The key features of SARS-CoV-2 leader and NSP1 required for viral escape of NSP1-mediated repression.

SARS-CoV-2, responsible for the ongoing global pandemic, must overcome a conundrum faced by all viruses. To achieve its own replication and spread, it simultaneously depends on and subverts cellular mechanisms. At the early stage of infection, SARS-CoV-2 expresses the viral nonstructural protein 1 (NSP1), which inhibits host translation by blocking the mRNA entry tunnel on the ribosome; this interferes with the binding of cellular mRNAs to the ribosome. Viral mRNAs, on the other hand, overcome this blockade. We show that NSP1 enhances expression of mRNAs containing the SARS-CoV-2 leader. The first stem-loop (SL1) in the viral leader is both necessary and sufficient for this enhancement mechanism. Our analysis pinpoints specific residues within SL1 (three cytosine residues at the positions 15, 19, and 20) and another within NSP1 (R124), which are required for viral evasion, and thus might present promising drug targets. We target SL1 with the antisense oligo (ASO) to efficiently and specifically down-regulate SARS-CoV-2 mRNA. Additionally, we carried out analysis of a functional interactome of NSP1 using BioID and identified components of antiviral defense pathways. Our analysis therefore suggests a mechanism by which NSP1 inhibits the expression of host genes while enhancing that of viral RNA. This analysis helps reconcile conflicting reports in the literature regarding the mechanisms by which the virus avoids NSP1 silencing.

Adaptation of and Protocol for the Validation of the Alcohol Use Disorders Identification Test (AUDIT) in the Russian Federation for Use in Primary Healthcare.

AIMS: To adapt and validate the Alcohol Use Disorders Identification Test (AUDIT) for use in the Russian Federation and countries with Russian-speaking populations by. METHODS: Systematic review of past use and validation of the Russian-language AUDIT. Interviews to be conducted with experts to identify problems encountered in the use of existing Russian-language AUDIT versions. A pilot study using a revised translation of the Russian-language AUDIT that incorporates country-specific drinking patterns in the Russian Federation. RESULTS AND CONCLUSIONS: The systematic review identified over 60 different Russian-language AUDIT versions without systematic validation studies. The main difficulties encountered with the use of the AUDIT in the Russian Federation were related to the lack of:A revised version of the Russian-language AUDIT was created based on the pilot studies, and was validated in primary healthcare facilities in all regions in 2019/2020.

Probabilistic Analysis of Dam Accidents Worldwide: Risk Assessment for Dams of Different Purposes in OECD and Non-OECD Countries with Focus on Time Trend Analysis.

This study presents probabilistic analysis of dam accidents worldwide in the period 1911-2016. The accidents are classified by the dam purpose and by the country cluster, where they occurred, distinguishing between the countries of the Organization for Economic Cooperation and Development (OECD) and nonmember countries (non-OECD without China). A Bayesian hierarchical approach is used to model distributions of frequency and severity for accidents. This approach treats accident data as a multilevel system with subsets sharing specific characteristics. To model accident probabilities for a particular dam characteristic, this approach samples data from the entire data set, borrowing the strength across data set and enabling to model distributions even for subsets with scarce data. The modelled frequencies and severities are combined in frequency-consequence curves, showing that accidents for all dam purposes are more frequent in non-OECD (without China) and their maximum consequences are larger than in OECD countries. Multipurpose dams also have higher frequencies and maximum consequences than single-purpose dams. In addition, the developed methodology explicitly models time dependence to identify trends in accident frequencies over the analyzed period. Downward trends are found for almost all dam purposes confirming that technological development and implementation of safety measures are likely to have a positive impact on dam safety. The results of the analysis provide insights for dam risk management and decision-making processes by identifying key risk factors related to country groups and dam purposes as well as changes over time.

A variant of LEAFY reveals its capacity to stimulate meristem development by inducing RAX1.

In indeterminate inflorescences, floral meristems develop on the flanks of the shoot apical meristem, at positions determined by auxin maxima. The floral identity of these meristems is conferred by a handful of genes called floral meristem identity genes, among which the LEAFY (LFY) transcription factor plays a prominent role. However, the molecular mechanism controlling the early emergence of floral meristems remains unknown. A body of evidence indicates that LFY may contribute to this developmental shift, but a direct effect of LFY on meristem emergence has not been demonstrated. We have generated a LFY allele with reduced floral function and revealed its ability to stimulate axillary meristem growth. This role is barely detectable in the lfy single mutant but becomes obvious in several double mutant backgrounds and plants ectopically expressing LFY. We show that this role requires the ability of LFY to bind DNA, and is mediated by direct induction of REGULATOR OF AXILLARY MERISTEMS1 (RAX1) by LFY. We propose that this function unifies the diverse roles described for LFY in multiple angiosperm species, ranging from monocot inflorescence identity to legume leaf development, and that it probably pre-dates the origin of angiosperms.

Translating and adapting the Alcohol Use Disorders Identification Test (AUDIT) for use in the Russian Federation: A multicentre pilot study to inform validation procedures.

