Expression of Regulatory Platelet MicroRNAs in Patients with Sickle Cell Disease

Background: Increased platelet activation in sickle cell disease (SCD) contributes to a state of hypercoagulability and confers a risk of thromboembolic complications. The role for post-transcriptional regulation of the platelet transcriptome by microRNAs (miRNAs) in SCD has not been previously explored. This is the first study to determine whether platelets from SCD exhibit an altered miRNA expression profile. Methods and Findings: We analyzed the expression of miRNAs isolated from platelets from a primary cohort (SCD = 19, controls = 10) and a validation cohort (SCD = 7, controls = 7) by hybridizing to the Agilent miRNA microarrays. A dramatic difference in miRNA expression profiles between patients and controls was noted in both cohorts separately. A total of 40 differentially expressed platelet miRNAs were identified as common in both cohorts (p-value 0.05, fold change>2) with 24 miRNAs downregulated. Interestingly, 14 of the 24 downregulated miRNAs were members of three families - miR-329, miR-376 and miR-154 - which localized to the epigenetically regulated, maternally imprinted chromosome 14q32 region. We validated the downregulated miRNAs, miR-376a and miR-409-3p, and an upregulated miR-1225-3p using qRT-PCR. Over-expression of the miR-1225-3p in the Meg01 cells was followed by mRNA expression profiling to identify mRNA targets. This resulted in significant transcriptional repression of 1605 transcripts. A combinatorial approach using Meg01 mRNA expression profiles following miR-1225-3p overexpression, a computational prediction analysis of miRNA target sequences and a previously published set of differentially expressed platelet transcripts from SCD patients, identified three novel platelet mRNA targets: PBXIP1, PLAGL2 and PHF20L1. Conclusions: We have identified significant differences in functionally active platelet miRNAs in patients with SCD as compared to controls. These data provide an important inventory of differentially expressed miRNAs in SCD patients and an experimental framework for future studies of miRNAs as regulators of biological pathways in platelets. © 2013 Jain et al

Reduced transcription of TCOF1 in adult cells of Treacher Collins syndrome patients

<p>Abstract</p> <p>Background</p> <p>Treacher Collins syndrome (TCS) is an autosomal dominant craniofacial disorder caused by frameshift deletions or duplications in the <it>TCOF1 </it>gene. These mutations cause premature termination codons, which are predicted to lead to mRNA degradation by nonsense mediated mRNA decay (NMD). Haploinsufficiency of the gene product (treacle) during embryonic development is the proposed molecular mechanism underlying TCS. However, it is still unknown if <it>TCOF1 </it>expression levels are decreased in post-embryonic human cells.</p> <p>Methods</p> <p>We have estimated <it>TCOF1 </it>transcript levels through real time PCR in mRNA obtained from leucocytes and mesenchymal cells of TCS patients (n = 23) and controls (n = 18). Mutational screening and analysis of NMD were performed by direct sequencing of gDNA and cDNA, respectively.</p> <p>Results</p> <p>All the 23 patients had typical clinical features of the syndrome and pathogenic mutations were detected in 19 of them. We demonstrated that the expression level of <it>TCOF1 </it>is 18-31% lower in patients than in controls (<it>p < 0.05</it>), even if we exclude the patients in whom we did not detect the pathogenic mutation. We also observed that the mutant allele is usually less abundant than the wild type one in mesenchymal cells.</p> <p>Conclusions</p> <p>This is the first study to report decreased expression levels of <it>TCOF1 </it>in TCS adult human cells, but it is still unknown if this finding is associated to any phenotype in adulthood. In addition, as we demonstrated that alleles harboring the pathogenic mutations have lower expression, we herein corroborate the current hypothesis of NMD of the mutant transcript as the explanation for diminished levels of <it>TCOF1 </it>expression. Further, considering that <it>TCOF1 </it>deficiency in adult cells could be associated to pathologic clinical findings, it will be important to verify if TCS patients have an impairment in adult stem cell properties, as this can reduce the efficiency of plastic surgery results during rehabilitation of these patients.</p

Fluorescence Dequenching Makes Haem-Free Soluble Guanylate Cyclase Detectable in Living Cells

