The incorporation of bioactive compounds onto polymer fibrous scaffolds with further control of drug release kinetics is essential to improve the functionality of scaffolds for personalized drug therapy and regenerative medicine. In this study, polymer and hybrid microcapsules were prepared and used as drug carriers, which are further deposited onto polymer microfiber scaffolds [polycaprolactone (PCL), poly(3-hydroxybutyrate) (PHB), and PHB doping with the conductive polyaniline (PANi) of 2 wt % (PHB-PANi)]. The number of immobilized microcapsules decreased with increase in their ζ-potential due to electrostatic repulsion with the negatively charged fiber surface, depending on the polymer used for the scaffold's fabrication. Additionally, the immobilization of the capsules in dynamic mechanical conditions at a frequency of 10 Hz resulted in an increase in the number of the capsules on the fibers with increase in the scaffold piezoelectric response in the order PCL < PHB < PHB-PANi, depending on the chemical composition of the capsules. The immobilization of microcapsules loaded with different bioactive molecules onto the scaffold surface enabled multimodal triggering by physical (ultrasound, laser radiation) and biological (enzymatic treatment) stimuli, providing controllable release of the cargo from scaffolds. Importantly, the microcapsules immobilized onto the surface of the scaffolds did not influence the cell growth, viability, and cell proliferation on the scaffolds. Moreover, the attachment of human mesenchymal stem cells (hMSCs) on the scaffolds revealed that the PHB and PHB-PANi scaffolds promoted adhesion of hMSCs compared to that of the PCL scaffolds. Two bioactive compounds, antibiotic ceftriaxone sodium (CS) and osteogenic factor dexamethasone (DEXA), were chosen to load the microcapsules and demonstrate the antimicrobial properties and osteogenesis of the scaffolds. The modified scaffolds had prolonged release of CS or DEXA, which provided an improved antimicrobial effect, as well as enhanced osteogenic differentiation and mineralization of the scaffolds modified with capsules compared to that of individual scaffolds soaked in CS solution or incubated in an osteogenic medium. Thus, the immobilization of microcapsules provides a simple, convenient way to incorporate bioactive compounds onto polymer scaffolds, which makes these multimodal materials suitable for personalized drug therapy and bone tissue engineering.

While DNA and messenger RNA (mRNA) based therapies are currently changing the biomedical field, the delivery of genetic material remains the key problem preventing the wide introduction of these methods...

On 26 November 2021 WHO designated a new variant of concern B.1.1.529 named Omicron. This variant has a large number of mutations, some of which are concerning. Preliminary evidence suggests an increased risk of reinfection with this variant and reduced neutralization by convalescent and vaccinated sera, as compared to other VOCs. Implementation of the high-throughput rRT-PCR screening for Omicron is of great importance for monitoring the spread of this VOC in the population, especially in resource-limited countries lacking sufficient sequencing capacity. Omicron lineage B.1.1.529 (BA.1) has some indels that turned out to be a good target for its detection. In the current protocol, we use ins214EPE for this purpose. Here we describe the 1-step quantitative multiplex RT-qPCR assay consisting of the newly developed Ins214EPE detection set and widely used Hong Kong University N gene assay for SARS-CoV-2 detection (Chu et al., 2020). The assay was validated on the Omicron variant RNA kindly provided by the Pathogenic Microorganisms Variability Laboratory (Dr. Vladimir Guschin, Gamaleya Institute, Moscow, Russia) and RNA from the collection of Smorodintsev Research Institute of Influenza. Omicron RNA specimens were positive in the assay as expected. Negative controls were found negative. 10-fold serial dilutions of Omicron RNA were used to assess ins214EPE assay amplification efficiency. The amplification efficiency was 98,9% (R2 = 0,99). The developed rRT-PCR assay demonstrates high specificity. It was tested on 26 clinical samples (RNA extracted from oropharyngeal swabs) with previously characterized viruses belonging to 8 different SARS-CoV-2 lineages (including Delta B.1.617.2+AY.*) Specific signal was detected only in samples with SARS-CoV-2 Omicron lineage RNA (confirmed by whole-genome sequencing). Specificity was additionally tested on clinical samples positive for other respiratory viruses from the collection of Smorodintsev Research Institute of Influenza - influenza, parainfluenza, human seasonal coronaviruses (OC43, NL63, 229E, HKU1), hRSV, rhinoviruses, bocaviruses, metapneumovirus (33 in total) - with no false-positive results. Ins214EPE Cq 6x B.1.1.7 2x B.1.351 5x AT.1 6x B.1.617.2 4x AY.122 P.1 B.1.1.529 28,72 B.1.1.529 26,29 virus RP Cq SARS Cq Ins214 Cq RSV A 28,76 RSV A 30,56 RSV A 27,70 RSV B 31,49 RSV B 30,98 RSV B 32,33 NL63 32,20 NL63 30,42 NL63 24,95 Oc43 30,34 Oc43 30,69 Oc43 28,64 HKU1 30,06 HKU1 28,30 HKU1 30,73 229E 29,11 229E 32,52 229E 29,37 BoV 32,26 BoV 30,75 BoV 27,25 Rv 32,85 Rv 33,76 Rv 27,75 Piv1 28,63 Piv2 24,72 Piv3 27,01 Piv4 23,90 Adv 29,47 MPV 30,12 HIV A 29,13 HIV A 28,45 HIV A 28,16 39,06 c+ 34,15 26,61 28,44 Analytical sensitivity determination is underway. We consider developed assay to be useful in wide populational RT-PCR screening to assess the spread of Omicron variant.

