Different approaches in microRNA analysis

MicroRNA might serve as a predictive biomarker for treatment response in stem cell treatment in knee osteoarthritis. Different sample types are going to be collected to enlighten the true biological role. MicroRNA analysis necessitates diverse approaches based on the sample type. In this study, we examined microRNA profiles in plasma samples, synovial fluid, and adipose-derived fat tissue. We conducted a comparative analysis of different microRNA analysis methods to assess the data. The first approach involved a series of steps, including adapter trimming, quality filtering, size filtering, and mapping of all reads to the human reference genome (GRCh38.p12). Subsequently, genome-mapped reads were aligned to known miRNA sequences from miRBase. Reads that did not match miRNAs were subjected to further classification using additional databases, such as RNAcentral. The second pipeline also encompassed adapter trimming, quality filtering, and size filtering. Additionally, it involved collapsing individual reads into repeat sequences, followed by alignment to the mature index of miRBase. Unaligned reads were classified as isomiRs based on their alignment to the hairpin index of miRBase. We processed sequences from three plasma samples, three adipose fat tissue samples, and three synovial fluid samples. Although there were slight variations in microRNA read counts, the average ratio between counts was 0.92 (SD=0.29). Notably, the second pipeline yielded higher read counts compared to the first pipeline. The results obtained from both microRNA bioinformatic pipelines demonstrated similar outcomes, suggesting that the choice of pipeline is unlikely to have a significant impact on the derived biological insights.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202

Feasibility of droplet digital PCR analysis of plasma cell-free DNA from kidney transplant patients

Increasing research demonstrates the potential of donor-derived cell-free DNA (dd-cfDNA) as a biomarker for monitoring the health of various solid organ transplants. Several methods have been proposed for cfDNA analysis, including real-time PCR, digital PCR, and next generation sequencing-based approaches. We sought to revise the droplet digital PCR (ddPCR)-based approach to quantify relative dd-cfDNA in plasma from kidney transplant (KTx) patients using a novel pilot set of assays targeting single nucleotide polymorphisms that have a very high potential to distinguish cfDNA from two individuals. The assays are capable of accurate quantification of down to 0.1% minor allele content when analyzing 165 ng of human DNA. We found no significant differences in the yield of extracted cfDNA using the three different commercial kits tested. More cfDNA was extracted from the plasma of KTx patients than from healthy volunteers, especially early after transplantation. The median level of donor-derived minor alleles in KTx samples was 0.35%. We found that ddPCR using the evaluated assays within specific range is suitable for analysis of KTx patientsʼ plasma but recommend prior genotyping of donor DNA and performing reliable preamplification of cfDNA

In-depth comparison of adeno-associated virus containing fractions after CsCl ultracentrifugation gradient separation

Recombinant adeno-associated viruses (rAAVs) play a pivotal role in the treatment of genetic diseases. However, current production and purification processes yield AAV-based preparations that often contain unwanted empty, partially filled or damaged viral particles and impurities, including residual host cell DNA and proteins, plasmid DNA, and viral aggregates. To precisely understand the composition of AAV preparations, we systematically compared four different single-stranded AAV (ssAAV) and self-complementary (scAAV) fractions extracted from the CsCl ultracentrifugation gradient using established methods (transduction efficiency, analytical ultracentrifugation (AUC), quantitative and digital droplet PCR (qPCR and ddPCR), transmission electron microscopy (TEM) and enzyme-linked immunosorbent assay (ELISA)) alongside newer techniques (multiplex ddPCR, multi-angle light-scattering coupled to size-exclusion chromatography (SEC-MALS), multi-angle dynamic light scattering (MADLS), and high-throughput sequencing (HTS)). Suboptimal particle separation within the fractions resulted in unexpectedly similar infectivity levels. No single technique could simultaneously provide comprehensive insights in the presence of both bioactive particles and contaminants. Notably, multiplex ddPCR revealed distinct vector genome fragmentation patterns, differing between ssAAV and scAAV. This highlights the urgent need for innovative analytical and production approaches to optimize AAV vector production and enhance therapeutic outcomes