In cardiovascular disease, the protective NO/sGC/cGMP signalling-pathway is impaired due to a decreased pool of NO-sensitive haem-containing sGC accompanied by a reciprocal increase in NO-insensitive haem-free sGC. However, no direct method to detect cellular haem-free sGC other than its activation by the new therapeutic class of haem mimetics, such as BAY 58-2667, is available. Here we show that fluorescence dequenching, based on the interaction of the optical active prosthetic haem group and the attached biarsenical fluorophor FlAsH can be used to detect changes in cellular sGC haem status. The partly overlap of the emission spectrum of haem and FlAsH allows energy transfer from the fluorophore to the haem which reduces the intensity of FlAsH fluorescence. Loss of the prosthetic group, e.g. by oxidative stress or by replacement with the haem mimetic BAY 58-2667, prevented the energy transfer resulting in increased fluorescence. Haem loss was corroborated by an observed decrease in NO-induced sGC activity, reduced sGC protein levels, and an increased effect of BAY 58-2667. The use of a haem-free sGC mutant and a biarsenical dye that was not quenched by haem as controls further validated that the increase in fluorescence was due to the loss of the prosthetic haem group. The present approach is based on the cellular expression of an engineered sGC variant limiting is applicability to recombinant expression systems. Nevertheless, it allows to monitor sGC's redox regulation in living cells and future enhancements might be able to extend this approach to in vivo conditions

Circulating IgM Requires Plasma Membrane Disruption to Bind Apoptotic and Non-Apoptotic Nucleated Cells and Erythrocytes

<div><p>Autoimmunity is associated with defective phagocytic clearance of apoptotic cells. IgM deficient mice exhibit an autoimmune phenotype consistent with a role for circulating IgM antibodies in apoptotic cell clearance. We have extensively characterised IgM binding to non-apoptotic and apoptotic mouse thymocytes and human Jurkat cells using flow cytometry, confocal imaging and electron microscopy. We demonstrate strong specific IgM binding to a subset of Annexin-V (AnnV)⁺PI (Propidium Iodide)⁺ apoptotic cells with disrupted cell membranes. Electron microscopy studies indicated that IgM⁺AnnV⁺PI⁺ apoptotic cells exhibited morphologically advanced apoptosis with marked plasma membrane disruption compared to IgM^-AnnV⁺PI⁺ apoptotic cells, suggesting that access to intracellular epitopes is required for IgM to bind. Strong and comparable binding of IgM to permeabilised non-apoptotic and apoptotic cells suggests that IgM bound epitopes are 'apoptosis independent' such that IgM may bind any cell with profound disruption of cell plasma membrane integrity. In addition, permeabilised erythrocytes exhibited significant IgM binding thus supporting the importance of cell membrane epitopes. These data suggest that IgM may recognize and tag damaged nucleated cells or erythrocytes that exhibit significant cell membrane disruption. The role of IgM <i>in vivo</i> in conditions characterized by severe cell damage such as ischemic injury, sepsis and thrombotic microangiopathies merits further exploration.</p></div

Real-Time Energy Storage Simulators for the Electricity Grid

\ua9 2024 IEEE. In this paper, a novel Distributed Real-Time Simulation Environment (DRTSE) which enables the coordinated control of multiple Real-Time Simulators (RTSs) positioned across the UK is introduced and demonstrated for an energy storage application. Using RTSs instead of physical energy storage assets enables the testing of different communication and control strategies, thereby reducing the risk of failure when the physical storage assets are deployed. In addition, the testing of different storage types (e.g. batteries, compressed air, flywheels, etc.) and storage locations can be conducted without expensive hardware modifications. In this paper, technical details of the RTSs are given, including the hardware and electrical storage models. The Central Controller (CC) and communication are also described, and results from the DRTSE presented

Assessing the Impact of Distributed Energy Storage in Future Distribution Grids

\ua9 The Institution of Engineering &amp; Technology 2023. The growth of distributed energy storage (DES) in the future power grid is driven by factors such as the integration of renewable energy sources, grid flexibility requirements, and the desire for energy independence. Grid operators have published future energy scenarios projecting the widespread adoption of DES, prompting the need to investigate its impact under different operational modes. This study develops case models and conducts case studies to explore the implications comprehensively. The findings highlight the importance of coordinating the operation of DES to address grid issues and mitigate charging during high carbon dioxide (CO2) emissions periods. Uncoordinated DES operation can lead to significant grid challenges and high emissions impacts. Conversely, when DES is aggregated and operated in a coordinated manner, it demonstrates the potential to alleviate grid problems and avoid charging during high-emission hours. The study highlights the existence of trade-offs among DES profit, network reinforcement cost, and CO2 emissions. Accounting for the network reinforcement costs has a 13% negative impact on DES aggregator profits, while accounting for CO2 costs could lead to a larger decrease of 35%. These findings highlight the need for proper regulation and policy implementation to ensure adequate compensation for DES aggregator and incentivise the coordination potential while balancing the trade-offs among these objectives