Evidence for the extracellular delivery of influenza NS1 protein

We constructed a reporter influenza A/Puerto Rico/8/1934 virus expressing truncated 124aa N-terminal NS1 protein fused to a luciferase reporter sequence (NanoLuc) without signal peptide. The reproduction activity of the vector correlated well with the luminescent activity in the lysates of infected cell cultures or mouse respiratory organ suspensions. Surprisingly, we found that luciferase enzymatic activity was present not only in the intracellular compartments but also in cell culture supernatants as well as in the sera or bronchiolar lavages of infected mice. This fact allowed us to formulate a working hypothesis about the extracellular delivery mechanism of the NS1 protein. To test this idea, we conducted co-transfection experiments in Vero cells with different combinations of plasmids encoding influenza genomic segments and chimeric NS1-NanoLuc encoding plasmid. We found that the emergence of the luciferase reporter in the extracellular compartment was promoted by the formation of the ribonucleoprotein complex (RNP) from the co-transfection of plasmids expressing PB1, PB2, PA, and NP proteins. Therefore, influenza NS1 protein may be delivered to the extracellular compartment together with the nascent RNP complexes during the maturation of virus particles.

Mutations designed to modify the NS gene mRNA secondary structure affect influenza A pathogenicity in vivo

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.

Clostridium difficile infection in pediatric patients of oncological hospital: cultivation of anaerobic intestinal flora and treatment

In recent years, the number of infectious diseases caused by Clostridium difficile in the world has grown with a significant increase in relapses and mortality in patients, particularly among the cancer patients in hospitals. There is also observed an increase in the resistance of Clostridium difficile to the first-line drugs, namely metronidazole and vancomycin, which makes the search for new methods of treatment and prevention of this infection even more urgent. In this review, we analyze the recent data on the methods of cultivation and isolation of the pure bacterial culture of Clostridium difficile and other anaerobic enteropathogens over the course of enterocolitis treatment with antimicrobial drugs in pediatric patients with oncopathology. Novel approaches to the therapy of this infection are discussed.

Monitoring of microbial contamination of the educational and scientific clean laboratory module

The production environment in pharmaceutical industry should not be a source of microbial contamination of the product. The purpose of this study was to examine the patterns of changes in the microbial contamination level in clean rooms. The study was conducted at the educational and scientific clean room module of the Biotechnology Department of the Obninsk Institute of Nuclear Power Engineering (IATE NRNU MEPhI). It was shown that the level of contamination of surfaces in production facility increases with the presence of personnel on the premises. The degree of microbial contamination of the air varies slightly depending on the type of activity and the number of personnel on premises. The compliance of the investigated clean room module with class D for clean rooms was confirmed. It was concluded that it is necessary to monitor microbial contamination of premises more often, as well as to implement the sanitary treatment in order to eliminate the sources of spore-forming microflora.

Membrane protein of SARS-CoV-2 plays a pivotal role in the availability of active testosterone through its interaction with AKR1C2 enzyme leading to the upregulation of TMPRSS2 protease expression

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease (COVID-19) and ongoing pandemic that has devastated humankind. During the COVID-19 pandemic, it was noticed that the mortality rate in men is higher than that in women. The membrane (M) protein of SARS-CoV-2 plays a pivotal role in the viral life cycle regulating intracellular trafficking and processing of spike (S) protein. In infected individuals, M protein inhibits the conversion of active testosterone to its inactive form through its interaction with Aldo-keto reductase family 1 member C2 (AKR1C2) protein. This leads to the high availability of active testosterone and boosts the formation of its complex with an androgen receptor that in turn promotes the transcription of the transmembrane protease serine 2 (TMPRSS2) gene. TMPRSS2 is known to play a pivotal role in the priming of S protein that is necessary for the SARS-CoV-2 entry into the host cell. Therefore, the interaction of the M protein of SARSCoV-2 with AKR1C2 eventually leads to the upregulation of the transcription of the TMPRSS2 gene that results in an enhanced viral infection and in turn higher mortality in men. The interaction of M protein with AKR1C2 could be a possible target for SARSCoV-2 antiviral drug design.

The risk of developing a Clostridium difficile infection from the administration of different classes of antibiotics and their combinations to children in an oncological hospital

Patients in pediatric oncological hospitals are at risk of developing a Clostridium difficile infection. The purpose of this study was to determine the risk of developing a Clostridium difficile infection in patients who are treated with antibiotics of different classes and combinations by way of a retrospective analysis of 122 patient records. It was shown that the administration of antibacterial chemotherapeutic drugs that belong to the classes of nitrofurans (enterofuryl), sulfonamides (biseptol), cephalosporins, and macrolides/azalides significantly increased the risk of developing a Clostridium difficile infection in pediatric patients. On the contrary, treatment with antibiotics of different classes, such as linezolid, colistin, and metronidazole, significantly reduced the risk of developing a Clostridium difficile infection. The use of penicillins, aminoglycosides, fluoroquinolones, glycopeptides, and carbapenems was not associated with the risk of developing a Clostridium difficile infection in pediatric patients. The administration of one or two antimicrobial drugs of different classes increased the risk of developing a Clostridium difficile infection while a combination of three different types of antimicrobial drugs lowered the rate of this infection in pediatric patients.