Both RNase E and RNase III control the stability of sodB mRNA upon translational inhibition by the small regulatory RNA RyhB

Previous work has demonstrated that iron-dependent variations in the steady-state concentration and translatability of sodB mRNA are modulated by the small regulatory RNA RyhB, the RNA chaperone Hfq and RNase E. In agreement with the proposed role of RNase E, we found that the decay of sodB mRNA is retarded upon inactivation of RNase E in vivo, and that the enzyme cleaves within the sodB 5′-untranslated region (5′-UTR) in vitro, thereby removing the 5′ stem–loop structure that facilitates Hfq and ribosome binding. Moreover, RNase E cleavage can also occur at a cryptic site that becomes available upon sodB 5′-UTR/RyhB base pairing. We show that while playing an important role in facilitating the interaction of RyhB with sodB mRNA, Hfq is not tightly retained by the RyhB–sodB mRNA complex and can be released from it through interaction with other RNAs added in trans. Unlike turnover of sodB mRNA, RyhB decay in vivo is mainly dependent on RNase III, and its cleavage by RNase III in vitro is facilitated upon base pairing with the sodB 5′-UTR. These data are discussed in terms of a model, which accounts for the observed roles of RNase E and RNase III in sodB mRNA turnover

Evaluating the Epidemic Risk Posed by Marek's Disease Virus in Russia

Background: The highly oncogenic avian alpha herpes virus known as Marek's disease virus (MDV) raises concerns about its potential oncogenicity in humans. In this article, authors examine the potential epidemic risks associated with MDV, taking into account the current epidemic and epizootic situation in the Russian Federation, with a particular focus on the modern practices of pig and poultry farming. Given its oncogenic properties, MDV remains a cause for concern in the field of avian herpesviruses.Methods: The study was conducted in 2010-2022 in poultry farms Russian Federation, Republic of Belarus and the Republic of Kazakhstan where authors monitored the rate of vesicular enteritis and MDV. In total, 155 epizootics were examined, including the analysis of 62 epizootics of vesicular enteritis and the population of the studied area exceeded 1 million people.Results: The authors of the study found that vesicular enteritis, distributed over poultry farms in the Russia since 2011, and its safety for the population remains unknown. The increasing incidence of vesicular enteritis was also accompanied by various health issues in contact persons and veterinary personnel, as well as abnormal and in sync increases in infectious laryngotracheitis and MDV in young birds.Conclusion: Authors come to the conclusion that the consequences of COVID-19 are accompanied by a deficiency of folic acid, which in turn leads to a risk of developing diseases associated with DNA viruses.It is concluded that MDV may contribute to the development of reproductive and breast cancers, highlighting the need for increased awareness and attention to the health risks associated with poultry farming and further research in this area.Keywords: Marek's disease virus; Vesicular enteritis; Herpesvirus infection; COVID 19   

Features of model coronaviruses distribution in feline organs and tissues in the context of COVID-19 pathogenesis study

To date, the reisreason to believe that, unlike classical a cuterespiratory virusinfections caused by adenoviruses, rhinoviruses, orthomyxoviruses, COVID-19 behaves completely differently. Firstly, the pathological process esare likely to be immune-mediated and the immun esystem quites lowly ensurest heelimination of the virus from the organism. Secondly, the dynamics of the diseases ymptom development and the duration of intestinal virusshedding after recovery give reason to believe that theSARS-CoV-2 infection is mainly localizedin the intestine. A possible reason isthat in the presence of proteolyticenzymes, viral particlesmature, hydrophilic aminoacids are removed from the surface of the virion, making it more hydrophobic and able to ad here to cells due to hydrophobic interactions. The presence of the ACE2 recept or mainly in the enterocytes of the ileumdoes not exclude the accumulation of coronavirusin lymphocytes, given that there are more lymphocytes in the gastrointestinal tract than anywhere else, this fact can beconsidered as another justification for the predominant accumulation of coronaviruses, including SARS-CoV-2 in the intestine. A distinctive feature of feline coronavirus infection and, in particular, infectious feline peritonitis, from human COVID-19 infection was considered to be the presence of effusion peritonitis as the main complication leading to death, while respiratory and card iovascular in sufficiency is more characteristic for humans. Never the less, cases of serous peritonitis in humans infected with COVID-19 have already been described. In the context of the analyzed model, theclinical case describedin the study allows principal possibility of exacerbation of chronic coronavirus infection in caseof re-infection (superinfection) and development of apredominantlylocal infection

