The role of polyamines in the regulation of macrophage polarization and function

Amino Acids - Naturally occurring polyamines are ubiquitously distributed and play important roles in cell development, amino acid and protein synthesis, oxidative DNA damage, proliferation, and...     

Synthesis,Crystal Structure,and in Vitro Biological Evaluation of C-6 Pyrimidine Derivatives: New Lead Structures for Monitoring Gene Expression in Vivo

Novel C-6 substituted pyrimidine derivatives are good substrates of herpes simplex virus type 1 thymidine kinase (HSV1-TK). Enzyme kinetic experiments showed that our lead compound, N-methyl DHBT (N-methyl-6-(1,3-dihydroxyisobutyl) thymine; N-Me DHBT), is phosphorylated at a similar rate compared to “gold standard” 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine, FHBG, (K _m = 10 ± 0.3 μM; k _cat = 0.036 ± 0.015 sec^?1). Additionally, it does not show cytotoxic properties on B16F1 cells up to a concentration of 10 mM. The x-ray analysis of the crystal structures of HSV1-TK with N-Me DHBT and of HSV1-TK with the fluorinated derivative N-Me FHBT confirmed the binding mode predicted by docking studies and their substrate characteristics. Moreover, the crystal structure of HSV1-TK with N-Me DHBT revealed an additional water-mediated H-bond interesting for the design of further analogues.     

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

Heligmosomoides polygyrus (formerly known as Nematospiroides dubius, and also referred to by some as H. bakeri) is a gastrointestinal helminth that employs multiple immunomodulatory mechanisms to establish chronic infection in mice and closely resembles prevalent human helminth infections. H. polygyrus has been studied extensively in the field of helminth-derived immune regulation and has been found to potently suppress experimental models of allergy and autoimmunity (both with active infection and isolated secreted products). The protocol described in this paper outlines management of the H. polygyrus life cycle for consistent production of L3 larvae, recovery of adult parasites, and collection of their excretory-secretory products (HES).     

Nutlin-3a: A Potential Therapeutic Opportunity for TP53 Wild-Type Ovarian Carcinomas

Epithelial ovarian cancer is a diverse molecular and clinical disease, yet standard treatment is the same for all subtypes. TP53 mutations represent a node of divergence in epithelial ovarian cancer histologic subtypes and may represent a therapeutic opportunity in subtypes expressing wild type, including most low-grade ovarian serous carcinomas, ovarian clear cell carcinomas and ovarian endometrioid carcinomas, which represent approximately 25% of all epithelial ovarian cancer. We therefore sought to investigate Nutlin-3a—a therapeutic which inhibits MDM2, activates wild-type p53, and induces apoptosis—as a therapeutic compound for TP53 wild-type ovarian carcinomas. Fifteen epithelial ovarian cancer cell lines of varying histologic subtypes were treated with Nutlin-3a with determination of IC₅₀ values. Western Blot (WB) and quantitative real-time polymerase chain reaction (qRT-PCR) analyses quantified MDM2, p53, and p21 expression after Nutlin-3a treatment. DNA from 15 cell lines was then sequenced for TP53 mutations in exons 2-11 including intron-exon boundaries. Responses to Nutlin-3a were dependent upon TP53 mutation status. By qRT-PCR and WB, levels of MDM2 and p21 were upregulated in wild-type TP53 sensitive cell lines, and p21 induction was reduced or absent in mutant cell lines. Annexin V assays demonstrated apoptosis in sensitive cell lines treated with Nutlin-3a. Thus, Nutlin-3a could be a potential therapeutic agent for ovarian carcinomas expressing wild-type TP53 and warrants further investigation.     

