Eclectic characterisation of chemically modified cell-derived matrices obtained by metabolic glycoengineering and re-assessment of commonly used methods

Azide-bearing cell-derived extracellular matrices (“clickECMs”) have emerged as a highly exciting new class of biomaterials. They conserve substantial characteristics of the natural extracellular matrix (ECM) and offer simultaneously small abiotic functional groups that enable bioorthogonal bioconjugation reactions. Despite their attractiveness, investigation of their biomolecular composition is very challenging due to the insoluble and highly complex nature of cell-derived matrices (CDMs). Yet, thorough qualitative and quantitative analysis of the overall material composition, organisation, localisation, and distribution of typical ECM-specific biomolecules is essential for consistent advancement of CDMs and the understanding of the prospective functions of the developed biomaterial. In this study, we evaluated frequently used methods for the analysis of complex CDMs. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and (immune)histochemical staining methods in combination with several microscopic techniques were found to be highly eligible. Commercially available colorimetric protein assays turned out to deliver inaccurate information on CDMs. In contrast, we determined the nitrogen content of CDMs by elementary analysis and converted it into total protein content using conversion factors which were calculated from matching amino acid compositions. The amount of insoluble collagens was assessed based on the hydroxyproline content. The Sircol™ assay was identified as a suitable method to quantify soluble collagens while the Blyscan™ assay was found to be well-suited for the quantification of sulphated glycosaminoglycans (sGAGs). Eventually, we propose a series of suitable methods to reliably characterise the biomolecular composition of fibroblast-derived clickECM.

Common characterisation methods for cell-derived extracellular matrices (ECMs) are compared using both unmodified and azide-bearing fibroblast-derived ECM.     

Short- and Medium-Term Efficacy of a Web-Based Computer-Tailored Nutrition Education Intervention for Adults Including Cognitive and Environmental Feedback: Randomized Controlled Trial

Background

Web-based, computer-tailored nutrition education interventions can be effective in modifying self-reported dietary behaviors. Traditional computer-tailored programs primarily targeted individual cognitions (knowledge, awareness, attitude, self-efficacy). Tailoring on additional variables such as self-regulation processes and environmental-level factors (the home food environment arrangement and perception of availability and prices of healthy food products in supermarkets) may improve efficacy and effect sizes (ES) of Web-based computer-tailored nutrition education interventions.

Objective

This study evaluated the short- and medium-term efficacy and educational differences in efficacy of a cognitive and environmental feedback version of a Web-based computer-tailored nutrition education intervention on self-reported fruit, vegetable, high-energy snack, and saturated fat intake compared to generic nutrition information in the total sample and among participants who did not comply with dietary guidelines (the risk groups).

Methods

A randomized controlled trial was conducted with a basic (tailored intervention targeting individual cognition and self-regulation processes; n=456), plus (basic intervention additionally targeting environmental-level factors; n=459), and control (generic nutrition information; n=434) group. Participants were recruited from the general population and randomly assigned to a study group. Self-reported fruit, vegetable, high-energy snack, and saturated fat intake were assessed at baseline and at 1- (T1) and 4-months (T2) postintervention using online questionnaires. Linear mixed model analyses examined group differences in change over time. Educational differences were examined with group×time×education interaction terms.

Results

In the total sample, the basic (T1: ES=–0.30; T2: ES=–0.18) and plus intervention groups (T1: ES=–0.29; T2: ES=–0.27) had larger decreases in high-energy snack intake than the control group. The basic version resulted in a larger decrease in saturated fat intake than the control intervention (T1: ES=–0.19; T2: ES=–0.17). In the risk groups, the basic version caused larger decreases in fat (T1: ES=–0.28; T2: ES=–0.28) and high-energy snack intake (T1: ES=–0.34; T2: ES=–0.20) than the control intervention. The plus version resulted in a larger increase in fruit (T1: ES=0.25; T2: ES=0.37) and a larger decrease in high-energy snack intake (T1: ES=–0.38; T2: ES=–0.32) than the control intervention. For high-energy snack intake, educational differences were found. Stratified analyses showed that the plus version was most effective for high-educated participants.

Conclusions

Both intervention versions were more effective in improving some of the self-reported dietary behaviors than generic nutrition information, especially in the risk groups, among both higher- and lower-educated participants. For fruit intake, only the plus version was more effective than providing generic nutrition information. Although feasible, incorporating environmental-level information is time-consuming. Therefore, the basic version may be more feasible for further implementation, although inclusion of feedback on the arrangement of the home food environment and on availability and prices may be considered for fruit and, for high-educated people, for high-energy snack intake.

Trial Registration

Netherlands Trial Registry NTR3396; http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=3396 (Archived by WebCite at http://www.webcitation.org/6VNZbdL6w).     

