Soluble Ligand of the Immune Checkpoint Receptor (sPD-L1) in Blood Serum of Patients with Renal Cell Carcinoma.

The content of the soluble ligand of the immune checkpoint receptor (sPD-L1) was determined in the blood serum of 106 patients with renal cell carcinoma and 11 patients with benign kidney tumors by direct ELISA (Human sPD-L1 Platinum ELISA; Affimetrix, eBioscience). The control group included 19 healthy men and 18 women. Serum level of sPD-L1 significantly surpassed the control values in both patients with primary renal cancer (p<0.0001) and in patients examined during disease progression (p<0.05). In patients with benign kidney tumors, the level of this marker was significantly higher than in the control (p<0.05), but lower than in patients with renal cell carcinoma. The sPD-L1 level significantly increased with disease stage (p<0.001); it was higher in the presence of metastases in regional lymph nodes irrespective of their number (N1 or N2) than in the absence of metastases (N0); it was also increased in patients with distant metastases (M1) and patients with grade III-IV tumors in comparison with grade III-IV tumors (p<0.05). The highest sPD-L1 levels were recorded in patients with tumor size corresponding to T2 and T3 and decreased in patients with T4 tumors. Thus, sPD-L1 level in patients with renal cell carcinoma correlated with tumor grade and metastasizing and can be considered as a promising marker in monitoring of the effect of anti-PD1/PD-L1 therapy.

Video-tactile pneumatic sensor for soft tissue elastic modulus estimation.

BACKGROUND: A new sensor for estimating elasticity of soft tissues such as a liver was developed for minimally invasive surgery application. METHODS: By measuring deformation and adjusting internal pressure of the pneumatic sensor head, the sensor can be used to do palpation (indentation) of tissues with wide range of stiffness. A video camera installed within the sensor shell is used to register the radius of the contact area. Based on finite element model simulations and the measured data, elastic modulus of the indented soft tissue can be calculated. RESULTS AND CONCLUSIONS: Three phantom materials, namely plastic, silicone and gelatin, with varied stiffness were tested. The experimental results demonstrated that the new sensor can obtain highly reliable data with error less than 5%. The new sensor might be served as an instrument in laparoscopic surgery for diagnosis of pathological tissues or internal organs.

SCIENTIFIC AND PRACTICAL STUDIES OF MALARIA IN THE CIS COUNTRIES AND GEORGIA.

The paper presents the scientific studies of malaria pathogens and vectors, which have been specially conducted in the endemic areas of the CIS countries and Georgia for use in an epidemiological surveillance system. The main ones investigate the structure of malaria foci and the level of G-6-PD deficiency among residents, determine the malariogenic potential. of the territory and the risk of infection in the population, and specify the taxonomy, systematics, and spread of major malaria vectors in .the countries ofWHO European Region. In addition, the time and magnitude of manifestations of long-term post-incubation tertian malaria were established; th6 susceptibility of P.vivax to antimalarials and the levels of resistance and irritability of malaria vectors to insecticides were studied. The experience in using a geographic information system for the epidemiological surveillance of malaria is given.

[Drosophila melanogaster Cell Culture as an Experimental Model to Study Recombination in Wolbachia pipientis].

Wolbachiapipientis is an obligate intracellular endosymbiont that commonly infects arthropods. Comparative genomic studies of Wolbachia reveal traces of numerous events of intergenic and intragenic recombination. The molecular mechanisms of recombination in Wolbachia are not currently known. We conducted experimental verification of the possibility of recombination of two strains of Wolbachia: wMel and wRi, after using these strains for double infection of the Dm2008Wb1 (D. melanogaster) cell culture clone permissive to Wolbachia. We obtained cell culture subclones with double Wolbachia infection and subclones infected only by strain wMel. Dual infection with the Wolbachia strains wMel and wRi has been stably maintained in the subclones for two years. Multilocus sequence typing (MLST) of the obtained subclones revealed the presence of dual infection for all five Wolbachia genes used for MLST Cloning and nucleotide sequence analysis of individual forms of the fbpA gene of Wolbachia from cell clones with dual infection showed intragenic recombination events between strains wMel and wRi, which occurred in the permanent D. melanogaster culture cell culture. The fact that putative recombination sites contain no insertions of nucleotide sequences of phages or IS elements, as well as the asymmetrical character of recombinants, favors the hypothesis that gene conversion is the most probable molecular mechanism of recombination in Wolbachia.