Studying formation of <I>Pseudomonas aeruginosa</I> biofilms grown under different cultivation conditions

The purpose of the present study is to assess how cultivation conditions influence growth and formation of Pseudomonas aeruginosa biofilms. The topic is of great importance due to high incidence of P. aeruginosa-caused infections and P. aeruginosa resistance associated with its ability to form biofilms. The paper analyzes factors that influence biofilm formation, i.e.: growth phase used for inoculation (log, stationary), volume of the growth medium (0.2 and 1.0 ml) and concentration of nutrients (liquid nutrient media diluted to concentrations of 50; 25; 12.5 and 6%) in the cultivation volume. As the research demonstrates, all these factors influence biofilm formation; and a P. aeruginosa growth phase before inoculation is a determining factor in the biofilm formation. When P. aeruginosa is inoculated at a stationary phase, biofilm formation shows non-linear dependence on concentration of nutrients and on their total amount in the cultivation volume. The linear dependence of biofilm formation on concentration of nutrients in the culture medium is more pronounced, when P. aeruginosa is inoculated at a log phase. The study shows that lower concentrations of nutrient media components lead to more noticeable differences in biofilm formation, and such differences are statistically significant. Two-fold dilution of the liquid nutrient medium does not affect the intensity of biofilm formation; however, a 4 to 8-folddecrease in concentration of nutrients in 0.2 ml of cultivation volume in habited the biofilms formation. In 1.0 ml of the culture medium, the biofilm forms evenly, and in 0.2 ml of 4–8-fold dilution of nutrient medium it grows slower. The slow growth rate is statistically significant. The cultivation volume is also of great importance. For example, cultures grown in 0.2 ml of nutrient medium at different concentrations of nutrients formed fewer biofilms than microorganisms cultivated in 1.0 ml. At the same time, when inoculating P. aeruginosa both at log and stationary growth phases, biofilm formation is more pronounced in wells containing more cultivation volume

Роль метилирования промоторов герпесвирусов в реализации патогенного потенциала на примере болезни Марека

Marek’s disease virus is ubiquitous and can harm not only poultry, but also be oncogenic for humans. VBM and malignant tumors induced by them are a convenient and accessible natural model for studying herpesvirus-associated carcinogenesis. To date, according to our observations, there are additional risks of human infection with the Marek’s disease virus - the disease began to appear in broiler chickens 30 days and older, i.e. contact with poultry meat carries a risk of infection. In addition, COVID-19 disease may be accompanied by folic acid deficiency, i.e. a violation of the folate cycle in humans, which increases the risk of manifestation of diseases associated with DNA viruses, since a violation of the folate cycle can reduce the activity of DNA methylation, incl. viral DNA. Methylation is carried out enzymatically in the first minutes after DNA replication, i.e. postreplicatively. Since the DNA nucleotide sequence does not change, methylation is essentially an epigenetic event. We have studied the relationship between the methylation of promoters of the Marek’s disease virus and the copy number of the virus. The assessment of the presence or absence of methylation, as well as partial methylation, was carried out on the basis of identifying the difference between the threshold cycles dC(t). The presence of unmethylated sites included in the studied promoter sequence was detected on the basis of the ability of methylsensitive restrictases AciI and GlaI. A correlation was found between the concentration of genomic DNA of the Marek’s disease virus serotype 1 strain CVI 988 in cell culture and the presence of demethylated CpG islands in the composition of promoters located at position 9413-9865 bp. and 127943 - 128193 b.p. genomic DNA of the virus. The data obtained make it possible to explain the mechanism of the increase in the pathogenicity of herpesvirus infections under conditions of a decrease in the activity of viral DNA methylation in the body.Вирус болезни Марека (ВБМ) имеет повсеместное распространение и может не только наносить вред домашней птице, но и обладать онкогенностью для человека. ВБМ и индуцируемые ими злокачественные опухоли являются удобной и доступной естественной моделью изучения канцерогенеза, ассоциированного с герпесвирусами. На сегодняшний день, по нашим наблюдениям, появились дополнительные риски заражения людей вирусом болезни Марека – болезнь начала появляться у цыплят-бройлеров 30 дней и старше, т.е. контакт с мясом птицы несет риск заражения. Помимо этого, заболевание COVID-19 может сопровождаться дефицитом фолиевой кислоты, т.е. нарушением фолатного цикла у людей, что повышает риск манифестации заболеваний, связанных с ДНК-вирусами, так как нарушение фолатного цикла способно снизить активность метилирования ДНК, в т.ч. вирусной ДНК. Метилирование осуществляется ферментативно в первые минуты после репликации ДНК, т.е. пострепликативно. Поскольку нуклеотидная последовательность ДНК при этом не меняется, метилирование по сути своей – событие эпигенетическое. Нами была изучена зависимость между метилированием промоторов вируса болезни Марека и копийностью вируса. Оценка наличия или отсутствия метилирования, так же как и частичного метилирования, осуществлялась на основе выявления разницы между пороговыми циклами dC(t). Наличие неметилированных сайтов, входящих в состав изучаемой промоторной последовательности, выявлялось на основе способности метилчувствительных рестриктаз AciI и GlaI. Была обнаружена корреляционная зависимость между концентрацией геномной ДНК вируса болезни Марека 1-го серотипа штамма CVI988 в культуре клеток и наличием деметилированных CpG-островков в составе промоторов, локализующихся в положении 9413–9865 и 127943–128193 п.н. геномной ДНК вируса. Полученные данные позволяют объяснить механизм повышения патогенности герпесвирусных инфекций в условиях снижения активности метилирования вирусной ДНК в организме