Expression and function of microRNA-188-5p in activated rheumatoid arthritis synovial fibroblasts

Activated synovial fibroblasts in rheumatoid arthritis (RASF) play a critical role in the pathology of rheumatoid arthritis (RA). Recent studies suggested that deregulation of microRNAs (miRs) affects the development and progression of RA. Therefore, we aimed to identify de-regulated miRs in RASF and to identify target genes that may contribute to the aggressive phenotype of RASF. Quantitative real-time PCR revealed a marked downregulation of miR-188-5p in synovial tissue samples of RA patients as well as in RASF. Exposure to the cytokine interleukine-1β lead to a further downregulation of miR-188-5p expression levels compared to control cells. Re-expression of miR-188-5p in RASF by transient transfection significantly inhibited cell migration. However, miR-188-5p re-expression had no effects on glycosaminoglycan degradation or expression of repellent factors, which have been previously shown to affect the invasive behavior of RASF. In search for target genes of miR-188-5p in RASF we performed gene expression profiling in RASF and found a strong regulatory effect of miR-188-5p on the hyaluronan binding protein KIAA1199 as well as collagens COL1A1 and COL12A1, which was confirmed by qRT-PCR. In silico analysis revealed that KIAA1199 carries a 3’UTR binding site for miR-188-5p. COL1A1 and COL12A1 showed no binding site in the mRNA region, suggesting an indirect regulation of these two genes by miR-188-5p. In summary, our study showed that miR-188-5p is down-regulated in RA in vitro and in vivo, most likely triggered by an inflammatory environment. MiR-188-5p expression is correlated to the activation state of RASF and inhibits migration of these cells. Furthermore, miR-188-5p is directly and indirectly regulating the expression of genes, which may play a role in extracellular matrix formation and destruction in RA. Herewith, this study identified potential novel therapeutic targets to inhibit the development and progression of RA.     

Expression and function of microRNA-188-5p in activated rheumatoid arthritis synovial fibroblasts

Activated synovial fibroblasts in rheumatoid arthritis (RASF) play a critical role in the pathology of rheumatoid arthritis (RA). Recent studies suggested that deregulation of microRNAs (miRs) affects the development and progression of RA. Therefore, we aimed to identify de-regulated miRs in RASF and to identify target genes that may contribute to the aggressive phenotype of RASF. Quantitative real-time PCR revealed a marked downregulation of miR-188-5p in synovial tissue samples of RA patients as well as in RASF. Exposure to the cytokine interleukine-1β lead to a further downregulation of miR-188-5p expression levels compared to control cells. Re-expression of miR-188-5p in RASF by transient transfection significantly inhibited cell migration. However, miR-188-5p re-expression had no effects on glycosaminoglycan degradation or expression of repellent factors, which have been previously shown to affect the invasive behavior of RASF. In search for target genes of miR-188-5p in RASF we performed gene expression profiling in RASF and found a strong regulatory effect of miR-188-5p on the hyaluronan binding protein KIAA1199 as well as collagens COL1A1 and COL12A1, which was confirmed by qRT-PCR. In silico analysis revealed that KIAA1199 carries a 3’UTR binding site for miR-188-5p. COL1A1and COL12A1 showed no binding site in the mRNA region, suggesting an indirect regulation of these two genes by miR-188-5p. In summary, our study showed that miR-188-5p is down-regulated in RA in vitro and in vivo, most likely triggered by an inflammatory environment. MiR-188-5p expression is correlated to the activation state of RASF and inhibits migration of these cells. Furthermore, miR-188-5p is directly and indirectly regulating the expression of genes, which may play a role in extracellular matrix formation and destruction in RA. Herewith, this study identified potential novel therapeutic targets to inhibit the development and progression of RA.     

Decision making about medical interventions in the end-of-life care of people with intellectual disabilities: A national survey of the considerations and beliefs of GPs,ID physicians and care staff

Objective

This paper explores the personal beliefs and specific considerations of professionals regarding decisions about potentially burdensome medical interventions in the end-of-life care for people with intellectual disabilities (ID).

Methods

A survey questionnaire covering decision making about potentially burdensome medical interventions was sent to nationally representative samples of 294 ID care staff-members, 273 ID physicians and 1000 GPs.

Results

Professionals predominantly believed that considerations about quality of life are most important. Quality of life and wellbeing were also frequently considered in both decisions to start/continue an intervention and decisions to forgo/withdraw an intervention. Seventy percent believed that people with ID should always be informed about interventions, and 61% would respect a refusal by the person. The family's wishes were explicitly considered more often than the wishes of the person with ID.

Conclusion

Although respondents agree that the quality of life is highly important, the wishes of people with ID (especially of those with severe/profound ID) were often not considered in decisions about potentially burdensome medical interventions.

Practice implications

To enhance the active involvement of people with ID in decision making we recommend that professionals integrate collaborative principles in decision making and make use of pictorial and easy reading resources.