[Genotypic Diversity of Wolbachia pipientis in Native and Invasive Harmonia axyridis Pall., 1773 (Coleoptera, Coccinellidae) Populations].

The distribution and variability of reproductive symbiotic Wolbachia pipientis bacteria were studied in seven native and six invasive H. axyridis populations. Wolbachia-infected individuals were found in two invasive and two native populations. We demonstrated for the first time an infection of invasive H. axyridis populations with Wolbachia. Two new molecular forms of Wolbachia were detected by a system of multilocus typing. The supergroup A Wolbachia was found for the first time in H. axyridis. The detected genotypic diversity of Wolbachia indicates repeated and independent infection events in the evolutionary past of H. axyridis.

[Identification of potentially invasive species of black flies [Diptera: Simuliidae] from Armenia based on an analysis of variability in the mtDNA barcode of the cox1 gene and chromosomal polymorphism].

Black flies (Diptera, Simuliidae) are well known for their medical, environmental, and veterinary importance. The simuliid fauna of Armenia includes 53 species. A number of dominant species are of ecological importance. Complex analysis, which involved morphometric, cytogenetic, and molecular genetic approaches, was conducted to characterize the species status of black flies inhabiting the territory of Armenia. It was shown that the predominant simuliid species, Simulium paraequinum and Simulium kiritshenkoi, belong to a group of species with minimal variability of the cox1 gene. The recently discovered species, Simulium noellery and Simulium [B.] erythrocephalum, which are new to Armenia, can be considered as potentially invasive, which is supported by the low level of variability of the cox1 gene.

Multi-analyte homogenous immunoassay based on quenching of quantum dots by functionalized graphene.

We propose a homogenous multi-analyte immunoassay based on the quenching of quantum dot (QD) fluorescence by means of graphene. Two QDs with emission maxima at 636 and 607 nm were bound to antibodies selective for mouse or chicken immunoglobulins, respectively, and graphene functionalized with carboxylic moieties was employed to covalently link the respective antigen. The antibody-antigen interaction led graphene close enough to QDs to quench the QD fluorescence by resonance energy transfer. The addition of free antigens that competed with those linked to graphene acted as a "turn-on" effect on QD fluorescence. Fluorescence emitted by the two QDs could be recorded simultaneously since the QDs emitted light at different wavelengths while being excited at the same wavelength and proved to be linearly correlated with free antigen concentration. The developed assay allows measuring both antigens over 2-3 orders of magnitude and showed estimated limits of detection in the nanomolar range. This approach is thus a promising universal strategy to develop homogenous immunoassays for diverse antigens (cells, proteins, low-molecular-mass analytes) in a multi-analyte configuration.

Quantum dot loaded liposomes as fluorescent labels for immunoassay.

Liposomes loaded with water-soluble and water-insoluble quantum dots (QD) were for the first time applied as labels in different heterogeneous immunoassays for the determination of food contaminants, using mycotoxin zearalenone (ZEN) as a model. A great deal of work was devoted to the optimal choice of phospholipids for the liposomes preparation and to the factors which are important for the stability and size of obtained liposomes. Thin-film hydration and reverse-phase evaporation techniques were evaluated in terms of stability of the obtained liposomes and their efficiency for QD loading. Conjugation of liposomes with proteins and the influence of cross-linkers to the nonspecific interaction of the obtained liposomes with the surface of microtiter plates and cartridges were investigated and 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester was found as the optimal cross-linker. The limits of detection (LOD) for ZEN of fluorescence-labeled immunosorbent assays were 0.6 µg kg(-1), 0.08 µg kg(-1), and 0.02 µg kg(-1), using QD, liposomes loaded with water-soluble QD, and water-insoluble QD, respectively. Similarly, the developed qualitative on-site tests using the different QD labels and taking into account the EU maximum residues level for ZEN in unprocessed cereals showed cutoff levels of 100, 50, and 20 µg kg(-1).

Liposomes loaded with quantum dots for ultrasensitive on-site determination of aflatoxin M1 in milk products.