Adaptive remodeling of the bacterial proteome by specific ribosomal modification regulates Pseudomonas infection and niche colonisation

Post-transcriptional control of protein abundance is a highly important, underexplored regulatory process by which organisms respond to their environments. Here we describe an important and previously unidentified regulatory pathway involving the ribosomal modification protein RimK, its regulator proteins RimA and RimB, and the widespread bacterial second messenger cyclic-di-GMP (cdG). Disruption of rimK affects motility and surface attachment in pathogenic and commensal Pseudomonas species, with rimK deletion significantly compromising rhizosphere colonisation by the commensal soil bacterium P. fluorescens, and plant infection by the pathogens P. syringae and P. aeruginosa. RimK functions as an ATP-dependent glutamyl ligase, adding glutamate residues to the C-terminus of ribosomal protein RpsF and inducing specific effects on both ribosome protein complement and function. Deletion of rimK in P. fluorescens leads to markedly reduced levels of multiple ribosomal proteins, and also of the key translational regulator Hfq. In turn, reduced Hfq levels induce specific downstream proteomic changes, with significant increases in multiple ABC transporters, stress response proteins and non-ribosomal peptide synthetases seen for both ΔrimK and Δhfq mutants. The activity of RimK is itself controlled by interactions with RimA, RimB and cdG. We propose that control of RimK activity represents a novel regulatory mechanism that dynamically influences interactions between bacteria and their hosts; translating environmental pressures into dynamic ribosomal changes, and consequently to an adaptive remodeling of the bacterial proteome

Translational activation of rpoS mRNA by the non-coding RNA DsrA and Hfq does not require ribosome binding

At low temperature, translational activation of rpoS mRNA, encoding the stationary phase sigma-factor, σS, involves the small regulatory RNA (sRNA) DsrA and the RNA chaperone Hfq. The Hfq-mediated DsrA-rpoS interaction relieves an intramolecular secondary structure that impedes ribosome access to the rpoS ribosome binding site. In addition, DsrA/rpoS duplex formation creates an RNase III cleavage site within the duplex. Previous biochemical studies suggested that DsrA and Hfq associate with the 30S ribosomal subunit protein S1, which implied a role for the ribosome in sRNA-mediated post-transcriptional regulation. Here, we show by ribosome profiling that Hfq partitions with the cytoplasmic fraction rather than with 30S subunits. Besides, by employing immunological techniques, no evidence for a physical interaction between Hfq and S1 was obtained. Similarly, in vitro studies did not reveal a direct interaction between DsrA and S1. By employing a ribosome binding deficient rpoS mRNA, and by using the RNase III clevage in the DsrA/rpoS duplex as a diagnostic marker, we provide in vivo evidence that the Hfq-mediated DsrA/rpoS interaction, and consequently the structural changes in rpoS mRNA precede ribosome binding. These data suggest a simple mechanistic model in which translational activation by DsrA provides a translationally competent rpoS mRNA to which 30S subunits can readily bind