Ineffective Degradation of Immunogenic Gluten Epitopes by Currently Available Digestive Enzyme Supplements

Background

Due to the high proline content of gluten molecules, gastrointestinal proteases are unable to fully degrade them leaving large proline-rich gluten fragments intact, including an immunogenic 33-mer from α-gliadin and a 26-mer from γ-gliadin. These latter peptides can trigger pro-inflammatory T cell responses resulting in tissue remodeling, malnutrition and a variety of other complications. A strict lifelong gluten-free diet is currently the only available treatment to cope with gluten intolerance. Post-proline cutting enzymes have been shown to effectively degrade the immunogenic gluten peptides and have been proposed as oral supplements. Several existing digestive enzyme supplements also claim to aid in gluten degradation. Here we investigate the effectiveness of such existing enzyme supplements in comparison with a well characterized post-proline cutting enzyme, Prolyl EndoPeptidase from Aspergillus niger (AN-PEP).

Methods

Five commercially available digestive enzyme supplements along with purified digestive enzymes were subjected to 1) enzyme assays and 2) mass spectrometric identification. Gluten epitope degradation was monitored by 1) R5 ELISA, 2) mass spectrometric analysis of the degradation products and 3) T cell proliferation assays.

Findings

The digestive enzyme supplements showed comparable proteolytic activities with near neutral pH optima and modest gluten detoxification properties as determined by ELISA. Mass spectrometric analysis revealed the presence of many different enzymes including amylases and a variety of different proteases with aminopeptidase and carboxypeptidase activity. The enzyme supplements leave the nine immunogenic epitopes of the 26-mer and 33-mer gliadin fragments largely intact. In contrast, the pure enzyme AN-PEP effectively degraded all nine epitopes in the pH range of the stomach at much lower dose. T cell proliferation assays confirmed the mass spectrometric data.

Conclusion

Currently available digestive enzyme supplements are ineffective in degrading immunogenic gluten epitopes.     

Impact of a Clinical Decision Model for Febrile Children at Risk for Serious Bacterial Infections at the Emergency Department: A Randomized Controlled Trial

Objectives

To assess the impact of a clinical decision model for febrile children at risk for serious bacterial infections (SBI) attending the emergency department (ED).

Methods

Randomized controlled trial with 439 febrile children, aged 1 month-16 years, attending the pediatric ED of a Dutch university hospital during 2010-2012. Febrile children were randomly assigned to the intervention (clinical decision model; n=219) or the control group (usual care; n=220). The clinical decision model included clinical symptoms, vital signs, and C-reactive protein and provided high/low-risks for “pneumonia” and “other SBI”. Nurses were guided by the intervention to initiate additional tests for high-risk children. The clinical decision model was evaluated by 1) area-under-the-receiver-operating-characteristic-curve (AUC) to indicate discriminative ability and 2) feasibility, to measure nurses’ compliance to model recommendations. Primary patient outcome was defined as correct SBI diagnoses. Secondary process outcomes were defined as length of stay; diagnostic tests; antibiotic treatment; hospital admission; revisits and medical costs.

Results

The decision model had good discriminative ability for both pneumonia (n=33; AUC 0.83 (95% CI 0.75-0.90)) and other SBI (n=22; AUC 0.81 (95% CI 0.72-0.90)). Compliance to model recommendations was high (86%). No differences in correct SBI determination were observed. Application of the clinical decision model resulted in less full-blood-counts (14% vs. 22%, p-value<0.05) and more urine-dipstick testing (71% vs. 61%, p-value<0.05).

Conclusions

In contrast to our expectations no substantial impact on patient outcome was perceived. The clinical decision model preserved, however, good discriminatory ability to detect SBI, achieved good compliance among nurses and resulted in a more standardized diagnostic approach towards febrile children, with less full blood-counts and more rightfully urine-dipstick testing.

Trial Registration

Nederlands Trial Register NTR2381     

Acute Effects of Whole Body Vibration on Inhibition in Healthy Children

Objectives

Whole Body Vibration (WBV) is a passive exercise method known to have beneficial effects on various physical measures. Studies on adults furthermore demonstrated beneficial effects of WBV treatment on cognition (e.g. inhibition). The present study replicated these findings in healthy children and examined acute effects of WBV treatment on inhibition.

