon (PTC) readthrough , as a means of reactivating endogenous APC. By quantifying the readthrough of 11 nonsense mutations in APC, we were able to identify those giving the highest levels of readthrough after treatment. For these mutations, we demonstrated that aminoglycoside or negamycin treatment led to a recovery of the biological activity of APC in cancer cell lines, and showed that the level of APC activity was proportional to the level of induced readthrough. These findings show that treatment with readthrough inducers should be considered as a potential strategy for treating cancers caused by nonsense mutations APC gene. They also provide a rational basis for identifying mutations responsive to readthrough inducers. The APC (adenomatous polyposis coli) tumor suppressor gene encodes a 311 kDa multidomain protein with binding sites for numerous partners, including microtubules, axin and beta-catenin In the last few years, certain antibiotics have been shown to interfere with the mammalian ribosome, inducing the readthrough of premature stop codons (PTCs). Aminoglycosides are the antibiotics most frequently studied in this respect, and multiple studies have suggested that these drugs could be used in innovative strategies for the treatment of genetic diseases caused by nonsense mutations  triggered apoptosis of cancer cells harboring a nonsense-mutated p53 gene We evaluated readthrough efficiency for 11 nonsense mutations involved in colorectal cancer, with a view to identifying the nonsense mutations in the APC gene the most responsive to readthrough-inducing treatments. For the mutations with the highest readthrough levels, including an endogenous mutation, antibiotic treatment resulted in recovery of the biological activity of APC.We studied 11 nonsense mutations found in 23% of cancer cells carrying a nonsense mutation in the APC gene . Readthrough efficiency has been shown to depend on the nature of the sequences surrounding the stop codon We assessed whether readthrough levels were similar in cells of colon origin, by also evaluating the readthrough directed by each nonsense mutation in the cancer colorectal DLD-1 cell line. The results obtained were similar to those for NIH3T3 cells (data not shown).For further characterization, we focused on the L360X and R1114X nonsense mutations, which displayed the highest levels of readthrough in the presence of the aminoglycoside gentamicin . For the two selected nonsense mutations, we quantified the readthrough levels obtained in the presence of various concentrations of G418 (geneticin), because this antibiotic is the most potent readthrough inducer of the aminoglycosides . Several studies have also demonstrated that only a few nonsense mutations give “significant” readthrough in the presence of gentamicin. In this study, only two of the eleven nonsense mutations tested, L360X and R1114X, displayed gentamicin-induced readthrough levels greater than 0.5%, but only L360X had a C residue in the +4 position. The R1114X mutation would therefore have been discarded if this had been the only criterion used to identify mutations responding well to readthrough inducers. Our approach thus provides a rational strategy for identifying patients likely to benefit from treatment with readthrough inducers.Readthrough level depends on the nature of the stop codon and the surrounding nucleotide context, but the rules governing readthrough level are complex and remain to be determined for mammalian cells. The presence of a C residue immediately after the stop codon (+4) is correlated with a high level of readthrough, although this does not seem to be the sole determinant of readthrough levelWe then investigated whether aminoglycoside treatment induced detectable levels of biological activity for an APC protein produced from a cDNA bearing the L360X mutation. Active APC mediates the sequestration of beta-catenin in the cytoplasm, thus preventing the interaction of this molecule with the transactivation factor TCF required for the expression of target genes. APC protein activity was evaluated by determining the ability of this protein to bind beta-catenin, using a reporter construct containing TCF/beta-catenin binding sites inserted upstream from the luciferase firefly gene (pTopGlow) DLD-1 cells devoid of functional APC protein (APC del 1 bp 1406) were transfected with the pCMV expression vector containing either WT or L360X mutant APC cDNA, together with pTopGlow . TransfeWe then investigated whether the aminoglycoside treatment of endogenous nonsense mutations would give similar results. LoVo cells (APC R1114X) were transfected with the reporter plasmid pTopGlow and left untreated or treated with G418 or negamycin. The activity of the luciferase firefly reporter gene was maximal in the absence of treatment and was set at 100%, reflecting the lack of activity of the APC protein. Treatment with negamycin and G418 decreased pTopGlow firefly luciferase activity to 50% and 18% the reference value, respectively . ActivitAs for the other mutation tested, luciferase expression from pFopGlow harboring mutated TCF/beta-catenin binding sites was not significantly affected by antibiotic treatment (data not shown). We checked that active APC protein was actually responsible for the observed decrease in firefly luciferase expression, using a siRNA specifically targeting the APC mRNA. We first used RT-PCR to check that the siRNA effectively decreased APC mRNA levels . LoVo ceAfter G418 treatment (200 µg/ml), the APC-targeting siRNA gave a decrease in luciferase activity two times lower than that observed in absence of siRNA, whereas the non specific siRNA had no effect on luciferase inhibition . Thus, tFor these two mutants, we investigated the ability of aminoglycoside treatment to restore the production of full-length APC protein. We were able to detect the full-length APC protein in HeLa cells (APC WT), but this protein was not detectable after drug treatment in either LoVo cells (R1114X) or DLD-1 cells transfected with the L360X cDNA . This laIn this study, we investigated the ability of readthrough-inducing molecules to induce the production of a functional APC protein in cells carrying various nonsense mutations in the APC gene. Biological activity was detected for the two nonsense mutations most responsive to aminoglycoside treatment (R1114X and L360X) and this activity was directly proportional to the readthrough levels measured with a dual reporter system. This study shows that readthrough inducers, such as aminoglycoside and negamycin, can reactivate the APC tumor suppressor gene in cancer cells. It provides a rational strategy for identifying patients likely to respond and therefore more likely to benefit from treatment with readthrough inducers. A recent study has provided the proof-of-principle that the re-expression of a full-length APC protein after treatment with readthrough inducers reduces tumor size in a xenograft mouse 2, at 37°C. NIH3T3 (kindly provided by Marc Sitbon) cells are embryonic mouse fibroblasts. LoVo , supplemented with 10% fetal calf serum  and 100 U/ml penicillin/streptomycin. Cells were incubated in a humidified atmosphere containing 5.5% COComplementary oligonucleotides corresponding to nonsense mutations embedded in their natural context . Directed mutagenesis was performed on a fragment of the APC gene to create the APC L360X nonsense mutation (pCMV APC L360X). The sequence of the re-inserted fragment was verified.LoVo cells were treated with G418 (200 µg/ml) for 72 h. The medium was replaced and fresh antibiotics were added each day. Cells were treated as previously described The biological activity of APC was estimated with the pTOPGlow vector .DLD-1 cells were cotransfected with pCMV L360X (4 µg), pTOPGlow or pFOPGlow (10 µg) and pCMVLacZ (2 µg), by the calcium phosphate method. Each DNA precipitate was split between two wells of a six-well plate. Antibiotics  were added immediately after transfection, except for G418, which was added the day after. The medium was replaced daily and fresh antibiotics were added. We prepared protein extracts 48 hours after transfection, and measured enzymatic activities.For LoVo cells, antibiotics  were added the day before transfection and on each subsequent day. LoVo cells were cotransfected, by the calcium phosphate method, with TOPGlow or FOPGlow (10 µg) and pCMVLacZ (2.8 µg) to normalize for transfection efficiency, cell viability and protein extraction variability. Each DNA precipitate was split between two wells of a six-well plate. Three days after transfection, protein extracts were prepared and enzymatic activities were measured.For each cell line, at least three independent transfection experiments were performed.In LoVo cells, protein activity was determined as described above. Cells were transfected with siRNA targeting mRNA APC (Dharmacon ON-TARGET plus J-003869-12) or NS, nontargeting (Dharmacon ON-TARGET plus control siRNA#1), in the presence of DharmaFECT Duo , 24 h after their initial transfection as described above. Transfected cells were immediately incubated in medium with or without G418 (200 µg/ml) supplementation. Three days after transfection, the cells were harvested and luciferase and beta-galactosidase activities were assayed as described above. At least four independent transfection experiments were performed.


