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

Developing bioinformatics pipeline for processing environmental DNA metabarcoding sequencing data

Environmental DNA (eDNA) is DNA present in an environmental sample, originating from any biological material released from organisms living in that environment. This DNA can be isolated, amplified, sequenced, and analyzed in order to examine the taxonomic richness and abundance of different organism groups in the targeted environment. Methods of eDNA metabarcoding thus offer a unique opportunity to systematically streamline and scale-up regular biological assessments across many different environments of interest. Recently, as a part of the project funded by European structural and investment funds, Labena d.o.o. company established a modern laboratory in Zagreb focused on the research and provision of services in the field of eDNA. In collaboration with the Institute Ruđer Bošković we have been working on developing tests for analysis of water quality based on the eDNA and, as part of the standardization and optimization of sample-to-results eDNA analysis process, we developed a custom bioinformatics pipeline to facilitate efficient and effective eDNA sequencing data analysis. The pipeline was was written in Bash and utilizes several different algorithms to filter, trim, merge, denoise and classify targeted eDNA sequences. Python-based scripts which allow automatically download, filter, and format the data available on various online platforms were included in the pipeline to facilitate the curation of custom reference databases needed for taxonomic classification of targeted organism groups. User-friendly and interactive pipeline report generation, comprised of both wet- and dry-lab step-bystep sample statistics and graphical representations or the main results, is supported using Rmarkdown and Plotly and DataTables libraries. The pipeline is containerized in Docker, allowing for easier environment building and pipeline deployment.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202

Non-coding RNAs in preeclampsia—molecular mechanisms and diagnostic potential

Preeclampsia (PE) is a leading cause of maternal and neonatal morbidity and mortality worldwide. Defects in trophoblast invasion, differentiation of extravillous trophoblasts and spiral artery remodeling are key factors in PE development. Currently there are no predictive biomarkers clinically available for PE. Recent technological advancements empowered transcriptome exploration and led to the discovery of numerous non-coding RNA species of which microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are the most investigated. They are implicated in the regulation of numerous cellular functions, and as such are being extensively explored as potential biomarkers for various diseases. Altered expression of numerous lncRNAs and miRNAs in placenta has been related to pathophysiological processes that occur in preeclampsia. In the following text we offer summary of the latest knowledge of the molecular mechanism by which lnRNAs and miRNAs (focusing on the chromosome 19 miRNA cluster (C19MC)) contribute to pathophysiology of PE development and their potential utility as biomarkers of PE, with special focus on sample selection and techniques for the quantification of lncRNAs and miRNAs in maternal circulation

Single cell 3’ transcriptome profiling

Whole 3’ transcriptome profiling at the single cell level opens up new abilities for researchers to answer complex questions. Thousands of individual cells per sample are Barcoded separately to index the transcriptome of each cell individually. It is done by partitioning thousands of cells into nanoliter-scale Gel Beads-in-emulsion (GEMs), where cells are delivered at a limiting dilution, such that the majority (~90-99%) of generated GEMs contain no cell. The 16 bp 10x Barcode and 12 bp UMI are encoded in Read 1, while the poly(dT) primers are used in this protocol for generating Single Cell 3’ Gene Expression libraries. After GEM generation, copartitioned cells are lysed and reverse transcription (RT) was performed after which all cDNA from single cell share a common Barcode. Full-length cDNA was amplified via PCR to generate sufficient mass for library construction. This is followed by enzymatic fragmentation and size selection to optimize the cDNA amplicon size. Library construction was finished via End Repair, A-tailing, Adaptor Ligation, and PCR. P5, P7, i7 and i5 sample index, and TruSeq Read 2 (read 2 primer sequence) were added. TruSeq Read 1 and TruSeq Read 2 are standard Illumina sequencing primer sites used in paired-end sequencing. The library prepared in this way, containing the P5 and P7 primers, is ready for Illumina amplification.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202