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.

Omicron lineage (B.1.1.529) – SARS-CoV-2 variant of concern designated on 26 November 2021 by WHO. This variant has a large number of mutations, some of which are concerning. There is already enough evidence that Omicron lineage connected with increased risk of reinfection with this variant and reduced neutralization by convalescent and vaccinated sera, as compared to other VOCs. Omicron subvariants includes several subvariants with strikingly different genetic characteristics: BA.1, BA.2, BA.3. We developed RT-qPCR specific to omicron lineage with detemination of BA.1 and BA.2/BA.3 for epidemiological reasons. Omicron lineage B.1.1.529 and its sublineages BA.1 and BA.2/BA.3 have some indels that turned out to be a good target for its detection. Using annotations of amino acid substitutions and indels for more than 5 million samples in 1513 Pango lineages from the GISAID database available on 2021-12-03, relative frequencies of substitutions and indels by each lineage were calculated with Python v3.9.2 programming language, Pandas v1.3.5 and Numpy v1.3.5 tools. Using this data we found indels highly specific for BA.1 and BA.2/BA.3: ERS31del for Omicron (B.1.1.529) lineage, including BA.1, BA.2 and BA.3; and Ins214EPE specific only to BA.1. For these targets have been developed assays, that were then multiplexed. Assay for detection BA.1 have been already published at protocols.io. Here we publish multiplex RT-qPCR detecting all Omicron subvariants with the opportunity to differentiate BA.1 and BA.2/BA.3. Characteristics and analysis of specificity of Ins214EPE were already published in previous protocol. We only can add that limit of detection of the test is 1000 copies/ml Here we describe features of newly introduced ERS31del assay. 10-fold serial dilutions of SARS-CoV-2 RNA of Omicron lineage were used to assess ERS31del assay amplification efficiency. The amplification efficiency was 105% (R2 = 0,99). Developed RT-qPCR assay demonstrates high specificity. It was tested on 27 clinical samples (RNA extracted from oropharyngeal swabs) with previously characterized viruses belonging to 10 different SARS-CoV-2 lineages. RP 2-SARS-ORF NdeI Ins214 1 c- 2 P.1 35,35 15,45 3 B.1.351 39,91 4 B.1 15,69 5 B.1 33,84 26,69 6 B.1 24,26 26,98 7 B.1.1.7 25,27 20,64 8 B.1.1.7 30,54 28,05 9 B.1.1.7 26,99 31,25 10 AT.1 30,30 33,07 11 AT.1 30,02 31,33 12 AT.1 28,00 33,00 13 B.1.1.523 29,12 27,09 14 B.1.1.523 27,39 23,46 15 B.1.1.523 30,02 27,09 16 B.1.1.317 25,22 25,31 17 B.1.1.317 27,58 22,71 18 B.1.1.317 28,70 23,91 19 B.1.617.2 26,66 34,03 20 B.1.617.2 25,72 28,43 21 B.1.617.2 26,78 25,42 22 AY.122 23,96 28,95 23 AY.122 31,16 35,05 24 AY.122 24,19 34,93 25 BA.1 27,98 22,20 23,57 22,08 26 BA.1 27,08 24,93 27,16 24,65 27 BA.1 25,19 27,48 29,83 25,57 28 BA.2 26,50 19,64 21,07 29 BA.2 24,70 20,81 22,70 Specific signal was detected only in samples with SARS-CoV-2 Omicron lineage RNA: BA.1 and BA.2 (confirmed by whole-genome sequencing). Specificity was additionally tested on clinical samples positive for other respiratory viruses from the collection of Smorodintsev Research Institute of Influenza - influenza A and B, human seasonal coronaviruses, hRSV, rhinoviruses - with no false-positive results. Analytical sensitivity Limit of detection of the test is 1000 copies/ml

No abstract

Viral nucleoprotein (NP) is an abundant essential protein of an influenza virus that has important functional and structural roles. It participates in genomic organization, nuclear trafficking, RNA transcription, and genome replication. From the research point of view, NP is an important protein that is used in the development of new diagnostic methods and vaccination protocols. NP is a promising target for antiviral chemotherapeutic drugs as well. Successful expression of codon-optimized NP genes in E. coli has been reported. In this study, we demonstrated the efficient expression and purification of soluble NPs of influenza A and B viruses in E. coli without the codon-optimization of DNA sequences. This procedure preserves the co-translational protein folding, protein configuration and function. Obtained NPs of influenza A and B viruses were monomers and reacted well with mouse specific antibodies according to Western blot analysis. Our results show that both influenza A and influenza B virus NPs can be efficiently expressed in E. coli without codon-optimization.

Whole genome sequencing (WGS) is considered the best instrument to track both virus evolution and the spread of new, emerging variants. However, WGS still does not allow the analysis of as many samples as qPCR does. Epidemiological and clinical research needs to develop advanced qPCR methods to identify emerging variants of SARS-CoV-2 while collecting data on their spreading in a faster and cheaper way, which is critical for introducing public health measures. This study aimed at designing a one-step RT-qPCR assay for multiplex detection of the Omicron lineage and providing additional data on its subvariants in clinical samples. The RT-qPCR assay demonstrated high sensitivity and specificity on multiple SARS-CoV-2 variants and was cross-validated by WGS.