Early initiation of antiretroviral therapy (ART) in eligible pregnant women is a key intervention for prevention of mother-to-child transmission (PMTCT) of HIV. However, in many settings in sub-Saharan Africa where ART-eligibility is determined by CD4 cell counts, limited access to laboratories presents a significant barrier to rapid ART initiation. Point-of-care (POC) CD4 cell count testing has been suggested as one approach to overcome this challenge, but there are few data on the agreement between POC CD4 cell enumeration and standard laboratory-based testing.TM Analyzer) to laboratory-based flow cytometry in consecutive HIV-positive pregnant women. Bland–Altman methods were used to compare the two methods, including analyses by subgroups of participant gestational age.Working in a large antenatal clinic in Cape Town, South Africa, we compared POC CD4 cell enumeration (using the Alere Pimap=0.04).Among the 521 women participating, the median gestational age was 23 weeks, and the median CD4 cell count according to POC and laboratory-based methods was 388 and 402 cells/µL, respectively. On average, the Pima POC test underestimated CD4 cell count relative to flow cytometry: the mean difference (laboratory test minus Pima POC) was 22.7 cells/µL , and the limits of agreement were −129.2 to 174.6 cells/µL. When analysed by gestational age categories, there was a trend towards increasing differences between laboratory and POC testing with increasing gestational age; in women more than 36 weeks’ gestation, the mean difference was 45.0 cells/µL (These data suggest reasonable overall agreement between Pima POC CD4 testing and laboratory-based flow cytometry among HIV-positive pregnant women. The finding for decreasing agreement with increasing gestational age requires further investigation, as does the operational role of POC CD4 testing to increase access to ART within PMTCT programmes. Initiation of lifelong antiretroviral therapy (ART) in eligible pregnant women is a critical intervention both to prevent the mother-to-child transmission (PMTCT) of HIV infection and to promote maternal health . HoweverPoint-of-care (POC) CD4 cell count testing has been suggested as one approach to overcome the challenges related to limited laboratory access in many parts of Africa . In adulAlthough POC CD4 cell count testing has the potential to enhance the identification of ART-eligible women within PMTCT services, there are few data on the test reliability of POC CD4 cell count testing in pregnancy. Previous evaluations of POC CD4 technologies have suggested reasonable agreement with laboratory-based CD4 cell enumeration , with some concerns raised around operational aspects of POC tests in real-world settings –14. In aThe study took place in a single large antenatal clinic in Cape Town, South Africa, with a heavy burden of antenatal HIV infection . At this clinic, all women making their first antenatal visit receive voluntary counselling and testing for HIV and undergo phlebotomy for routine antenatal screening. Venous specimens for laboratory CD4 testing were taken from HIV-positive women at this first antenatal visit. Routine CD4 cell count enumeration was conducted at the South African National Health Laboratory Services (NHLS) via Beckman-Coulter flow cytometry using panleucogated methodology, the standard of care in this setting and described in detail previously . ResultsTM Analyzer  into routine PMTCT services in this setting from June 2012. POC testing was conducted by either a nurse–midwife or a trained counsellor, both working in the PMTCT service, and both trained by the manufacturer. Testing used the same venous specimen that was sent for laboratory CD4 testing, with sampling using capillary tubes supplied by the manufacturer. Test procedures for the Alere Pima Analyzer followed manufacturer's guidelines, including the use of daily quality control beads and routine machine maintenance. Results are returned to patients 20–30 minutes later.We implemented the POC Alere Pimat-tests were used to compare means; all statistical tests are two-sided at α=0.05. Results are presented with 95% confidence intervals (CI) throughout.Data on women's age, prior ART use and gestation at the time of testing was abstracted from routine health care records, with approval by the Research Ethics Committee of the University of Cape Town. In analysis, we compared laboratory versus POC CD4 test results using Bland–Altman analysis for the mean difference and limits of agreement for laboratory versus POC testing . FurtherA total of 546 women were tested using the POC Alere Pima Analyzer. During this period 629 test runs were conducted, with 61 women requiring multiple attempts at POC testing (four women did not receive POC CD4 testing due to repeated machine errors). The most common errors were related to inadequate or inappropriate specimen collection and/or cartridge use . Of these women, 521 women also had laboratory testing data available and are included in this analysis; there were no significant differences between those excluded and those included in the analysis. Three machines were used during this time, though the majority of tests (61%) were done with a single machine.The median age of women participating was 27 years , and 13% of women were on ART at the time of testing . The medThe overall correlation between the Alere Pima Analyzer and laboratory-based CD4 cell counts was high  . The Blap=0.04).p=0.47).These data suggest that in the context of pregnancy, the POC Alere Pima Analyzer appears to slightly underestimate laboratory-based flow cytometry in CD4 cell enumeration. In keeping with this, the overall sensitivity of this POC test in detecting women who are ART-eligible based on laboratory CD4 cell counts ≤350 cells/µL is high (92%). These data point to the potential role that this POC CD4 test could play in enhanced identification of ART-eligibility within PMTCT services.This is the largest evaluation to date of POC CD4 testing in HIV-positive pregnant women, using venous blood specimens in the Alere Pima Analyzer rather than capillary blood. In smaller studies using capillary blood specimens from pregnant women, similar mean biases were documented  , 15. GenOf note, the agreement in these data between the Alere Pima Analyzer and laboratory-based flow cytometry appears to decline with increasing gestational age at the time of testing, from a mean bias of 6 cells/µL in the first trimester to 45 cells/µL in women tested after 36 weeks’ gestation. The interpretation of absolute CD4 cell counts during gestation is a known concern due to haemodilution of pregnancy , 21, butThese data are subject to several limitations. The results come from an operational evaluation conducted at a single, large primary-care antenatal clinic, with tests using three Alere Pima Analyzer machines. While all tests were conducted using manufacturer's equipment by trained individuals, the results should still be generalized with caution. In addition, we had access only to routine patient data, so potentially important information (e.g. other haematology parameters that may help to interpret the agreement between the CD4 tests) was not available. In addition, we did not assess the assay precision (repeatability) of the Alere Pima Analyzer in this study design, and additional investigations of assay precision are