Two additive mechanisms impair the differentiation of 'substrate-selective' p38 inhibitors from classical p38 inhibitors in vitro

<p>Abstract</p> <p>Background</p> <p>The success of anti-TNF biologics for the treatment of rheumatoid arthritis has highlighted the importance of understanding the intracellular pathways that regulate TNF production in the quest for an orally-available small molecule inhibitor. p38 is known to strongly regulate TNF production via MK2. The failure of several p38 inhibitors in the clinic suggests the importance of other downstream pathways in normal cell function. Recent work has described a 'substrate-selective' p38 inhibitor that is able to preferentially block the activity of p38 against one substrate (MK2) versus another (ATF2). Using a combined experimental and computational approach, we have examined this mechanism in greater detail for two p38 substrates, MK2 and ATF2.</p> <p>Results</p> <p>We found that in a dual (MK2 and ATF2) substrate assay, MK2-p38 interaction reduced the activity of p38 against ATF2. We further constructed a detailed kinetic mechanistic model of p38 phosphorylation in the presence of multiple substrates and successfully predicted the performance of classical and so-called 'substrate-selective' p38 inhibitors in the dual substrate assay. Importantly, it was found that excess MK2 results in a stoichiometric effect in which the formation of p38-MK2-inhibitor complex prevents the phosphorylation of ATF2, despite the preference of the compound for the p38-MK2 complex over the p38-ATF2 complex. MK2 and p38 protein expression levels were quantified in U937, Thp-1 and PBMCs and found that [MK2] > [p38].</p> <p>Conclusion</p> <p>Our integrated mechanistic modeling and experimental validation provides an example of how systems biology approaches can be applied to drug discovery and provide a basis for decision-making with limited chemical matter. We find that, given our current understanding, it is unlikely that 'substrate-selective' inhibitors of p38 will work as originally intended when placed in the context of more complex cellular environments, largely due to a stoichiometric excess of MK2 relative to p38.</p

The influence of methylphenidate on the power spectrum of ADHD children – an MEG study

BACKGROUND: The present study was dedicated to investigate the influence of Methylphenidate (MPH) on cortical processing of children who were diagnosed with different subtypes of Attention Deficit Hyperactivity Disorder (ADHD). As all of the previous studies investigating power differences in different frequency bands have been using EEG, mostly with a relatively small number of electrodes our aim was to obtain new aspects using high density magnetoencephalography (MEG). METHODS: 35 children (6 female, 29 male) participated in this study. Mean age was 11.7 years (± 1.92 years). 17 children were diagnosed of having an Attention-Deficit/Hyperactivity Disorder of the combined type (ADHDcom, DSM IV code 314.01); the other 18 were diagnosed for ADHD of the predominantly inattentive type (ADHDin, DSM IV code 314.0). We measured the MEG during a 5 minute resting period with a 148-channel magnetometer system (MAGNES™ 2500 WH, 4D Neuroimaging, San Diego, USA). Power values were averaged for 5 bands: Delta (D, 1.5–3.5 Hz), Theta (T, 3.5–7.5 Hz), Alpha (A, 7.5–12.5 Hz), Beta (B, 12.5–25 Hz) and Global (GL, 1.5–25 Hz).). Additionally, attention was measured behaviourally using the D2 test of attention with and without medication. RESULTS: The global power of the frequency band from 1.5 to 25 Hz increased with MPH. Relative Theta was found to be higher in the left hemisphere after administration of MPH than before. A positive correlation was found between D2 test improvement and MPH-induced power changes in the Theta band over the left frontal region. A linear regression was computed and confirmed that the larger the improvement in D2 test performance, the larger the increase in Theta after MPH application. CONCLUSION: Main effects induced by medication were found in frontal regions. Theta band activity increased over the left hemisphere after MPH application. This finding contradicts EEG results of several groups who found lower levels of Theta power after MPH application. As relative Theta correlates with D2 test improvement we conclude that MEG provide complementary and therefore important new insights to ADHD

Understanding Streptococcus suis serotype 2 infection in pigs through a transcriptional approach

<p>Abstract</p> <p>Background</p> <p><it>Streptococcus suis </it>serotype 2 (<it>S. suis </it>2) is an important pathogen of pigs. <it>S suis 2 </it>infections have high mortality rates and are characterized by meningitis, septicemia and pneumonia. <it>S. suis </it>2 is also an emerging zoonotic agent and can infect humans that are exposed to pigs or their by-products. To increase our knowledge of the pathogenesis of meningitis, septicemia and pneumonia in pigs caused by <it>S. suis </it>2, we profiled the response of peripheral blood mononuclear cells <b>(</b>PBMC), brain and lung tissues to infection with <it>S. suis </it>2 strain SC19 using the Affymetrix Porcine Genome Array.</p> <p>Results</p> <p>A total of 3,002 differentially expressed transcripts were identified in the three tissues, including 417 unique genes in brain, 210 in lung and 213 in PBMC. These genes showed differential expression (DE) patterns on analysis by visualization and integrated discovery (DAVID). The DE genes involved in the immune response included genes related to the inflammatory response (CD163), the innate immune response (TLR2, TLR4, MYD88, TIRAP), cell adhesion (CD34, SELE, SELL, SELP, ICAM-1, ICAM-2, VCAM-1), antigen processing and presentation (MHC protein complex) and angiogenesis (VEGF), together with genes encoding cytokines (interleukins). Five selected genes were validated by qRT-PCR analysis.</p> <p>Conclusions</p> <p>We studied the response to infection with <it>S. suis </it>2 strain SC19 by microarray analysis. Our findings confirmed some genes identified in previous studies and discovered numerous additional genes that potentially function in <it>S. suis </it>2 infections in vivo. This new information will form the foundation of future investigations into the pathogenesis of <it>S. suis</it>.</p

