Whole-exome sequencing is an attractive alternative to microarray analysis because of the low cost and potential ability to detect copy number variations (CNV) of various sizes (from 1–2 exons to several Mb). Previous comparison of the most popular CNV calling tools showed a high portion of false-positive calls. Moreover, due to a lack of a gold standard CNV set, the results are limited and incomparable. Here, we aimed to perform a comprehensive analysis of tools capable of germline CNV calling available at the moment using a single CNV standard and reference sample set. Compiling variants from previous studies with Bayesian estimation approach, we constructed an internal standard for NA12878 sample (pilot National Institute of Standards and Technology Reference Material) including 110,050 CNV or non-CNV exons. The standard was used to evaluate the performance of 16 germline CNV calling tools on the NA12878 sample and 10 correlated exomes as a reference set with respect to length distribution, concordance, and efficiency. Each algorithm had a certain range of detected lengths and showed low concordance with other tools. Most tools are focused on detection of a limited number of CNVs one to seven exons long with a false-positive rate below 50%. EXCAVATOR2, exomeCopy, and FishingCNV focused on detection of a wide range of variations but showed low precision. Upon unified comparison, the tools were not equivalent. The analysis performed allows choosing algorithms or ensembles of algorithms most suitable for a specific goal, e.g. population studies or medical genetics.

Copy number variations (CNVs) are the predominant class of structural genomic variations involved in the processes of evolutionary adaptation, genomic disorders, and disease progression. Compared with single-nucleotide variants, there have been challenges associated with the detection of CNVs owing to their diverse sizes. However, the field has seen significant progress in the past 20–30 years. This has been made possible due to the rapid development of molecular diagnostic methods which ensure a more detailed view of the genome structure, further complemented by recent advances in computational methods. Here, we review the major approaches that have been used to routinely detect CNVs, ranging from cytogenetics to the latest sequencing technologies, and then cover their specific features.

Over the past decade, tools of omics technologies have generated a large amount of data in various repositories, which are of interest for meta-analysis today. Now, researchers in the field of...

The crystal structure of bacterial oligopeptidase B from Serratia proteamaculans (SpOpB) in complex with a chloromethyl ketone inhibitor was determined at 2.2 Å resolution. SpOpB was crystallized in a closed (catalytically active) conformation. A single inhibitor molecule bound simultaneously to the catalytic residues S532 and H652 mimicked a tetrahedral intermediate of the catalytic reaction. A comparative analysis of the obtained structure and the structure of OpB from Trypanosoma brucei (TbOpB) in a closed conformation showed that in both enzymes, the stabilization of the D-loop (carrying the catalytic D) in a position favorable for the formation of a tetrahedral complex occurs due to interaction with the neighboring loop from the β-propeller. However, the modes of interdomain interactions were significantly different for bacterial and protozoan OpBs. Instead of a salt bridge (as in TbOpB), in SpOpB, a pair of polar residues following the catalytic D617 and a pair of neighboring arginine residues from the β-propeller domain formed complementary oppositely charged surfaces. Bioinformatics analysis and structural modeling show that all bacterial OpBs can be divided into two large groups according to these two modes of D-loop stabilization in closed conformations.

Introduction. The use of cellular constructs based on human pluripotent stem cells (hPSCs) is associated with a number of challenges, including the need to standardize methods for cultivating chondrocyte-like hPSCs derivatives to produce a cartilage tissue similar to natural hyaline cartilage. Glycosaminoglycans (GAGs) are the basis of the extracellular matrix (ECM) of cartilage tissue; therefore, a qualitative and quantitative analysis of the GAG composition of cartilage-like tissue engineering structures is an important step in the final assessment of their potential therapeutic effectiveness.Objective. To determine the composition of GAGs synthesized in vitro by chondrocytes of various origins using enzyme-linked immunoassay (ELISA) and liquid chromatography with tandem mass spectrometry (LC–MS/MS), as well as to evaluate the effect of 2D and 3D culturing on their synthesis.Materials and methods. We analyzed the GAG levels in 2D and 3D tissue-engineered structures obtained from the cartilage tissue of five donors, chondrocyte-like cells differentiated from two hPSC lines. Cellular spheroids were obtained by aggregation in microlunar plates and cultured in mini-bioreactors. The analysis of the GAG content in cell culture samples and spheroids was carried out using ELISA and LC–MS/MS. The Kruskal–Wallis and Dunn tests were used to assess the statistical significance of the differences between the samples.Results. The ELISA study revealed statistically significant differences (p < 0.0021), confirming higher levels of GAGs synthesized in 3D cultures of native chondrocytes compared to 2D cultures (108.67 ng/mL and 1099.87 ng/mL, respectively). The average number of spectra of the chondroitin sulfate proteoglycan 4 protein, determined using LC–MS/MS, was also higher in 3D cultures, amounting to 41.75 spectra compared to 2.24 spectra in 2D culture samples. The levels of aggrecan, biglican, and decorin did not differ between cultures. 3D cultures of chondrocyte-like cells from hPSC showed no significant differences in the content of GAG compared to 2D cultures, which indicates the need to optimize the conditions for their differentiation.Conclusions. In our study, the composition of the GAGs synthesized in vitro by chondrocytes of various origins was determined using ELISA and LC–MS/MS. The the effect of 2D and 3D cultivation on their synthesis was evaluated. The results showed that 3D culture media create favorable conditions for a more complete formation of chondrocytic ECM in native chondrocyte samples. Despite this, the obtained spheroids of chondrocyte-like hPSCs derivatives fail to achieve functional identity with natural cartilage tissue, even after completion of differentiation protocols, thus not representing optimal tissue engineering structures for correcting cartilage defects.