warranted.Recently, the World Health Organization recommended consideration of an approach to PMTCT based on universal ART initiation of all HIV-positive pregnant women regardless of CD4 cell count, with either cessation of ART at the end of breastfeeding for women with high CD4 cell counts (“Option B”) or continuation of ART as lifelong treatment (“Option B+”) . Under tPOC CD4 testing may play an important role in PMTCT programmes in two different settings. First, POC CD4 testing provides a practical alternative in settings where there is no regular access to laboratory-based CD4 cell enumeration. Second, in settings that have access to laboratory CD4 cell enumeration but where delays in providing CD4 results hinder PMTCT services, POC CD4 testing may provide a rapid testing strategy: by determining eligibility on the same day as HIV testing, it may be possible to expedite substantially ART initiation. These data suggest sufficient agreement between the Pima POC CD4 test and laboratory-based testing for either of these roles, but additional research is required to understand the role that POC CD4 testing can play in each of these settings. In addition, from an operational perspective, we found that 83 additional tests were required on the Alere Pima Analyzer to provide CD4 results on 546 women, leading to an excess testing proportion of approximately 15%. This error rate is consistent with a previous evaluation of the Pima CD4 test in an antenatal setting (ranging from 10 to 20%) . The errIn summary, these data suggest reasonable overall agreement between Pima POC CD4 testing and laboratory-based flow cytometry among HIV-positive pregnant women. The preliminary findings for decreasing agreement with increasing gestational age require further investigation, as does the operational role of POC CD4 testing to increase access to ART within PMTCT programmes.


Intrauterine growth restriction (IUGR) is the single largest contributing factor to perinatal mortality in non-anomalous fetuses. Advances in antenatal and neonatal critical care have resulted in a reduction in neonatal deaths over the past decades, while stillbirth rates have remained unchanged. Antenatal detection rates of fetal growth failure are low, and these pregnancies carry a high risk of perinatal death.th centile, between 24+0 and 36+6 weeks gestation. All recruited fetuses underwent serial sonographic assessment of fetal weight and multi-vessel Doppler studies until birth. Perinatal outcomes were recorded for all pregnancies. Case records of the perinatal deaths from this prospectively recruited IUGR cohort were reviewed, their pregnancy details and outcome were analysed descriptively and compared to the entire cohort.The Prospective Observational Trial to Optimize Paediatric Health in IUGR (PORTO) Study recruited 1,200 ultrasound-dated singleton IUGR pregnancies, defined as EFW <10th centile, 6 resulted in perinatal deaths including 3 stillbirths and 3 early neonatal deaths. Perinatal deaths occurred between 24+6 and 35+0 weeks gestation corresponding to birthweights ranging from 460 to 2260 grams. Perinatal deaths occurred more commonly in pregnancies with severe growth restriction (EFW <3rd centile) and associated abnormal Doppler findings resulting in earlier gestational ages at delivery and lower birthweights. All of the described pregnancies were complicated by either significant maternal comorbidities, e.g. hypertension, systemic lupus erythematosus (SLE) or diabetes, or poor obstetric histories, e.g. prior perinatal death, mid-trimester or recurrent pregnancy loss. Five of the 6 mortalities occurred in women of non-Irish ethnic backgrounds. All perinatal deaths showed abnormalities on placental histopathological evaluation.Of 1,116 non-anomalous singleton infants with EFW <10The PNMR in this cohort of prenatally identified IUGR cases was 5.4/1,000 and compares favourably to the overall national rate of 4.1/1,000 births, which can be attributed to increased surveillance and timely delivery. Despite antenatal recognition of IUGR and associated maternal risk factors, not all perinatal deaths can be prevented. The perinatal outcome of IUGR fetuses is largely dependent on the severity of growth restriction with those below the 3 outcome .Advances in obstetrical and critical neonatal care are reflected in a substantial decrease in the overall perinatal mortality rate (PNMR) in high income countries. While this effect is mainly seen in a reduction of early neonatal deaths, stillbirth rates have remained largely unchanged over the past years ,4. In adth customised centile and only 30% of those were suspected antenatally [Ireland consistently reports highest birth rates among European countries with 16.8 births per 1,000 population  . Stillbienatally .Current antenatal detection rates of IUGR are reported at 25 to 36% ,9. Thereth centile [This paper describes the 6 perinatal mortality cases which occurred in the setting of a multicentre prospective study of 1,200 pregnancies with prenatally identified IUGR defined as EFW below the 10th centile based on sonographic measurements of the fetal head circumference (HC), bi-parietal diameter (BPD), abdominal circumference (AC) and femur length (FL) according to population-based growth standards (Hadlock-4) [+0 and 36+6 weeks’ and an EFW equal or greater than 500 grams. Fetuses with congenital structural or chromosomal abnormalities were excluded from the final analysis. Ethical approval was obtained at each participating site, and a written informed consent was given by all study participants.The Prospective Observational Trial to Optimise Paediatric Health in IUGR (PORTO) is a national prospective study conducted at seven largest maternity units in Ireland. Intrauterine growth restriction (IUGR) was defined as EFW below the 10dlock-4) . BetweenWomen with IUGR pregnancies underwent serial sonographic evaluation of fetal weight at 2-weekly intervals until birth including evaluation of amniotic fluid volume and multi-vessel Doppler assessment. Details on Doppler assessment and reference ranges are described in a previous publication . In caseThe perinatal mortality rate (PNMR) was calculated by the number of stillbirths and neonatal deaths per 1,000 live births and stillbirths . The PNMR was corrected for congenital abnormalities.All cases were prospectively recruited and medical records of all mortalities were reviewed retrospectively for this descriptive analysis. Comparisons were made to the entire cohort, and demographic and pregnancy course and outcome data were recorded along with a review of placental histopathology.Placental abnormalities were categorised according to Redline criteria  into vilDue to the small sample size, formal statistical comparisons and modelling (such as logistic regression) are underpowered. Therefore, statistically non-significant findings have the potential to be misleading. Furthermore, due to the biases associated with retrospective selection of controls and the potential for overmatching with a small case-set, the mortalities are presented as a case-series rather than as a case-control study. Consequently, the cases have been described individually  and associated abnormal Doppler findings resulting in earlier gestational ages at delivery and lower birthweights.There were 6 mortalities including 3 stillbirths (SB) and 3 early neonatal deaths (NND) within