Copy Number Variation of CCL3-like Genes Affects Rate of Progression to Simian-AIDS in Rhesus Macaques (Macaca mulatta)

Variation in genes underlying host immunity can lead to marked differences in susceptibility to HIV infection among humans. Despite heavy reliance on non-human primates as models for HIV/AIDS, little is known about which host factors are shared and which are unique to a given primate lineage. Here, we investigate whether copy number variation (CNV) at CCL3-like genes (CCL3L), a key genetic host factor for HIV/AIDS susceptibility and cell-mediated immune response in humans, is also a determinant of time until onset of simian-AIDS in rhesus macaques. Using a retrospective study of 57 rhesus macaques experimentally infected with SIVmac, we find that CCL3L CNV explains approximately 18% of the variance in time to simian-AIDS (p<0.001) with lower CCL3L copy number associating with more rapid disease course. We also find that CCL3L copy number varies significantly (p<10−6) among rhesus subpopulations, with Indian-origin macaques having, on average, half as many CCL3L gene copies as Chinese-origin macaques. Lastly, we confirm that CCL3L shows variable copy number in humans and chimpanzees and report on CCL3L CNV within and among three additional primate species. On the basis of our findings we suggest that (1) the difference in population level copy number may explain previously reported observations of longer post-infection survivorship of Chinese-origin rhesus macaques, (2) stratification by CCL3L copy number in rhesus SIV vaccine trials will increase power and reduce noise due to non-vaccine-related differences in survival, and (3) CCL3L CNV is an ancestral component of the primate immune response and, therefore, copy number variation has not been driven by HIV or SIV per se

Luminal-Applied Flagellin Is Internalized by Polarized Intestinal Epithelial Cells and Elicits Immune Responses via the TLR5 Dependent Mechanism

Bacteria release flagellin that elicits innate responses via Toll-like receptor 5 (TLR5). Here, we investigated the fate of apically administrated full length flagellin from virulent and avirulent bacteria, along with truncated recombinant flagellin proteins in intestinal epithelial cells and cellular responses. Flagellin was internalized by intestinal epithelial cell (IEC) monolayers of IEC-18. Additionally, apically applied flagellin was internalized by polarized human Caco-2BBe and T-84 cells in a TLR5 dependent mechanism. More, flagellin exposure did not affect the integrity of intestinal monolayers. With immunofluorescent staining, internalized flagellin was detected in both early endosomes as well as lysosomes. We found that apical exposure of polarized Caco-2BBe and T-84 to flagellin from purified Salmonella, Escherichia coli O83:H1 (isolate from Crohn’s lesion) or avirulent E. coli K12 induced comparable levels of basolateral IL-8 secretion. A recombinant protein representing the conserved amino (N) and carboxyl (C) domains (D) of the flagellin protein (ND1/2ECHCD2/1) induced IL-8 secretion from IEC similar to levels elicited by full-length flagellins. However, a recombinant flagellin protein containing only the D3 hypervariable region elicited no IL-8 secretion in both cell lines compared to un-stimulated controls. Silencing or blocking TLR5 in Caco-2BBe cells resulted in a lack of flagellin internalization and decreased IL-8 secretion. Furthermore, apical exposure to flagellin stimulated transepithelial migration of neutrophils and dendritic cells. The novel findings in this study show that luminal-applied flagellin is internalized by normal IEC via TLR5 and co-localizes to endosomal and lysosomal compartments where it is likely degraded as flagellin was not detected on the basolateral side of IEC cultures

Competitive Tendering In The Netherlands: Central Planning Or Functional Specifications?

Institute of Transport and Logistics Studies. Faculty of Economics and Business. The University of Sydne

DNA methylation and methyl-CpG binding proteins: developmental requirements and function

DNA methylation is a major epigenetic modification in the genomes of higher eukaryotes. In vertebrates, DNA methylation occurs predominantly on the CpG dinucleotide, and approximately 60% to 90% of these dinucleotides are modified. Distinct DNA methylation patterns, which can vary between different tissues and developmental stages, exist on specific loci. Sites of DNA methylation are occupied by various proteins, including methyl-CpG binding domain (MBD) proteins which recruit the enzymatic machinery to establish silent chromatin. Mutations in the MBD family member MeCP2 are the cause of Rett syndrome, a severe neurodevelopmental disorder, whereas other MBDs are known to bind sites of hypermethylation in human cancer cell lines. Here, we review the advances in our understanding of the function of DNA methylation, DNA methyltransferases, and methyl-CpG binding proteins in vertebrate embryonic development. MBDs function in transcriptional repression and long-range interactions in chromatin and also appear to play a role in genomic stability, neural signaling, and transcriptional activation. DNA methylation makes an essential and versatile epigenetic contribution to genome integrity and function

ICAR: endoscopic skull‐base surgery

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