A quantitative fluorescence-labeled immunosorbent assay and qualitative on-site column tests were developed for the determination of aflatoxin M1 in milk products. The use of liposomes loaded with quantum dots as a label significantly increased the assay sensitivity by encapsulating multiple quantum dots in a single liposome and, therefore, amplifying the analytical signal. Two different techniques were compared to obtain aflatoxin-protein conjugates, used for further coupling with the liposomes. The influence of nonspecific interactions of the liposome-labeled conjugates obtained with the surface of microtiter plates and column cartridges was evaluated and discussed. The limit of detection for fluorescence-labeled immunosorbent assay was 0.014 µg kg(-1). For qualitative on-site tests, the cutoff was set at 0.05µg kg(-1), taking into account the EU maximum level for aflatoxin M1 in raw milk, heat-treated milk, and milk for the manufacture of milk-based products. The direct addition of labeled conjugate to the milk samples resulted in an additional decrease of analysis time. An intralaboratory validation was performed with sterilized milk and cream samples artificially spiked with aflatoxin M1 at concentrations less than, equal to and greater than the cutoff level. It is shown that milk products can be analyzed without any sample preparation, just diluted with the buffer. The rates for false-positive and false-negative results were below 5% (2.6% and 3.3%, respectively).

Fluorescent polymer markers photoconvertible with a 532 nm laser for individual cell labeling.

Fluorescent photoconvertible materials and molecules have been successfully exploited as bioimaging markers and cell trackers. Recently, the novel fluorescent photoconvertible polymer markers have been developed that allow the long-term tracking of individual labeled cells. However, it is still necessary to study the functionality of this type of fluorescent labels for various operating conditions, in particular for commonly used discrete wavelength lasers. In this article, the photoconversion of fluorescent polymer labels with both pulsed and continuous-wave lasers with 532 nm-irradiation wavelength, and under different laser power densities were studied. The photoconversion process was described and its possible mechanism was proposed. The peculiarities of fluorescent polymer capsules performance as an aqueous suspension and as a single capsule were described. We performed the successful nondestructivity marker photoconversion inside RAW 264.7 monocyte/macrophage cells under continuous-wave laser with 532 nm-irradiation wavelength, showing prospects of these fluorescent markers for long-term live cell labeling.

Quantum dot based rapid tests for zearalenone detection.

Three different kinds of immunosorbent assays with luminescence detection were developed for the determination of zearalenone (ZEN), a secondary toxic metabolite of Fusarium fungi. CdSe/ZnS core/shell quantum dots (QDs) were used as a label in quantitative micro-well plate immunoassays (fluorescent-labeled immunosorbent assay, FLISA) and in qualitative column test methods. As carriers for QD-based column tests, sepharose gel (for covalent binding of antibody) and polyethylene frits (for physical absorption of antibody) were used and compared. The application of QDs as a label resulted in a fourfold decrease in the IC(50) value with FLISA (0.1 ng mL(-1)) with a detection limit of 0.03 ng mL(-1) when compared with the traditional immunosorbent assay which makes use of horseradish peroxidase as the enzyme label. The cutoff levels for both qualitative column test methods were selected based on the maximum level for ZEN in unprocessed cereals established by the European Commission (100 µg kg(-1)) as 5 ng mL(-1) taking into account extraction and dilution. The different developed immumoassays were tested for ZEN determination in raw wheat samples. As a confirmatory method, liquid chromatography coupled to tandem mass spectrometry was used. The obtained results allow using FLISA and both qualitative column test methods for the analysis of analytes with very low established maximum limits, even in very complicated food matrices, owing to the high dilution of the sample extract.

Multiplex flow-through immunoassay formats for screening of mycotoxins in a variety of food matrices.

Two multi-analyte flow-through immunoassay formats for rapid detection of mycotoxins in a variety of food matrices (peanut cake, maize, and cassava flour) were developed and evaluated. The selected food matrices are typical staple foods and export products for most low-income communities around the world. The assay formats included gel-based and membrane-based flow-through assays and were based on the principle of indirect enzyme-linked immunosorbent assay. Using the same immunoreagents, the performance characteristics of both assays were compared. To the best of our knowledge, this is the first report on such a comparison. The gel-based format was developed to screen for ochratoxin A, fumonisin B(1), deoxynivalenol, and zearalenone detection at cut-off values of 3, 1,250, 1,000, and 200 µg kg(-1), respectively, while the membrane-based format can be used to screen ochratoxin A, aflatoxin B(1,) deoxynivalenol, and zearalenone at the following cut-offs: 3, 5, 700, and 175 µg kg(-1), respectively. The applicability of these assay formats was demonstrated by evaluating the performance characteristics of both tests through performing multiple experiments on different days. Both assays were further evaluated by analyzing naturally contaminated samples in the laboratory and also in the field under tropical conditions (Cameroon, West Africa). The false-negative rate with both formats was less than 5%, which is in good agreement with Commission Decision 2002/657/EC regarding the performance of analytical methods intended for screening purposes.