the entire cohort of 1,116 non-anomalous singleton pregnancies completing the study protocol. This corresponds to a PNMR of 5.4/1,000. The maternal demographics, baseline characteristics and sonographic findings are described in Table +6 and 31+3 weeks with birthweights ranging from 460 to 740 grams (mean 607 grams). All stillbirths had estimated weights below the 3rd centile and AEDF in the umbilical artery, and they occurred during inpatient care. The median time from study recruitment to diagnosis of intrauterine demise was 12 days (range 0-45 days). One woman had placenta praevia associated with recurrent antepartum haemorrhages and one woman had severe pre-eclampsia on the background of essential hypertension. Two women had previous pregnancies that resulted in perinatal deaths , and all women with stillbirths were non-Irish nationals.Stillbirths occurred at gestational ages between 24+4 and 35+0 weeks gestation corresponding to birthweights between 600 and 2260 grams (mean 1147 grams). The median time of antenatal surveillance was 25 days (range 24-34 days). As outlined in Figure rd centile and umbilical artery AEDF in case #5. The mother received inpatient care for active SLE and the neonate died on day 3 of life. Neonatal death #6 resulted from a recurrent placental abruption at 35 weeks gestation one week prior to her planned delivery; in this case neonatal intensive care was withdrawn after 4 days due to grade 3 hypoxic ischemic encephalopathy (HIE) and multi-organ failure.All cases of neonatal deaths were delivered by emergency caesarean section between 27ams mean 147 gramsFigure rd centile and abnormal UA Doppler velocimetries in the umbilical artery. The 2 perinatal deaths with normal UA Doppler were attributed to pulmonary hypoplasia and histologically confirmed chorioamnionitis due to prolonged preterm rupture of the membranes which and to a recurrent placental abruption at 35 weeks gestation.The majority of perinatal deaths (n = 5) were associated with an EFW < 3The striking and common feature of all perinatal death cases in our study is that they more commonly occurred in non-Irish national women attending for antenatal care with poor obstetric histories and coexistent maternal co-morbidities. These findings are in agreement with other reports which found that ethnic minorities are an overrepresented group among mothers who experience perinatal deaths ,13. In prd centile and/or abnormal UA Doppler are a greater risk of poor perinatal outcome therefore making this a more appropriate cut-off for the definition of pathological growth restriction [Perinatal deaths occurred more commonly among infants with severe growth restriction and associated abnormal umbilical artery Doppler values which highlights the fact that fetuses with EFW <3triction .A detailed evaluation of the cord and placenta is useful in determining the underlying causes which have led to an IUGR diagnosis. Histopathological examination was carried out for all perinatal deaths in our study and interestingly showed abnormal findings in 100% of cases including maternal and fetal vascular injuries, placental developmental abnormalities or inflammatory lesions. Unfortunately, there are no specific antenatal screening or preventative strategies for cord and placental abnormalities. Together with maternal factors, this information can however be utilised when caring for women in a subsequent pregnancies.The PNMR in this IUGR cohort compares favourably to the national corrected PNMR of 4.1/1,000. The relatively low PNMR in this high risk population of IUGR fetuses can be explained by (i) the intensive prenatal surveillance within a research setting attributed to the Hawthorne effect, which has been described as a confounding factor for performance in clinical observational studies and (ii) the exclusion of pregnancies at most severe spectrum of disease (i.e. EFW <500 grams at or over 24 weeks gestation). Despite the antenatal recognition of maternal risk factors and diagnosis of IUGR with initiation of increased surveillance and inpatient management, not all perinatal deaths in our study were prevented. Some of these deaths occurred during inpatient care or were due to unforeseen acute complications such as placental abruption, antepartum haemorrhage in the presence of placenta praevia or acute chorioamnionitis. However the increased surveillance certainly benefited the majority of women and babies enrolled in PORTO reflected in the low PNMR in this high risk cohort which was comparable to the overall national rate.th centile cut-off, this approach would also allow to comment on growth trajectories which have been identified as an important factor in the prediction of morbidity and mortality outcomes [A limitation of our study concerns the inability to comment on antenatal detection rates of IUGR in Irish maternity units as data on this was not recorded. Prenatal recognition of IUGR remains the main challenge in daily obstetric practice. Current detection rates of IUGR in the antenatal setting are at best 36% and future research needs to urgently focus on how we can improve these detection rates. Clinical examination, abdominal palpation and fundal height measurement have limited accuracy in identifying IUGR prenatally and serial ultrasound scanning from 26 to 28 weeks of gestation has been proposed in patients with risk factors . A care outcomes . The reloutcomes .Fetal growth restriction is the single most important contributor to perinatal mortality in non-anomalous fetuses . SignifiVigilance towards antenatal risk factors for poor pregnancy outcome is important for the optimal management of IUGR pregnancies. The focus of future research needs to urgently address how we can improve the antenatal detection of IUGR. Prenatal recognition of IUGR dramatically improves pregnancy outcomes allowing for close monitoring and timely delivery. Nevertheless, even if recognised, not all perinatal deaths can be prevented.IUGR: Intrauterine growth restriction; PORTO: Prospective Observational Trial to Optimize Paediatric Health in IUGR; SB: Stillbirth; IUD: Intrauterine death; UtA: Uterine artery; UA: Umbilical artery; NND: Neonatal death; APH: Antepartum haemorrhage; BW: Birthweight; Dx: Diagnosis; PET: Pre-eclampsia; PPROM: Prolonged preterm rupture of membranes; AEDF: Absent end-diastolic in the UA; REDF: Reversed end-diastolic flow in the UA; CTG: Computerised short term variation cardiotocograph; HIE: Hypoxic ischaemic encephalopathy; PNMR: Perinatal mortality rate.The authors declare that they have no competing interests. The study was funded by the Health Research Board (HRB) and Friends of the Rotunda.All authors have made substantial contributions to conception, design and data acquisition of the PORTO study. KOD had the idea for this particular analysis. JU drafted the manuscript. All co-authors revised it critically for important intellectual content. PD performed the statistical analysis. All authors read and approved the final manuscript.The PORTO Study was conducted by the Perinatal Ireland Research Consortium, a nationwide collaborative research network comprising of the seven largest academic obstetric centres in Ireland. Those include the Rotunda Hospital Dublin, the Coombe Women’s and Infants University Hospital Dublin, the National Maternity Hospital Dublin, the Cork University Maternity Hospital, the Royal Jubilee Maternity Hospital Belfast, the Galway University Hospital and the Mid-Western Regional Maternity Hospital Limerick. FDM is the Chair of Perinatal Ireland. JU is the lead researcher of the PORTO study and clinical PhD student under the supervision of KOD and FDM.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2393/14/63/prepub


