Saprolegnia parasitica causes saprolegniosis, a disease responsible for significant economic losses in aquaculture and declines of fish populations in the wild, but the knowledge of its distribution and prevalence in the environment is limited. We developed a fast, sensitive and specific S. parasitica droplet digital PCR (ddPCR) assay and demonstrated its applicability for the detection and quantification of the pathogen in environmental samples: swab DNA collected from the host (trout skin, surface of eggs) and environmental DNA extracted from water. The developed assay was used to assess how abiotic (i.e. physico-chemical parameters of the water) and biotic (health status of the host) factors influence the S. parasitica load in the environment. The pathogen load in water samples was positively correlated with some site-specific abiotic parameters such as electrical conductivity (EC) and calcium, while fluorides were negatively correlated, suggesting that physico-chemical parameters are important for determining S. parasitica load in natural waters. Furthermore, skin swabs of injured trout had significantly higher pathogen load than swabs collected from healthy fish, confirming that S. parasitica is a widespread opportunistic pathogen. Our results provide new insights into various environmental factors that influence the distribution and abundance of S. parasitica.

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.

The construction and building sector is responsible for a large share of energy and material used during the life cycle of a building. It is therefore crucial to apply a circular economy model within the process wherever possible to minimize the impact on the environment. In this paper, the possibility of producing thermal and acoustic boards from industrial nonwoven waste textile is studied and presented. The nonwoven polyester textile obtained directly from the production line in the form of strips and bales was first shredded into smaller fractions and then in the form of pile compressed with a hot press to form compact thermal insulation boards. The first set of specimens was prepared only from waste polyester nonwoven textile, whereas the second set was treated with sodium silicate in order to check the material’s reaction to fire performance. The experimental work was conducted to define the acoustic properties, reaction to fire behavior and thermal conductivity of the produced specimens. The obtained results show that the thermal conductivity coefficient of specimens without added water glass dissolution is near to the values of conventional materials used as thermal insulation in buildings. The reaction to fire testing proved that the addition of water glass actually propagates the progressive flame over the entire product. It can be concluded that the presented thermal insulation can be used as an adequate and sustainable solution for building construction purposes.

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.

Liquid biopsy and cell-free DNA (cfDNA) show great promise in cancer diagnostics. In this study, we designed a custom droplet digital PCR (ddPCR) assay for the quantification and quality control of cfDNA isolated from serum. The assay was validated on a group of locally advanced colorectal cancer (CRC) patients and two control groups—patients with hemorrhoids and healthy individuals. The assay shows a high correlation with Qubit measurement (r = 0.976) but offers a higher dynamic range. Mean concentrations of cfDNA were 12.36 ng/µL, 5.17 ng/µL, and 0.29 ng/µL for CRC, hemorrhoid patients, and healthy controls, respectively. The quality of cfDNA was assessed with the measurement of B-cell DNA contamination. On a subset of CRC patients, we compared the mutation status on KRAS (G12A, G12D, G12V, G13D) and BRAF (V600E) genes in the primary tumor and cfDNA isolated from the serum. A total of 70.6% of primary tumor samples were mutated, and the mean fractional abundance of mutations was 9.50%. The matching serum samples were mutated in 38% cases with an average fractional abundance of 0.23%. We conclude that any decisions based solely on the amount of cfDNA present in patient serum must be interpreted carefully and in the context of co-morbidities. This study explores the potential of ddPCR somatic mutations detection from liquid biopsy as a supplement to tissue biopsy in targeted personalized CRC patient management.

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.

IntroductionMicroRNAs have a significant role in the pathogenesis of preeclampsia. Circulating microRNAs could represent a potential biomarker for preeclampsia. The aim of this study was to evaluate plasma miR210-3p and miR518b in preeclampsia and healthy pregnancy for the first time by digital droplet PCR (ddPCR). MethodsThirty-six pregnant women (seventeen healthy pregnancies, nineteen preeclampsia patients) were involved from the Clinic for Gynaecology and Obstetrics “Narodni front” in Belgrade, Serbia. Plasma miR210-3p, miR518b and cel-miR-39 as a spike-in control were measured by ddPCR. ResultsMiR518b was significantly elevated in preeclampsia compared to a healthy pregnancy (P = 0.034; 0.302(0.217–0.421) vs. 0.171(0.110–0.266)). MiR210-3p showed no significant difference between the two groups (P = 0.951). The adjustment of miR518b was made for a gestational age and smoking status and the difference between the preeclampsia and healthy pregnancy group was more significant (P = 0.026; 0.300(0.216–0.419) vs. 0.172(0.121–0.245)).Plasma miR-518b was significantly higher in the group of preeclampsia patients with proteinuria above the 75th percentile for the group (P = 0.033), in women who smoked (P = 0.039), and was positively related to uric acid in preeclampsia (P = 0.018, r = 0.536). Plasma miR518b was able to significantly discriminate between preeclampsia and healthy pregnancy, yielding AUC of 0.712 (95%CI:0.539–0.891), P = 0.028. ConclusionsIn this study plasma microRNA were measured for the first time in preeclampsia and healthy pregnancies with ddPCR. Placenta-specific miR-518b could serve as a potential biomarker for discriminating preeclampsia and healthy pregnancy, which should be confirmed on a larger study population. This study has failed to confirm the same potential for miR210-3p.

BackgroundThe thermostable serine protease pernisine originates from the hyperthermophilic Archaeaon Aeropyrum pernix and has valuable industrial applications. Due to its properties, A. pernix cannot be cultivated in standard industrial fermentation facilities. Furthermore, pernisine is a demanding target for heterologous expression in mesophilic heterologous hosts due to the relatively complex processing step involved in its activation.ResultsWe achieved production of active extracellular pernisine in a Streptomyces rimosus host through heterologous expression of the codon-optimised gene by applying step-by-step protein engineering approaches. To ensure secretion of fully active enzyme, the srT signal sequence from the S. rimosus protease was fused to pernisine. To promote correct processing and folding of pernisine, the srT functional cleavage site motif was fused directly to the core pernisine sequence, this way omitting the proregion. Comparative biochemical analysis of the wild-type and recombinant pernisine confirmed that the enzyme produced by S. rimosus retained all of the desired properties of native pernisine. Importantly, the recombinant pernisine also degraded cellular and infectious bovine prion proteins, which is one of the particular applications of this protease.ConclusionFunctional pernisine that retains all of the advantageous properties of the native enzyme from the thermophilic host was successfully produced in a S. rimosus heterologous host. Importantly, we achieved extracellular production of active pernisine, which significantly simplifies further downstream procedures and also omits the need for any pre-processing step for its activation. We demonstrate that S. rimosus can be used as an attractive host for industrial production of recombinant proteins that originate from thermophilic organisms.