Предложен способ модификации мишени для МАLDI-МS-анализа, позволяющий селективно выделять аналиты из биологических образцов непосредственно на поверхности мишени, как альтернатива классическим методам. Для модификации мишени суспензию металл-аффинного сорбента на основе оксида железа(III) в 50% водно-метанольном растворе электрораспыляли в бескапельном режиме c динамическим делением потока жидкости при атмосферном давлении в нормальных условиях. МАLDI-мишень выступала в качестве противоэлектрода. На МАLDI-мишень наносился слой сорбента в виде пятна, частицы которого в дальнейшем устойчивы к воздействию растворителей. На модифицированной МАLDI-мишени проводилось металл-аффинное обогащение фосфорилированного пептида с аминокислотной последовательностью SSNGHV(pY)EKLSSI из образца триптического гидролизата глобина человека. МАLDI-масс-спектр записывали с пятна сорбента. Такая методика создана в качестве альтернативы трудоемкой пробоподготовке биопроб и позволяет ограничиться минимальными объемами образца и растворителей.
The influenza A virus genome consists of eight segments of negative-sense RNA that encode up to 18 proteins. During the process of viral replication, positive-sense (+)RNA (cRNA) or messenger RNA (mRNA) is synthesized. Today, there is only a partial understanding of the function of several secondary structures within vRNA and cRNA promoters, and splice sites in the M and NS genes. The most precise secondary structure of (+)RNA has been determined for the NS segment of influenza A virus. The influenza A virus NS gene features two regions with a conserved mRNA secondary structure located near splice sites. Here, we compared 4 variants of the A/Puerto Rico/8/1934 strain featuring different combinations of secondary structures at the NS segment (+)RNA regions 82-148 and 497-564. We found that RNA structures did not affect viral replication in cell culture. However, one of the viruses demonstrated lower NS1 and NEP expression levels during early stage cell infection as well as reduced pathogenicity in mice compared to other variants. In particular, this virus is characterized by an RNA hairpin in the 82-148 region and a stable hairpin in the 497-564 region.
Type III interferons exhibit antiviral activity against influenza viruses, coronaviruses, rotaviruses, and others. In addition, this type of interferon theoretically has therapeutic advantages, in comparison with type I interferons, due to its ability to activate a narrower group of genes in a relatively small group of target cells. Hence, it can elicit more targeted antiviral or immunomodulatory responses. Obtaining biologically-active interferon lambda (hIFN-λ1) is fraught with difficulties at the stage of expression in soluble form or, in the case of expression in the form of inclusion bodies, at the stage of refolding. In this work, hIFN-λ1 was expressed in the form of inclusion bodies, and a simple, effective refolding method was developed. Efficient and scalable methods for chromatographic purification of recombinant hIFN-λ1 were also developed. High-yield, high-purity product was obtained through optimization of several processes including: recombinant protein expression; metal affinity chromatography; cation exchange chromatography; and an intermediate protein refolding stage. The obtained protein was shown to have expected, specific biological activity in line with published effects: induction of MxA gene expression in A549 cells.
Respiratory infections, collectively, are one of the world’s most common and serious illness groups. As recent observations have shown, the most severe courses of acute respiratory infection, often leading to death, are due to uncontrolled cytokine production (hypercytokinemia). The research presented is devoted to assessment of mRNA expression of specific cytokines (IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-18, TNF-α, IFN-λ) and MxA in whole blood leukocytes, by means of real-time PCR. This study involved 364 patients with respiratory illness being treated in clinics in St. Petersburg (Russia) in 2018-2019 and 30 healthy subjects. In 70% of patients, bacterial or viral pathogens were identified, with influenza viral infections (types A, B) prevailing. Cytokine analysis was carried out in the acute phase of illness (2-3 days from onset of initial symptoms) and in the stage of recovery (days 9-10). Significant increases in the expression of IL-18, TNF, and IL-10 were observed, relative to controls, only with influenza viral infections. We have shown a difference in IL-6 mRNA expression in patients with bacterial or viral pathogens. No significant difference was found in WBC IL-4 expression levels between patients and healthy subjects. Investigation of the nuances of systemic cytokine production, in response to specific viral and bacterial pathogens, makes it possible to: assess the risks of developing hypercytokinemia during respiratory infection with agents circulating in the human population; and to predict the pathogenicity and virulence of circulating threats.
This paper considers the evaluation of uncertainty of quantitative gel electrophoresis. To date, such uncertainty estimation presented in the literature are based on the multiple measurements performed for assessing the intra- and interlaboratory reproducibility using standard samples. This paper shows how to estimate the uncertainty in cases where we cannot study scattering components of the results. The first point is dedicated to a case where we have standard samples (the direct expressions are shown). The second point considers the situation when standard samples are absent (the algorithm for estimating the lower bound for uncertainty is discussed). The role of the data processing algorithm is demonstrated.
One of the main functions of alpha-2-macroglobulin (A2M) in human blood serum is the binding of all classes of protease. It is known that trypsin, after such interaction, possesses modified proteolytic activity. Trypsin first hydrolyzes two bonds in A2M’s ‘bait region’, and the peptide 705VGFYESDVMGR715 is released from A2M. In this work, specifics of the A2M-trypsin interaction were used to determine A2M concentration directly in human blood serum using MALDI mass-spectrometry. Following exogenous addition of trypsin to human blood serum in vitro, the concentration of the VGFYESDVMGR peptide was measured, using its isotopically-labeled analogue (18O), and A2M concentration was calculated. The optimized mass spectrometric approach was verified using a standard method for A2M concentration determination (ELISA) and the relevant statistical analysis methods. It was also shown that trypsin’s modified proteolytic activity in the presence of serum A2M can be used to analyze other serum proteins, including potential biomarkers of pathological processes. Thus, this work describes a promising approach to serum biomarker analysis that can be technically extended in several useful directions.
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