Mitochondrial dysfunction, lipid accumulation, insulin resistance and metabolic inflexibility have been implicated in the etiology of type 2 diabetes (T2D), yet their interrelationship remains speculative. We investigated these interrelationships in a group of T2D and obese normoglycemic control subjects.In vivo mitochondrial function was measured by PCr recovery in 30 T2D patients and 31 control subjects.49 non-insulin dependent male T2D patients and 54 male control subjects were enrolled, and a hyperinsulinemic-euglycemic clamp and indirect calorimetry were performed. A muscle biopsy was taken and intramyocellular lipid (IMCL) was measured. 2 = 0.18, p<0.05); whereas WGD predicted both insulin-stimulated RER  and metabolic flexibility .Fasting NEFA levels were significantly elevated in T2D patients compared with controls, but IMCL was not different. Mitochondrial function in T2D patients was compromised by 12.5% (p<0.01). Whole body glucose disposal (WGD) was higher at baseline and lower after insulin stimulation. Metabolic flexibility (ΔRER) was lower in the type 2 diabetic patients . Mitochondrial function was the sole predictor of basal respiratory exchange ratio (RER) (Rin vivo mitochondrial function in type 2 diabetic patients are only reflected in basal substrate oxidation and highlight the importance of glucose disposal rate as a determinant of substrate utilization in response to insulin.These results indicate that defects in skeletal muscle  Type 2 diabetes is characterized by both diminished rates of fatty acid oxidation in the fasting state and the inability to efficiently switch to glucose oxidation in the post-prandial state inflexibility inflexibility is merely a reflection of insulin resistance in obesity and type 2 diabetes. The study concluded that diminished glucose disposal rate was the main determinant of blunted metabolic flexibility in individuals with type 2 diabetes. Glucose disposal rate, however, comprises two distinct parts: oxidative and non-oxidative glucose metabolism. Both of these processes are aberrantly affected in type 2 diabetes Multiple mechanisms have recently emerged as potential contributors to insulin resistance and type 2 diabetes progression, among them metabolic in vivo mitochondrial function, metabolic flexibility and insulin resistance remains unclear.Given the prominent role of skeletal muscle in substrate selection, muscle mitochondrial function is likely to have a prominent role in metabolic flexibility. However, the connection between The relationship between metabolic flexibility and insulin resistance may also be confounded by elevated plasma fatty acids and muscle lipid accumulation. These factors have been suggested to interfere with muscle insulin sensitivity but may also affect mitochondrial function or vice versa Therefore, the interrelationships among mitochondrial function, metabolic flexibility and lipid metabolism in the setting of insulin resistance and type 2 diabetes warrants more investigation. In the current study, we assembled data from clamp studies performed at our research center between 2004 and 2011 to assess these interrelationships.The data in the present study is gathered from clamp studies previously performed in our research center 2/min) clamp was performed according to DeFronzo Ra, or endogenous glucose production, EGP) and disposal  as previously described In all subjects a 3-h hyperinsulinemic-euglycemic . The gas analyzer system was calibrated by an alcohol combustion test before every experiment. From oxygen consumption and carbondioxide production levels, whole body glucose oxidation and fat oxidation rates could be calculated using stoichiometric equations according to Frayn 31P-MRS for in vivo mitochondrial function as previously described In a subgroup of 30 T2D patients and 31 control subjects PCr-recovery was measured by Blood collected in tubes containing EDTA was immediately centrifuged and plasma stored at −80°C until assayed. Plasma non-esterified fatty acids (NEFAs) and glucose were measured with enzymatic assays on a Cobas Fara/Mira . Insulin concentration was determined using a radioimmunoassay . Isotopic enrichment of plasma glucose was determined by electron ionization gas chromatography – mass spectrometry and expressed as tracer-to-tracee ratio.Steele's single-pool non–steady-state equations were used to calculate glucose endogenous glucose production (EGP) and whole body glucose disposal (WGD) a priori at p<0.05.Data are reported as means ± SE. Statistical analyses were performed using SPSS version 16.0.2 for Mac OS X  and JMP version 8.0 . Differences between groups were analyzed by one-way ANOVA. Since all variables were normally distributed, correlations were performed in a pair wise fashion using the Pearson product moment statistic. To investigate the relationships among metabolic flexibility and mitochondrial function with other metabolic characteristics, we performed stepwise linear regression analyses. Dependent variables were selected based on simple regression analyses and biological importance in the linear regression analysis. Statistical significance was set The subject characteristics are presented in Oxidative and non-oxidative glucose uptake data are presented in p<0.01). This was reflected in a higher fasting glucose oxidation in T2D patients , whereas non-oxidative glucose disposal (NOGD) was not significantly different between groups . Basal endogenous glucose production (EGP), reflecting hepatic glucose output, was higher in T2D patients compared with control subjects .Basal whole body glucose disposal (WGD) was significantly higher in T2D patients compared to control subjects , although glucose oxidation during insulin infusion was also significantly lower in T2D patients . EGP was almost completely suppressed during insulin infusion in both groups, although T2D patients still had significantly higher EGP during insulin stimulation .Insulin-stimulated WGD was significantly lower in T2D patients compared with control subjects . This was mainly reflected in a reduced non-oxidative glucose uptake during insulin infusion in T2D patients . Insulin stimulation significantly decreased lipid oxidation in both groups, although to a lesser extent in the T2D patients compared to controls .EE did not significantly change upon insulin stimulation in the type 2 diabetic patients nor in the obese controls. Basal EE also did not differ between the groups see .Respiratory exchange ratios in basal and insulin-stimulated states are presented in in vivo mitochondrial function in T2D patients. None of the subjects showed significant acidification at the end of the exercise protocol, and the pH decreased similarly from rest to end-exercise in both groups .Mean PCr recovery half-time (PCr-t1/2) was prolonged by 12.5% in T2D patients compared with control subjects  and inversely related to in vivo PCr-recovery half-time . PCr-recovery rates were not significantly associated with IMCL (data not shown), but they were inversely related