Phosphorylation of C-terminal tyrosine residue 526 in FUS impairs its nuclear import

Aberrant cytoplasmic aggregation of FUS, which is caused by its mutations primarily in the C- terminal nuclear localization signal, is associated with 3% of familial amyotrophic lateral sclerosis (ALS). FUS aggregates are also pathognomonic for 10% of all frontotemporal lobar degeneration cases (FTLD-FUS), however they are not associated with its mutation. This implicates differences in the mechanisms driving inclusion formation of FUS in ALS and FTLD. Here we show that C-terminal tyrosine at position 526 of FUS is crucial for normal nuclear import. This tyrosine is subjected to phosphorylation, which reduces interaction with transportin 1 and may consequentially affect transport of FUS into the nucleus. Furthermore, we show that this phosphorylation can occur through the activity of Src family of kinases. Our study implicates phosphorylation as an additional mechanism by which nuclear transport of FUS may be regulated and potentially perturbed in ALS and FTLD.</jats:p

Extracellular vesicle-bound DNA in urine is indicative of kidney allograft injury

Extracellular vesicle-bound DNA (evDNA) is an understudied extracellular vesicle (EV) cargo, particularly in cancer-unrelated research. Although evDNA has been detected in urine, little is known about its characteristics, localization, and biomarker potential for kidney pathologies. To address this, we enriched EVs from urine of well-characterized kidney transplant recipients undergoing allograft biopsy, characterized their evDNA and its association to allograft injury. The SEC-based method enriched pure EVs from urine of kidney transplant recipients, regardless of the allograft injury. Urinary evDNA represented up to 29.2 ± 8% (mean ± SD) of cell-free DNA (cfDNA) and correlated with cfDNA in several characteristics but was less fragmented (P < 0.001). Importantly, using DNase treatment and immunogold labelling TEM, we demonstrated that evDNA was bound to the surface of urinary EVs. Normalised evDNA yield (P = 0.042) and evDNA copy number (P = 0.027) significantly differed between patients with normal histology, rejection injury and non-rejection injury, the later groups having significantly larger uEVs (mean diameter, P = 0.045) and more DNA bound per uEV. ddDNA is detectable in uEV samples of kidney allograft recipients, but its quantity is highly variable. In a proof-of-principle study, several evDNA characteristics correlated with clinical and histological parameters (P = 0.040), supporting that the potential of evDNA as a biomarker for kidney allograft injury should be further investigated

Nuclear RNA foci from C9ORF72 expansion mutation form paraspeckle-like bodies

The GGGGCC (G(4)C(2)) repeat expansion mutation in the C9ORF72 gene is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Transcription of the repeat and formation of nuclear RNA foci, which sequester specific RNA-binding proteins, is one of the possible pathological mechanisms. Here, we show that (G(4)C(2))(n) repeat RNA predominantly associates with essential paraspeckle proteins SFPQ, NONO, RBM14, FUS and hnRNPH and colocalizes with known paraspeckle-associated RNA hLinc-p21. As formation of paraspeckles in motor neurons has been associated with early phases of ALS, we investigated the extent of similarity between paraspeckles and (G(4)C(2))(n) RNA foci. Overexpression of (G(4)C(2))(72) RNA results in their increased number and colocalization with SFPQ-stained nuclear bodies. These paraspeckle-like (G(4)C(2))(72) RNA foci form independently of the known paraspeckle scaffold, the long non-coding RNA NEAT1. Moreover, the knockdown of SFPQ protein in C9ORF72 expansion mutation-positive fibroblasts significantly reduces the number of (G(4)C(2))(n) RNA foci. In conclusion, (G(4)C(2))(n) RNA foci have characteristics of paraspeckles, which suggests that both RNA foci and paraspeckles play roles in FTD and ALS, and implies approaches for regulation of their formation