[Mitochondrial genome variation in domesticated sable (Martes zibellina)].

The first comparison of mitochondrial variations in sables from captive and natural populations of the Urals, Central Siberia, Yakutia, Kamchatka, and Japan has been performed. The object of comparative analysis was a 427-bp 5' fragment of the mitochondrial control region, including the D-loop. Two main haplogroups of the sable mitochondrial genome have been found, which provides new data for reconstruction of the spread of the sable over its current range. Asymmetry of the haplotype abundances in the captive populations of sables has been detected. The mitochondrial haplotypes characteristic of sable breeds have been identified. The possible role of the frequent mitochondrial haplotypes of the captive population in the sable adaptation to the conditions of captivity is discussed.

Inferring the origin of populations introduced from a genetically structured native range by approximate Bayesian computation: case study of the invasive ladybird Harmonia axyridis.

Correct identification of the source population of an invasive species is a prerequisite for testing hypotheses concerning the factors responsible for biological invasions. The native area of invasive species may be large, poorly known and/or genetically structured. Because the actual source population may not have been sampled, studies based on molecular markers may generate incorrect conclusions about the origin of introduced populations. In this study, we characterized the genetic structure of the invasive ladybird Harmonia axyridis in its native area using various population genetic statistics and methods. We found that native area of H. axyridis most probably consisted of two geographically distinct genetic clusters located in eastern and western Asia. We then performed approximate Bayesian computation (ABC) analyses on controlled simulated microsatellite data sets to evaluate (i) the risk of selecting incorrect introduction scenarios, including admixture between sources, when the populations of the native area are genetically structured and sampling is incomplete and (ii) the ability of ABC analysis to minimize such risks by explicitly including unsampled populations in the scenarios compared. Finally, we performed additional ABC analyses on real microsatellite data sets to retrace the origin of biocontrol and invasive populations of H. axyridis, taking into account the possibility that the structured native area may have been incompletely sampled. We found that the invasive population in eastern North America, which has served as the bridgehead for worldwide invasion by H. axyridis, was probably formed by an admixture between the eastern and western native clusters. This admixture may have facilitated adaptation of the bridgehead population.

Novel gel-based rapid test for non-instrumental detection of ochratoxin A in beer.

A rapid easy-to-use immunoassay was optimised for the non-instrumental detection of ochratoxin A (OTA) in beer. The analytical method involves preconcentration on the immunoaffinity layer inside a column followed by direct competitive ELISA detection in the same layer. The visual cut-off value, i.e. the lowest OTA concentration resulting in no colour development, was 0.2 microg L(-1). Assay validation was performed using samples spiked with OTA. Thirty-seven naturally contaminated samples were screened with the gel-based method developed and no false-negative results were obtained. The method described offers a simple, rapid and cost-effective screening tool, thus contributing to better health protection of consumers.

Intragenomic heterogeneity of rDNA internal transcribed spacer 2 in Anopheles messeae (Diptera: Culicidae).

Because Anopheles messeae Falleroni (Diptera: Culicidae) is one of the main vectors of malaria in Russia, studying its genetic markers is important for reliable identification of this species. This species is distributed nearly throughout the Palearctic region, and it exhibits high genetic variability. We investigated polymorphism of the rDNA internal transcribed spacer (ITS) 2 of An. messeae in various regions of Russia, and we found intragenomic heterogeneity of ITS2 copies verified by chromatograms, polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis, and cloning PCR products. In total, we found nine different ITS2 variants. ITS2 variants that were considered specific to An. messeae and Anopheles daciae Linton, Nicolescu & Harbach were simultaneously present in one individual. These findings improve methods of species identification of An. messeae, and they do not support the species status of An. daciae.