to WGD during insulin stimulation in all subjects . This effect seemed to be mainly influenced by non-oxidative glucose disposal, as this, in contrast to glucose oxidation, negatively correlated with PCr-t1/2 .To assess the relationship between the distinct parameters, we performed a simple bivariate correlation analysis. This analysis revealed that metabolic flexibility was positively associated with WGD during insulin stimulation  was related to metabolic flexibility in simple bivariate correlation analysis, it was not a significant predictor of metabolic flexibility  and in the insulin-stimulated state ); therefore it might be plausible that the influence on metabolic flexibility is primarily substrate driven, as proposed by the Randle cycle theory Besides glucose disposal rates, also plasma NEFA levels, seemed an important determinant of metabolic flexibility. Both whole body glucose disposal and plasma NEFA levels during insulin stimulation significantly contributed to the prediction of metabolic flexibility, which also supports the findings of Galgani et al in vivo mitochondrial function remained significantly reduced in the type 2 diabetic patients compared to their controls (data not shown). This suggests that this defect is more than just a difference in training status in these patients. Even so, one can question why in vivo mitochondrial function measured by PCr recovery – reflecting ATP synthetic capacity – would affect basal substrate oxidation. Thus, a reduced capacity to synthesize ATP could be due to increased mitochondrial uncoupling, which might be compensated by increased oxidation in the basal state. However, it should be noted that uncoupling only accounts for a small part of the total oxidation rates in skeletal muscle, and as energy expenditure was not significantly different, the effect of muscle mitochondrial uncoupling on the whole body oxidation rates in this study probably is small. Actually, if anything, earlier results indicated that mitochondrial uncoupling would be decreased in diabetes rather than increased It might be speculated that reduced mitochondrial function in diabetic patients is influenced by differences in physical activity levels between the groups In conclusion, the current study advances the concept that mitochondrial function is a main determinant of basal substrate handling, whereas glucose disposal rate is the primary determinant of insulin-stimulated substrate handling. Furthermore, plasma free fatty acid levels are significantly elevated in type 2 diabetes when compared to obese subjects with similar age, BMI and fat mass, and may contribute to the defects consistently observed in metabolic flexibility in individuals with type 2 diabetes.


These enzymes degrade heme to generate biliverdin, free iron, and carbon monoxide (CO), and this reaction appears to be crucial in a number of diverse biological systems. Decreasing free heme in itself is beneficial . Stimul+/− KO mice I/R caused significantly greater cardiac damage than wild-type mice  caused significantly greater right ventricular hypertrophy, oxidative damage, and pulmonary hypertension   provides clear beneficial effects against the damage of I/R brain injury and ischemic stroke . Indeed, we reported recently that such effects of CO may account for some of the deleterious neurological effects of CO poisoning: we monitored Ca2+ homeostasis in human neuroblastoma (SH-SY5Y) cells and noted that exposure of cells to the CO donor CORM-2 caused an apparent increase in both voltage gated Ca2+ entry and prolonged receptor-mediated rises of [Ca2+]i arising from mobilization of Ca2+ from endoplasmic reticulum stores and subsequent capacitative Ca2+ entry. These potentially deleterious effects of CO were abolished either by an antioxidant or by inhibition of NO formation with L-NAME  in both SH-SY5Y cells and also in whole brain homogenates from rats exposed to 1000 or 3000 ppm CO for 40 mins  is not without risk, in part because it can lead to formation of the highly damaging ROS species peroxynitrite (ONOOin vivo, the cellular effects on PMCA were manifest using CORM-2 at 10–30 μM. Such concentrations are comparable to other reported effects of CO/CORMs which have been regarded, rightly or wrongly, as beneficial/physiological effects. Most electrophysiological studies have employed CORMs at concentrations of 1–100 μM (Peers, + current (Dallas et al., in vitro as potentially beneficial or detrimental. Such information is important in the progression of CO therapy.The example studies cited here raise the question of whether beneficial effects of CO differ from deleterious effects (shown schematically in Figure M Peers, , often a


Posttraumatic stress disorder (PTSD) is characterized by acute and chronic changes in the stress response, manifested as conditioned fear memory. Previously formed memories that are susceptible to disruption immediately after retrieval undergo a protein synthesis-dependent process to become persistent, termed reconsolidation, a process that is regulated by many distinct molecular mechanisms that control gene expression. Increasing evidence supports the participation of the transcription factor NF-κB in the different phases of memory. Here, we demonstrate that inhibition of NF-κB in the basolateral amygdala (BLA), but not central nucleus of the amygdala, after memory reactivation impairs the retention of amygdala-dependent auditory fear conditioning (AFC). We used two independent pharmacological strategies to disrupt the reconsolidation of AFC. Bilateral intra-BLA infusion of sulfasalazine, an inhibitor of IκB kinase that activates NF-κB, and bilateral intra-BLA infusion of SN50, a direct inhibitor of the NF-κB DNA-binding complex, immediately after retrieval disrupted the reconsolidation of AFC. We also found that systemic pretreatment with sodium butyrate, a histone deacetylase inhibitor that enhances histone acetylation, in the amygdala rescued the disruption of reconsolidation induced by NF-κB inhibition in the BLA. These findings indicate that NF-κB activity in the BLA is required for memory reconsolidation in AFC, suggesting that NF-κB might be a potential pharmacotherapy target for posttraumatic stress disorder. Posttraumatic stress disorder (PTSD) is characterized by acute and chronic changes in the stress response, manifested as a conditioned fear memory The NF-κB family of dimeric transcription factors consists of five mammalian members: RelA (p65), RelB, c-Rel, NF-κB1 (p105/p50), and NF-κB2 (p100/p52) Chasmagnathus, in which memory formation is highly correlated with NF-κB activation −/− mice showed spatial learning impairments Previous evidence indicated that NF-κB is activated during memory consolidation in the context signal memory paradigm in the crab The amygdala is the central structure involved in both fear conditioning and extinction To assess the effect of inhibiting NF-κB signaling on memory recall, we infused sulfasalazine (SSZ), a direct pharmacological inhibitor of IKK To evaluated whether NF-κB activation in the BLA is specific to auditory retrieval induced by reexposure to a shock-paired tone in another context (context B), the following experiment was conducted. Twenty-four hours after post-conditioning fear test, vehicle or SSZ (2.5 mM/side) was infused into the BLA, similarly to Experiment 1, with the exception that the