Methods

Fifty-five healthy children (aged 8–13) participated in this within-subject design study. WBV treatment was applied by having the children sit on a chair mounted to a vibrating platform. After each condition (vibration vs. non-vibration), inhibition was measured by using the Stroop Color-Word Interference Test. Repeated measures analyses were applied in order to explore the effects of WBV treatment on inhibition, and correlations were computed between the treatment effect and participant characteristics in order to explore individual differences in treatment sensitivity.

Results

Three-minute WBV treatments had significant beneficial effects on inhibition in this sample of healthy children. Especially the repeated application (three times) of WBV treatment appeared beneficial for cognition. Stronger WBV treatment effects were correlated with higher intelligence and younger age, but not with symptoms of Attention Deficit Hyperactivity Disorder (ADHD).

Conclusions

This study demonstrates that especially repeated WBV treatment improves inhibition in healthy children. As this cognitive function is often impaired in children with developmental disorders (e.g. ADHD), future studies should further explore the effects, working mechanism and potential applicability of WBV treatment for this target group.     

In Search for the Genetic Basis of Quality of Life in Healthy Swedish Women—A GWAS Study Using the iCOGS Custom Genotyping Array

Background

Quality of life (QoL) is increasingly measured in both research and clinical practice. QoL-assessments are built on a long, empirically-based, and stringent approach. There is ample evidence that QoL is, in part, heritable. We therefore performed a GWAS relating genetic variation to QoL in healthy females.

Methods

In 5,142 healthy females, background characteristics (e.g. demographic, clinical, lifestyle and psychological factors) and QoL by means of the EORTC QLQ-C30 were measured. Moreover, women were genotyped using a custom array including ~210,000 single nucleotide polymorphisms (SNPs). Initially, SNPs were related to each QoL-domain, by means of partially adjusted (controlling for age and population stratification) and fully adjusted (controlling for age, population stratification, and background characteristics) regression analyses. Additionally, gene-based analyses were performed relating the combined effect of SNPs within each gene to QoL using the statistical software package VEGAS.

Results

None of the associations between QoL and genetic variation (i.e. individual SNPs and genes) reached the bonferroni corrected significance level.

Conclusion

Reasons for a lack of association between genetic markers and QoL could be low variation in QoL-scores; selecting genetic markers not tagging QoL; or that the genetic effect that impacts one’s QoL is mediated through biological pathways rather than the effect of single SNPs or genes. Therefore, we opt for a pathway-based or system biology approach as a complementary and powerful approach to analyze the combined effect of genes and their biological implications in future studies focusing on QoL-issues.     

A Metabolic Probe-Enabled Strategy Reveals Uptake and Protein Targets of Polyunsaturated Aldehydes in the Diatom Phaeodactylum tricornutum

Diatoms are unicellular algae of crucial importance as they belong to the main primary producers in aquatic ecosystems. Several diatom species produce polyunsaturated aldehydes (PUAs) that have been made responsible for chemically mediated interactions in the plankton. PUA-effects include chemical defense by reducing the reproductive success of grazing copepods, allelochemical activity by interfering with the growth of competing phytoplankton and cell to cell signaling. We applied a PUA-derived molecular probe, based on the biologically highly active 2,4-decadienal, with the aim to reveal protein targets of PUAs and affected metabolic pathways. By using fluorescence microscopy, we observed a substantial uptake of the PUA probe into cells of the diatom Phaeodactylum tricornutum in comparison to the uptake of a structurally closely related control probe based on a saturated aldehyde. The specific uptake motivated a chemoproteomic approach to generate a qualitative inventory of proteins covalently targeted by the α,β,γ,δ-unsaturated aldehyde structure element. Activity-based protein profiling revealed selective covalent modification of target proteins by the PUA probe. Analysis of the labeled proteins gave insights into putative affected molecular functions and biological processes such as photosynthesis including ATP generation and catalytic activity in the Calvin cycle or the pentose phosphate pathway. The mechanism of action of PUAs involves covalent reactions with proteins that may result in protein dysfunction and interference of involved pathways.