Luminescent quantum dots for miRNA detection.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that are involved in nearly all developmental processes and human pathologies. MiRNAs are considered to be promising biomarkers, since their dysregulation correlates with the development and progress of many diseases. Short length, sequence homology among family members, susceptibility to degradation, and low abundance in total RNA samples make miRNA analysis a challenging task. Photoluminescent semiconductor nanoparticles (quantum dots, QDs) possess unique properties such as bright photoluminescence, photostability and narrow emission peaks, wide possibilities for surface modification and bioconjugation, which serve as the basis for the development of different analytical methods for variety of analytes. Relatively small size of QDs' and their narrow distribution are especially important for miRNA assay. The combination of QD-based biosensors with amplification techniques makes it possible to identify the target miRNA at a single-particle level with the detection limit at the attomolar scale. This review describes the principles of signal generation: direct intensity measurements, different "signal on" and "signal off" mechanisms as well as electro-chemiluminescence. Special attention is paid to the FRET-based techniques. According to our knowledge this is the first review related to QDs application for miRNA detection.

Genetic characterization of Plasmodium vivax in the Kyrgyz Republic.

At the end of 2016, Kyrgyz Republic was certified by the World Health Organization as a malaria-free country, while only a decade ago this disease posed a serious health threat. The progress achieved by Kyrgyz Republic provides a unique example of tertian (Plasmodium vivax) malaria elimination. This success was based on an integrated approach, including measures for the treatment of infected people and disease prevention, vector control and the development of an effective national epidemiological surveillance system. Lower P. vivax msp-1, msp-3α, csp and dbpII genes polymorphism was revealed in Kyrgyz Republic in compare with that in Tajikistan. Molecular characterization of the causative agent found that P. vivax populations in Kyrgyz Republic was comprised by several lineages, highly divergent in the south-western and genetically homogeneous in the northern regions of Kyrgyz Republic, d. Such profile in the northern regions was compatible with several recent introductions rather than a long-term endemic circulation of the parasite. A low level of genetic variability suggested that the parasitic systems of tertian malaria, were not adapted, which, along with other factors, largely determined the possibility of malaria elimination in northern Kyrgyz Republic. Other determinants included environmental, social, and epidemiological factors that limited the spread of malaria. South-western Kyrgyz Republic, a region with a high level of interstate migration, requires considerable attention to prevent the spread of malaria.

Lanthanide-to-quantum dot Förster resonance energy transfer (FRET): Application for immunoassay.

Förster resonance energy transfer (FRET) between lanthanide ion complexes (L) acting as donors and luminescent semiconductor quantum dots (QD) acting as acceptors is discussed in the terms of advantages and disadvantages for its application in immunoassay. L-QD-FRET is potentially a powerful tool that can be used to detect and confirm formation of immunocomplexes, but until now it had very limited practical analytical application. Therefore, the main aim of this review is to analyze all possibilities, advantages, and disadvantages of L-QD-FRET in immunoassay applications. Considering L and QD respectively applied as donor and acceptor, the most advantageous properties for analytical purposes are large decay time of L complexes and the high absorption of QD. L complexes' extremely long decay times make it possible to directly detect FRET through enhancement of QDs decay time as a result of energy transfer. Very high QD absorption predetermines extremely large Förster radii (ca. 10nm), which means that FRET can be utilized for proteins and protein complexes, such as antigen-antibody systems.

A fluorescent immunochromatographic strip test using Quantum Dots for fumonisins detection.

A fluorescent immunochromatographic strip test (ICST) based on the use of Quantum Dots (QD) was developed and applied to detect fumonisins in maize samples. A limit of detection for fumonisin B1 of 2.8 µg L(-1) was achieved, with an analytical working range of 3-350 µg L(-1), corresponding to 30-3500 µg kg(-1) in maize flour samples, according with the extraction procedure. The time required to perform the analysis was 22 min, including sample preparation. Recovery values in the range from 91.4% to 105.4% with coefficients of variation not exceeding 5% were obtained for fortified and naturally contaminated maize flour samples. To evaluate the possible improvements due to the use of QD for ICST technology, we performed a direct comparison of the proposed QD-ICST to a gold nanoparticles- and a chemiluminescent-ICST previously developed for fumonisins detection, in which the same immunoreagents were employed.