Inflammation-Mediated Regulation of MicroRNA Expression in Transplanted Pancreatic Islets

Nonspecific inflammation in the transplant microenvironment results in β-cell dysfunction and death influencing negatively graft outcome. MicroRNA (miRNA) expression and gene target regulation in transplanted islets are not yet well characterized. We evaluated the impact of inflammation on miRNA expression in transplanted rat islets. Islets exposed in vitro to proinflammatory cytokines and explanted syngeneic islet grafts were evaluated by miRNA arrays. A subset of 26 islet miRNAs was affected by inflammation both in vivo and in vitro. Induction of miRNAs was dependent on NF-κB, a pathway linked with cytokine-mediated islet cell death. RT-PCR confirmed expression of 8 miRNAs. The association between these miRNAs and mRNA target-predicting algorithms in genome-wide RNA studies of β-cell inflammation identified 238 potential miRNA gene targets. Several genes were ontologically associated with regulation of insulin signaling and secretion, diabetes, and islet physiology. One of the most activated miRNAs was miR-21. Overexpression of miR-21 in insulin-secreting MIN6 cells downregulated endogenous expression of the tumor suppressor Pdcd4 and of Pclo, a Ca2+ sensor protein involved in insulin secretion. Bioinformatics identified both as potential targets. The integrated analysis of miRNA and mRNA expression profiles revealed potential targets that may identify molecular targets for therapeutic interventions

TAT-Mediated Transduction of MafA Protein In Utero Results in Enhanced Pancreatic Insulin Expression and Changes in Islet Morphology

Alongside Pdx1 and Beta2/NeuroD, the transcription factor MafA has been shown to be instrumental in the maintenance of the beta cell phenotype. Indeed, a combination of MafA, Pdx1 and Ngn3 (an upstream regulator of Beta2/NeuroD) was recently reported to lead to the effective reprogramming of acinar cells into insulin-producing beta cells. These experiments set the stage for the development of new strategies to address the impairment of glycemic control in diabetic patients. However, the clinical applicability of reprogramming in this context is deemed to be poor due to the need to use viral vehicles for the delivery of the above factors. Here we describe a recombinant transducible version of the MafA protein (TAT-MafA) that penetrates across cell membranes with an efficiency of 100% and binds to the insulin promoter in vitro. When injected in utero into living mouse embryos, TAT-MafA significantly up-regulates target genes and induces enhanced insulin production as well as cytoarchitectural changes consistent with faster islet maturation. As the latest addition to our armamentarium of transducible proteins (which already includes Pdx1 and Ngn3), the purification and characterization of a functional TAT-MafA protein opens the door to prospective therapeutic uses that circumvent the use of viral delivery. To our knowledge, this is also the first report on the use of protein transduction in utero

Position Paper on Water, Energy, Food and Ecosystem (WEFE) Nexus and Sustainable development Goals (SDGs)

The EU and the international community is realising that the Water, Energy, Food and Ecosystem components are interlinked and require a joint planning in order to meet the daunting global challenges related to Water, Energy and Food security and maintaining the ecosystem health and in this way, reach the SDGs. If not dealt with, the world will not be able to meet the demand for water, energy and food in a not too far future and, in any case, in a not sustainable way. The strain on the ecosystems resulting from unsustainable single-sector planning will lead to increasing poverty, inequality and instability. The Nexus approach is fully aligned with and supportive of the EU Consensus on Development. Key elements of the Consensus will require collaborative efforts across sectors in ways that can be supported/implemented by a Nexus approach. In this way, transparent and accountable decision-making, involving the civil society is key and common to the European Consensus on Development and the Nexus approach. The Nexus approach will support the implementation of the SDG in particular SDG 2 (Food), SDG 6 (Water) and SDG 7 (Energy), but most SDGs have elements that link to food, water and energy in one or other way, and will benefit from a Nexus approach. The SDGs are designed to be cross-cutting and be implemented together, which is also reflected in a WEFE Nexus approach. A Nexus approach offers a sustainable way of addressing the effects of Climate Change and increase resilience. The WEFE Nexus has in it the main drivers of climate change (water, energy and food security) and the main affected sectors (water and the environment). Decisions around policy, infrastructure, … developed based on the WEFE Nexus assessments will be suitable as elements of climate change mitigation and adaptation. In fact, it is difficult to imagine solutions to the climate change issue that are not built on a form of Nexus approach. The Nexus approach is being implemented around the world, as examples in the literature demonstrate. These examples together with more examples from EU and member state development cooperation will help build experience that can be consolidated and become an important contribution to a Toolkit for WEFE Nexus Implementation. From the expert discussions, it appears that because of the novelty of the approach, a Toolkit will be an important element in getting the Nexus approach widely used. This should build on experiences from practical examples of NEXUS projects or similar inter-sectorial collaboration projects; and, there are already policy, regulation and practical experience to allow institutions and countries to start applying the Nexus concept.JRC.D.2-Water and Marine Resource

Quantification of ribozyme target RNA using real-time PCR

An important part of the ribozyme efficiency-screening process is to have a fast and accurate way to measure steady-state levels of the target RNA. Here, we describe the use of real-time polymerase chain reaction (PCR) for quantification of ribozyme target transcripts. In contrast to classical quantitative PCR, real-time PCR does not require extensive manipulation or generation of relatively complex reagents, thus reducing the risk of contamination. PCR products generated by Taq polymerase in the presence of SYBR Green dye I can be monitored each cycle by collecting fluorescence signals emitted only as the double-stranded DNA is formed. The temperature at which the fluorescent data used for quantification are collected is based on the melting-curve analysis of the amplified product. After constructing a standard curve by plotting the log of the standards' copy number vs their fractional cycle number, the copy number of the unknown samples is automatically determined by interpolation of this curve. However it is very important to validate the melting curve profile with standard gel electrophoresis, particularly while setting up the technique. Real-time PCR is fast and reproducible. Excluding the isolation of RNA and synthesis of cDNA, the results can be obtained in less than 1 h. The coefficient of variance is 15% in the range of 104-106 gene copies

Instability of miRNA and cDNAs derivatives in RNA preparations

Micro RNAs (miRNAs) are small RNA molecules, which function as important regulators of gene expression. We found that RNA preparation methods commonly utilized for miRNA expression studies yield highly unstable miRNAs. We studied the stability of four miRNAs belonging to different miRNAs families. A significant degradation of these molecules may be observed already three days after RNA isolation. Moreover, the respective cDNAs are highly unstable as well. Our findings indicate that instability of miRNAs and their cDNAs should be considered when designing miRNA expression studies