A validated microRNA profile with predictive potential in glioblastoma patients treated with bevacizumab

Purpose: We investigated whether microRNA expression data from glioblastoma could be used to produce a profile that defines a bevacizumab responsive group of patients. Patients and Methods: TCGA microRNA expression data from tumors resected at first diagnosis of glioblastoma in patients treated with bevacizumab at any time during the course of their disease were randomly separated into training (n=50) and test (n=37) groups for model generation. MicroRNA-seq data for 51 patients whose treatment included bevacizumab in the BELOB trial were used as an independent validation cohort. Results: Using penalized regression we identified 8 microRNAs as potential predictors of overall survival in the training set. We dichotomized the response score based on the most prognostic minimum of a density plot of the response scores (log-rank HR=0.16, p=1.2e-5) and validated the profile in the test cohort (one-sided log-rank HR=0.34, p=0.026). Analysis of the profile using all samples in the TCGA glioblastoma dataset, regardless of treatment received, (n=473) showed that the prediction of patient benefit was not significant (HR=0.84, p=0.083) suggesting the profile is specific to bevacizumab. Further independent validation of our microRNA profile in RNA-seq data from patients treated with bevacizumab (alone or in combination with CCNU) at glioblastoma recurrence in the BELOB trial confirmed that our microRNA profile predicted patient benefit from bevacizumab (HR=0.59, p=0.043). Conclusion: We have identified and validated an 8-microRNA profile that predicts overall survival in patients with glioblastoma treated with bevacizumab. This may be useful for identifying patients who are likely to benefit from this agent

Crystal structure of Hfq from Bacillus subtilis in complex with SELEX-derived RNA aptamer: insight into RNA-binding properties of bacterial Hfq

Bacterial Hfq is a protein that plays an important role in the regulation of genes in cooperation with sRNAs. Escherichia coli Hfq (EcHfq) has two or more sites that bind RNA(s) including U-rich and/or the poly(A) tail of mRNA. However, functional and structural information about Bacillus subtilis Hfq (BsHfq) including the RNA sequences that specifically bind to it remain unknown. Here, we describe RNA aptamers including fragment (AG)3A that are recognized by BsHfq and crystal structures of the BsHfq–(AG)3A complex at 2.2 Å resolution. Mutational and structural studies revealed that the RNA fragment binds to the distal site, one of the two binding sites on Hfq, and identified amino acid residues that are critical for sequence-specific interactions between BsHfq and (AG)3A. In particular, R32 appears to interact with G bases in (AG)3A. Poly(A) also binds to the distal site of EcHfq, but the overall RNA structure and protein–RNA interaction patterns engaged in the R32 residues of BsHfq–(AG)3A differ from those of EcHfq–poly(A). These findings provide novel insight into how the Hfq homologue recognizes RNA

Recognition and discrimination of target mRNAs by Sib RNAs, a cis-encoded sRNA family

Five Sib antitoxin RNAs, members of a family of cis-encoded small regulatory RNAs (sRNAs) in Escherichia coli, repress their target mRNAs, which encode Ibs toxins. This target repression occurs only between cognate sRNA–mRNA pairs with an exception of ibsA. We performed co-transformation assays to assess the ability of SibC derivatives to repress ibsC expression, thereby revealing the regions of SibC that are essential for ibsC mRNA recognition. SibC has two target recognition domains, TRD1 and TRD2, which function independently. The target site for TRD1 is located within the ORF of ibsC, whereas the target site for TRD2 is located in the translational initiation region. The TRD1 sequence is sufficient to repress ibsC expression. In contrast, TRD2 requires a specific structure in addition to the recognition sequence. An in vitro structural probing analysis showed that the initial interactions at these two recognition sites allowed base-pairing to progress into the flanking sequences. Displacement of the TRD1 and TRD2 domains of SibC by the corresponding domains of SibD changed the target specificity of SibC from ibsC to ibsD, suggesting that these two elements modulate the cognate target recognition of each Sib RNA by discriminating among non-cognate ibs mRNAs