OligoPrime

With the increasing number of molecular biology techniques, large numbers of oligonucleotides are frequently involved in individual research projects. Thus, a dedicated electronic oligonucleotide management system is expected to provide several benefits such as increased oligonucleotide traceability, facilitated sharing of oligonucleotides between laboratories, and simplified (bulk) ordering of oligonucleotides. Herein, we describe OligoPrime, an information system for oligonucleotide management, which presents a computational support for all steps in an oligonucleotide lifecycle, namely, from its ordering and storage to its application, and disposal. OligoPrime is easy to use since it is accessible via a web browser and does not require any installation from the end user’s perspective. It allows filtering and search of oligonucleotides by various parameters, which include the exact location of an oligonucleotide, its sequence, and availability. The oligonucleotide database behind the system is shared among the researchers working in the same laboratory or research group. Users might have different roles which define the access permissions and range from students to researchers and primary investigators. Furthermore, OligoPrime is easy to manage and install and is based on open-source software solutions. Its code is freely available at https://github.com/OligoPrime. Moreover, an implementation of OligoPrime, which can be used for testing is available at http://oligoprime.xyz/. To our knowledge, OligoPrime is the only software solution dedicated specifically to oligonucleotide management. We strongly believe that it has a large potential to enhance the transparency of use and to simplify the management of oligonucleotides in academic laboratories and research groups

Novel insights into biological roles of inducible cAMP early repressor ICER

ICER corresponds to a group of alternatively spliced Inducible cAMP Early Repressors with high similarity, but multiple roles, including in circadian rhythm, and are involved in attenuation of cAMPdependent gene expression. We present experimental and in silico data revealing biological differences between the isoforms with exon gamma (ICER) or without it (ICERγ). Both isoforms are expressed in the liver and the adrenal glands and can derive from differential splicing. In adrenals the expression is circadian, with maximum at ZT12 and higher amplitude of Icerγ. In the liver, the expression of Icerγ is lower than Icer in the 24 h time frame. Icer mRNA has a delayed early response to forskolin. The longer ICER protein binds to three DNA grooves of the Per1 promoter, while ICERγ only to two, as deduced by molecular modelling. This is in line with gel shift competition assays showing stronger binding of ICER to Per1 promotor. Only Icerγ siRNA provoked an increase of Per1 expression. In conclusion, we show that ICER and ICERγ have distinct biochemical properties in tissue expression, DNA binding, and response to forskolin. Data are in favour of ICERγ as the physiologically important form in hepatic cells where weaker binding of repressor might be preferred in guiding the cAMP-dependent response

Detection of fish pathogen Saprolegnia parasitica in environmental DNA samples by droplet digital PCR