retrieval trial was not conducted . No signThis experiment assessed the effect of bilateral intra-BLA infusion of SSZ on the expression of conditioned fear memory. Twenty-four hours after post-conditioning fear test in context A, the animals received bilateral intra-BLA infusion of SSZ or vehicle 20 min before the freezing test in context B. 1,16F = 6.803, p<0.05, This experiment assessed whether direct inhibition of the NF-κB DNA-binding complex by SN50 (10 µg/side) 2 hours prior to retrieval impairs AFC memory This experiment evaluated whether the impairment induced by bilateral intra-BLA infusion of SN50 is specific to reexposure to AFC. Twenty-four hours after post-conditioning fear test, the animals received bilateral intra-BLA infusion of vehicle or SN50 at the same time as in Experiment 4 but without a retrieval trial . No sign3,37F = 5.741, p<0.05; This experiment evaluated whether augmenting chromatin remodeling through activation of histone acetylation reverses the impairment of reconsolidation induced by inhibition of NF-κB in the BLA immediately after retrieval. We used the HDAC inhibitor sodium butyrate (NaB) to enhance histone acetylation and determined whether this manipulation could rescue the impaired long-term AFC memory induced by IKK inhibition in the BLA immediately after retrieval. The animals were trained as described previously. Twenty-four hours after post-conditioning fear test, the animals received an injection of vehicle or NaB  1 h prior to reexposure to the training cue in context B . One houThe present study identified the participation of the IKK/NF-κB signal pathway in the reconsolidation of AFC in rats. We found that inhibition of the NF-κB signaling cascade at the level of IKK protein kinase or the NF-κB DNA binding complex in the BLA, but not CeA, blocked the reconsolidation of long-term AFC memory and that inhibition of the NF-κB signaling cascade at the level of IKK protein kinase had no effect on the expression of AFC. Additionally, the impairment of the reconsolidation of long-term AFC induced by inhibition of the NF-κB signaling cascade in the BLA was rescued by pretreatment with a histone deacetylation inhibitor.Previous studies have shown that the amygdala is a critical part of emotional processing, supported by a large body of evidence from fear conditioning investigations. The amygdala is a central structure that plays pivotal roles in fear memory formation and reconsolidation Activation of the NF-κB signaling pathway requires upregulation of the IKK complex in hippocampus-dependent contextual fear memory In the present study, SSZ and SN50 were used to inhibit the activity of NF-κB. SN50 is a direct inhibitor of the NF-κB DNA-binding complex, and SSZ is a direct pharmacological inhibitor of IKK, both of which inhibit the activity of NF-κB Previous studies have shown that histone acetyltransferases (HATs) were activated in the hippocampus during memory reconsolidation In the present study, NaB, an inhibitor of HDAC that enhances histone acetylation, was used to explore whether the augmentation of chromatin remodeling induced by increasing histone acetylation can rescue the impairment of long-term AFC induced by inhibition of NF-κB activity in the BLA during reconsolidation phase. The results showed that the impairment of long-term AFC induced by inhibition of NF-κB activity in the BLA during the reconsolidation phase was reversed by systemic pretreatment with NaB, suggesting that freezing behavior deficits induced by IKK inhibition in the BLA after memory recall was rescued by enhancing histone acetylation induced by pretreatment with NaB. Although we found that HDAC inhibition rescues memory deficits caused by NF-κB blockade, the straightforward molecular mechanism underlying this effect needs to be further studied.According to a previous study, increased DNA binding activity of NF-κB induced by HDAC inhibitor enhanced fear-potentiated startle, which indicated that the interaction between histone acetylation and NF-kB pathway may be functional In conclusion, our results suggest that the NF-κB signaling pathway in the BLA is required for the reconsolidation of AFC through the regulation of gene transcription mechanisms. Additionally, fear amnesia induced by blocking NF-κB activity in the BLA during the reconsolidation phase could be rescued by pretreatment with an HDAC inhibitor. Activation of the NF-κB pathway is required for the reconsolidation of AFC memory, and histone deacetylation may be an intra-nuclear molecular switch that culminates in the termination of the NF-κB transcriptional response in long-term memory. Our findings may provide a novel target to help people release traumatic memories and a novel pharmacological intervention for the treatment of PTSD.One hundred eighty Sprague-Dawley rats  were obtained from the Peking University Experimental Animal Center. The animals were housed under a 12 h/12 h light/dark cycle and allowed ad libitum access to rodent chow and water. The room temperature was maintained at 21–23°C and 45–50% relative humidity. The rats were handled daily during the first week after arrival. All experimental procedures in the current study were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by Biomedical Ethics Committee of Peking University of animal use and protection.During surgery, each rat was implanted with a 23-gauge stainless steel cannula  into the BLA  or CeA  1 h prior to the retrieval session. After all of the behavioral tests, the rats were anesthetized with sodium pentobarbital  and transcardially perfused with paraformaldehyde . Cannula placements were assessed using Nissl staining with a section thickness of 40 µm under light microscopy. The rats with misplaced cannulae were excluded For the experiments that investigated the effect of inhibiting the NF-κB signaling pathway on long-term memory after recall, the animals received an infusion of either vehicle or SSZ into the BLA  or CeA (2.5 mM/side). Vehicle was saline solution with 10 mM HEPES (pH 7.6) plus 20% dimethyl sulfoxide  and SSZ was freshly dissolved in vehicle. All infusions were performed immediately following the retrieval session in chamber B. For the experiments that investigated the effect of inhibiting the NF-κB DNA-binding complex on long-term memory after recall, the SN50 active peptide (10 µg/side) or saline was infused into the BLA. SN50 infusions were performed 2 h prior to the retrieval session in chamber B. SN50 was dissolved in saline. SSZ or SN50 were bilaterally infused into BLA or CeA with Hamilton syringes connected to 30 gauge injectors (PlasticsOne). The infusion volume was 0.5 µl, and the drug was injected bilaterally over 1 min; the injection needle was kept in place for an additional 1 min to allow for drug diffusion Fear conditioning and tone testing were conducted in different chambers. For conditioning, the rats were placed in a Plexiglas rodent conditioning chamber (context A) with a grid floor and a white light. For tone testing, the rats were placed in a different Plexiglas chamber (context B) that was brightly lit with three house lights and had a black floor that had been washed with peppermint soap. A micro-video camera was mounted at the top of the chamber so that the rats could be videotaped during testing.The animals were handled for 5 days after surgery. On the day of the experiments, they were transported to the laboratory at least 2 h prior to fear conditioning. The animals were placed into training chamber A and allowed to explore it for 10 min. Fear conditioning was then conducted with three 20 s, 5 kHz, 80 dB tones (conditioned stimulus [CS]), each co-terminating with a 1 s, 1 mA footshock . Data in all experiments were analyzed with two-way analysis of variance (ANOVA) followed by the Tukey


