Project description:Mature human red blood cells (RBCs) are terminally differentiated anuclear cells. While initially thought to lack any nucleic acids, human RBCs are found to contain abundant and diverse species of RNA transcripts with functional relevance. Given the absence of novel transcription, RBCs may provide an interesting cellular context to study RNA metabolism over time. One clinically relevant context is the ex vivo storage of RBCs in blood banks for use in blood transfusion. Some studies have indicated that the transfusion of “old” or aged stored RBCs may be associated with adverse outcomes due to various storage changes termed “storage lesions”. However, other studies do not support these effects, and much remains unknown about the relevant changes associated with RBC storage. Here, we employed the NanoString nCounter assay for global miRNA profiling to comprehensively define the miRNA turnover during ex vivo RBC storage. This profiling demonstrates that the abundance of most RBC miRNAs did not change significantly during the 42 days of refrigerated storage, indicating extremely long decay half-lives. Unexpectedly, miR-720, a cleavage product of tRNAThr, increased dramatically in the first two weeks and persisted during storage. Furthermore, we present evidence for a role of angiogenin in tRNA cleavage to generate miR-720 during RBC storage. The dramatic increase in miR-720 may serve as a new characteristic for storage lesion and may be used to monitor transfused RBCs in clinical patients and athletes performing blood doping.
Project description:Investigation of miRNA expression level changes in RBCs stored for 20 days, RBCs stored for 20 days with 1uM SNP, compared to healthy,male type O donors, and to explore the mechanism of storage lesions of RBC.
Project description:Gene expression profiling of cells isolated ex vivo is a unique tool to assess gene expression in vivo. Exemplified for CD4+CD45RO+ effector/memory T helper (T E/M) lymphocytes of human peripheral blood, we have analyzed different isolation procedures and storage conditions for the introduction of bias.
Project description:Normothermic ex-vivo kidney perfusion (NEVKP) has demonstrated superior outcomes for donation-after-cardiovascular death (DCD) grafts compared to static cold storage (SCS). To determine the mechanisms responsible for this, we performed an unbiased genome-wide microarray analysis. Kidneys from 30kg-Yorkshire pigs were subjected to 30min of warm ischemia followed by 8hrs of NEVKP or SCS, or no storage (NS), prior to auto-transplantation. mRNA expression was analyzed on POD3 renal biopsies.
Project description:Prospective, open labelled, multicenter trial to evaluate the feasibility of ex vivo culture 3D (chemogram obtaining) on biopsies in order to estimate the predictive value of this technique for treatment response in patients treated by two different chemotherapies (FOLFOX or FOLFIRI) for colorectal cancer.
Project description:Deep RNA sequencing analysis revealed that compared to control (no cold storage/ischemia), 6h-cold storage led to 266 differentially expressed genes, many of which were implicated in modulating mitochondrial performance, oxidative stress response, myocardial function, and apoptosis. Either bone marrow- (BM) or adipose tissue-derived (Ad) mesencymal stromal cell conditioned medium (MSC-CM) restored these gene expression towards control.
Project description:Human in vivo skin wound: Non-wounded skin was obtained by taking punch biopsies from three healthy donors (donor 1,2 and 3). The samples were termed 'skin day 0 in vivo wound'. Skin wound samples were retrieved by making new punch biopsies from the edge of the original biopsies after four days. These samples were termed 'skin day 4 in vivo wound'. As much dermal tissue as possible was removed by dissection to make sure mainly epidermis was present in the samples. The samples were washed in NaCl to possible remove infiltrating inflammatory cells before RNA isolation. Ex vivo skin wounds: Skin was obtained from three healthy donors following reduction surgery (donor 1, 2, and 3). As much dermal tissue as possible was removed dissection. These samples were termed 'skin day 0 ex vivo wound'. Skin was sliced into 1x10 mm slices and incubated in keratinocyte medium for four days with either 1:1000 fold dilution of DMSO or 10 micromolar AG-1478 (dissolved in DMSO). Again as much dermal tissue was removed by dissection as possible before RNA was isolated. These samples were termed 'skin day 4 ex vivo wound' and 'skin day 4 AG-1478 ex vivo wound'. By comparing the gene expression day 4 in ex vivo wound with in vivo wounds it was possible to see which part of the gene expression in wounded skin that was due to the epidermal reaction to injury and how much was due to stimuli from infiltrating inflammatory cells absent in the ex vivo skin wounds. By comparing the data from ex vivo skin wounds day 4 with and without the EGFR-inhibitor AG-1478, it was possible to look at the importance of the EGF-receptor of EGFR for the gene expression in ex vivo wounded skin.