Oomycetes are fungal-like microorganisms parasitic towards a large number of plant and animal species. Genera from order Saprolegniales, such as Saprolegnia and Aphanomyces, cause devastating infections of freshwater animals. Saprolegnia parasitica is a widely distributed oomycete pathogen that causes saprolegniosis, a disease responsible for significant economic losses in aquaculture, as well as declines of natural populations of fish and other freshwater organisms. Despite its negative impact, no monitoring protocol for S. parasitica has been established to date. Thus, we aimed to develop a droplet digital PCR (ddPCR) assay for the detection and quantification of S. parasitica in environmental DNA samples.Saprolegnia parasitica-specific primers were designed to target internal transcribed spacer region 2 (ITS 2), based on the alignment of ITS sequences of S. parasitica and a range of Saprolegnia spp., as well as other oomycetes. The specificity of primers was tested using genomic DNA of S. parasitica (as positive control) and DNA of non–S. parasitica oomycete isolates, as well as trout/crayfish DNA (as negative control). The primers specifically amplified a segment of the ITS region of oomycete pathogen S. parasitica, while no amplification (i.e. no positive droplets) was obtained for closely related Saprolegnia spp. (e.g. Saprolegnia sp. 1 and S. ferax) and other more distantly related oomycetes. Next, the limit of detection (LOD) of the assay was established by using serial dilutions of the S. parasitica genomic DNA. The determined sensitivity of the assay was high: LOD was 15 fg of pathogen’s genomic DNA per µL of the reaction mixture.Assay performance was further assessed with environmental DNA samples isolated from water from the trout farms and natural environments, as well as (ii) biofilm from the host surface (swab samples). Water samples were collected from 21 different locations in Croatia, while swab samples were collected from S. parasitica host/carrier species: (i) skin and eggs of the rainbow trout (Oncorhynchus mykiss Walbaum, 1792) and brown trout (Salmo trutta Linnaeus, 1758), and (ii) cuticle of signal crayfish (Pacifastacus leniusculus Dana, 1852) and narrow clawed crayfish (Pontastacus leptodactylus Eschscholtz, 1823). Samples were classified into agent levels A0 to A6, depending of the number of S. parasitica ITS copies per ng of total DNA.Saprolegnia parasitica was detected in 76 % of water samples (16/21) and the range of pathogen’s ITS copies in positive samples was between 0.02 and 14 copies/ng of total DNA (agent levels A1 to A3). Regarding the swab samples, S. parasitica load was significantly higher in diseased trout than in those with healthy appearance: 9375 vs 3.28 S. parasitica copies/ng of total swab DNA (average agent level A6 vs. A2, respectively). Despite the fact that none of the sampled crayfish had signs of infection, the pathogen was detected in all tested cuticle swabs. Swabs of P. leniusculus, a known S. parasitica host, had significantly higher S. parasitica load than swabs of P. leptodactylus, S. parasitica carrier: 390 vs 83 S. parasitica copies/ng (agent level A5 vs. A4, respectively).In conclusion, our results demonstrate the applicability of the newly developed ddPCR assay in monitoring and early detection of S. parasitica in aquaculture facilities and natural freshwater environments. This would help in a better understanding of S. parasitica ecology and its effects on the host populations

Environmental DNA in subterranean biology update: from “Where?” to “How many?”

Recent records of Proteus anguinus outside its historically known range (Gorički et al. 2017), discovered through detection of its DNA dissolved in groundwater (environmental DNA or eDNA), mark the beginning of a new era in the study and conservation of cryptic subterranean biodiversity. An upgraded technology, droplet digital PCR (ddPCR), initially developed for studies of gene expression, detection of genetically modified organisms and in medical diagnostics, is being tested for improved detection of the much smaller and rare stygobiont, the cave clam Congeria jalzici. In parallel to eDNA assay development for various stygobiotic species of the Dinaric Karst, a groundwater-sample library is being created. The samples will be available for future analysis of their species composition and will also serve as a source of information on any changes in species distribution over time. In another line of eDNA research, the utility of ddPCR for direct quantification of eDNA molecules in groundwater is being explored by using the large, accessible and well-characterized (Zakšek and Trontelj 2017) natural Proteus population in the Planina Cave (Slovenia) as a model. The eDNA methodology may in the future be applied in estimation and monitoring of Proteus population sizes without having to see, mark or otherwise disturb the animals themselves

Cytokines and Chemokines Involved in Osteoarthritis Pathogenesis

Osteoarthritis is a common cause of disability worldwide. Although commonly referred to as a disease of the joint cartilage, osteoarthritis affects all joint tissues equally. The pathogenesis of this degenerative process is not completely understood; however, a low-grade inflammation leading to an imbalance between anabolic and katabolic processes is a well-established factor. The complex network of cytokines regulating these processes and cell communication has a central role in the development and progression of osteoarthritis. Concentrations of both proinflammatory and anti-inflammatory cytokines were found to be altered depending on the osteoarthritis stage and activity. In this review, we analyzed individual cytokines involved in the immune processes with an emphasis on their function in osteoarthritis