Endoglin is a transmembrane auxiliary receptor for transforming growth factor-beta (TGF-beta) that is predominantly expressed on proliferating endothelial cells. It plays a wide range of physiological roles but its importance on energy balance or insulin sensitivity has been unexplored. Endoglin deficient mice die during midgestation due to cardiovascular defects. Here we report for first time that heterozygous endoglin deficiency in mice decreases high fat diet-induced hepatic triglyceride content and insulin levels. Importantly, these effects are independent of changes in body weight or adiposity. At molecular level, we failed to detect relevant changes in the insulin signalling pathway at basal levels in liver, muscle or adipose tissues that could explain the insulin-dependent effect. However, we found decreased triglyceride content in the liver of endoglin heterozygous mice fed a high fat diet in comparison to their wild type littermates. Overall, our findings indicate that endoglin is a potentially important physiological mediator of insulin levels and hepatic lipid metabolism. Endoglin (Eng) is a transmembrane homodimeric glycoprotein (180 kDa) identified in human vascular endothelial cells where it is highly expressed Eng modulates processes mainly related to vascular physiology and pathophysiology +/−Eng) in normal conditions or when challenged with high fat diet.Mutations in the endoglin gene leading to endoglin haploinsufficiency are the cause of the Hereditary Hemorrhagic Telangiectasia (HHT) type 1 +/−Eng mice on a C57Bl/6 background was previously described +/−Eng and +/+Eng littermate male mice of 4–6 months of age (20–25 g). After weaning, mice were fed a standard chow diet, and after 8 weeks, the diet was changed to high fat diet  during 16 weeks. All animal procedures performed were approved by the University of Salamanca Animal Care and Use Committee and by the Animal Committee at the University of Santiago de Compostela. All the experiments were performed in agreement with the Rules of Laboratory Animal Care and International Law on Animal Experimentation.Generation and genotyping of Whole body composition was measured using NMR imaging . Animals were monitored in a custom 12-cage indirect calorimetry, food intake and locomotor activity monitoring system  as previously described 5′-GAA GGG TCG CAT GGC AAA-3′, 5′-FAM-AGG GCA AGA TCA TCA TGC ACG ACC C-TAM-3′. For the analysis of the data, the input value of the gene expression was standardized to the 18S value for each sample of each group and was expressed compared with the average value for the control group.RNA was extracted using Trizol® reagent (Invitrogen) according to the manufacturer's instructions and two micrograms of total RNA were used for each RT reaction and cDNA synthesis was performed using SuperScript™ First-Strand Synthesis System (Invitrogen) and random primers as previously described Western blots were performed as previously described Blood glucose levels were measured with an Accucheck glucometer (Roche) after an intraperitoneal injection of either 2 mg/g D-glucose (Sigma) or 0.75 U/kg insulin (Sigma-Aldrich) 2SO4 was added, samples were centrifuged, and the organic bottom layer was collected. The organic solvent was dried using a Speed Vac and redissolved in chloroform. TG  content of each sample was measured in duplicate after evaporation of the organic solvent using an enzymatic method.The extraction procedure for tissue TG was adapted from methods described previously Plasma glucose was measured by the glucose oxidase method . Plasma nonesterified fatty acids (NEFA) concentrations were determined using a kit from Wako (US); triacylglycerol (TG) and cholesterol were determined using a kit from Randox Laboratories . Plasma insulin levels were measured by a previously described RIA Values are plotted as the mean ± SEM for each genotype. Statistical significance was determined by Student's t -test. A P value less than 0.05 was considered statistically significant.+/−Eng mice of 4 weeks of age were maintained on standard diet for 8 weeks to assess their metabolic phenotypes. No body weight differences were found between both genotypes  after an overnight fasting, we did not find any alteration in the tolerance to glucose as compared to WT littermates  for 16 wk to assess their metabolic phenotypes. No differences in body weight were found between +/−Eng and WT mice when they were fed a HFD , pAKT, AKT, and phosphatase and tensin homolog (PTEN) protein levels in liver, skeletal muscle and adipose tissue samples. In the liver we found decreased levels of AKT phosphorylation (Ser473) and the ratio pAKT/AKT, whereas no changes were detected in NFkB, AKT or PTEN  and phosphoenolpyruvate carboxykinase (PEPCK)  content in the liver of +/−Eng mice fed a HFD was significantly lower than in WT mice  , but fai WT mice .Eng caused a significant decrease in insulin Eng die during the embryonary phase due to defective angiogenesis +/−Eng mice are viable, breed normally and represent a useful tool to assess the physiological role of endoglin +/Eng mice does not lead to significant changes in body weight, adiposity or food intake. Since HFD induces non-alcoholic fatty liver and causes hyperinsulinemia in mice, a remarkable observation is that after 16 weeks on HFD, +/−Eng mice showed decreased hepatic triglyceride content and lower insulin levels compared with WT mice.Mice lacking Eng  respect to cells from healthy subjects were involved in general metabolism SLC2A1) and Glut-4 (SLC2A4). Thus, in HHT1 cells, electroneutral cation-Cl cotransporter SLC12A2 (Na-K-Cl cotransporter), mitochondrial carrier SLC25A29 , fatty acid transport protein SLC27A3 (fatty acid transport protein 3), nucleoside-sugar transporter SLC35A5 (UDP-sugar transporter protein) and basolateral iron transporter SCL40A1 (ferroportin 1) were downregulated, whereas nucleoside-sugar transporters SLC35B2 (3′-phosphoadenosine 5′-phosphosulfate transporter) and SLC35D3 (fringe connection-like protein 1) were upregulated respect to controls. In addition to the evident involvement of these carrier proteins in the general metabolism, some of them have been reported to be involved in insulin-dependent metabolic pathways At the cellular level, insulin stimulates glucose uptake by inducing the translocation of the glucose transporter 4 (GLUT4) from intracellular storage sites to the plasma membrane, where the transporter facilitates the diffusion of glucose into muscle and adipocytes In summary, we conclude that Eng has a physiological role in the regulation of insulin levels and hepatic lipid content, particularly under challenged environmental conditions. The decreased insulin levels and lower hepatic lipid content seem to be independent of changes in body weight or adiposity. These findings expand our knowledge on the physiological effects controlled by Eng, and identify Eng as a potentially important physiological mediator of metabolism.