Aims: The Alcohol Use Disorders Identification Test (AUDIT) is one of the most widely used screening instruments worldwide. Although it was translated into many languages, not many country-specific adaptations exist, and a formal validation procedure of the Russian version has been carried out only recently. The present contribution documents the different steps taken to formally translate and adapt a Russian-specific version of the AUDIT (RUS-AUDIT). Methods: The AUDIT was translated into Russian following an established protocol, revised and adapted to the country context using an expert panel, and field-tested in an iterative approach, in line with WHO rules on instrument translation and adaptation A total of three pilot phases were carried out on 134 patients from primary healthcare (PHC) and 33 patients from specialised alcohol treatment facilities (narcology), guided by a specially established advisory board. Changes in each version were informed by the findings of the previous pilot phase and a thorough panel discussion. Results: Based on the findings of three different pilot phases, the RUS-AUDIT was developed as a paper-and-pencil interview for PHC professionals. Since various issues with representation and counting of standard drinks for the second test item arose, a special show card was developed to support the assessment. Preliminary AUDIT-C scores indicated that more than one-third of the screened women (34.2%) and about half of the screened men (50.9%) from PHC facilities have exceeded risk thresholds. Conclusions: The RUS-AUDIT was constructed as a feasible assessment tool for interviewers and patients. The large number of PHC patients who exceed the risk threshold has corroborated the need for formal validation and Russia-specific cut-off scores, considering the specific drinking patterns.

Pilot study to evaluate usability and acceptability of the 'Animated Alcohol Assessment Tool' in Russian primary healthcare.

BACKGROUND AND AIMS: Accurate and user-friendly assessment tools quantifying alcohol consumption are a prerequisite to effective prevention and treatment programmes, including Screening and Brief Intervention. Digital tools offer new potential in this field. We developed the 'Animated Alcohol Assessment Tool' (AAA-Tool), a mobile app providing an interactive version of the World Health Organization's Alcohol Use Disorders Identification Test (AUDIT) that facilitates the description of individual alcohol consumption via culturally informed animation features. This pilot study evaluated the Russia-specific version of the Animated Alcohol Assessment Tool with regard to (1) its usability and acceptability in a primary healthcare setting, (2) the plausibility of its alcohol consumption assessment results and (3) the adequacy of its Russia-specific vessel and beverage selection. METHODS: Convenience samples of 55 patients (47% female) and 15 healthcare practitioners (80% female) in 2 Russian primary healthcare facilities self-administered the Animated Alcohol Assessment Tool and rated their experience on the Mobile Application Rating Scale - User Version. Usage data was automatically collected during app usage, and additional feedback on regional content was elicited in semi-structured interviews. RESULTS: On average, patients completed the Animated Alcohol Assessment Tool in 6:38 min (SD = 2.49, range = 3.00-17.16). User satisfaction was good, with all subscale Mobile Application Rating Scale - User Version scores averaging >3 out of 5 points. A majority of patients (53%) and practitioners (93%) would recommend the tool to 'many people' or 'everyone'. Assessed alcohol consumption was plausible, with a low number (14%) of logically impossible entries. Most patients reported the Animated Alcohol Assessment Tool to reflect all vessels (78%) and all beverages (71%) they typically used. CONCLUSION: High acceptability ratings by patients and healthcare practitioners, acceptable completion time, plausible alcohol usage assessment results and perceived adequacy of region-specific content underline the Animated Alcohol Assessment Tool's potential to provide a novel approach to alcohol assessment in primary healthcare. After its validation, the Animated Alcohol Assessment Tool might contribute to reducing alcohol-related harm by facilitating Screening and Brief Intervention implementation in Russia and beyond.

A novel phytase from Citrobactergillenii: characterization and expression in Pichia pastoris (Komagataella pastoris).

The phyCg gene encoding a new phytase from Citrobacter gillenii was optimized, synthesized, cloned and expressed in Pichia pastoris. Analysis of the amino acid sequence of the enzyme showed that it belongs to the histidine acid phosphatase family. The amino acid sequence of the PhyCg phytase has the highest homology (73.49%) with a phytase sequence from Citrobacter braakii. The main characteristics for the purified recombinant phytase were established. The optimum pH and temperature were 4.5 and 50°C, respectively. The specific activity of the enzyme was 1577 U/mg. The Michaelis constant (Km) and the maximum reaction rate (Vmax) for sodium phytate were 0.185 mM and 2185 U/mg, respectively. The enzyme showed the pH and trypsin stability and had a high activity over a wide pH range.

Protocol and Rationale for the Russian-Japanese "Tackle Obesity and Metabolic Syndrome Outcome by Diet, Activities and Checking Body Weight Intervention" (RJ-TOMODACHI) Randomized Controlled Trial.

Background: The prevalence of obesity in Russia has increased sharply since the mid-1990s. Interestingly, the prevalence of obesity in Japan is lower than in many Western countries. Japan has implemented different types of weight control programs using a smart device to monitor patients remotely. New health promotion methods from Japan are now being used in Russia. The Russian-Japanese "Tackle Obesity and Metabolic Syndrome Outcome by Diet, Activities and Checking Body Weight Intervention" (RJ-TOMODACHI) study aims to evaluate a preventive intervention using Japanese health monitoring technology in reducing excess body weight, compared with standard care, in Russia. Methods and Results: The trial is a single-center, 3-armed, parallel group randomized controlled trial conducted among overweight/obese adults. It has been designed to compare the effectiveness of 2 newly developed interventions against standard care for 6 months. Participants in the low- and high-intensity intervention groups will have 3 and 6 consultations over the study period, respectively. In all, 260 adults were screened at baseline; 65 did not participate in the trial for various reasons. The remaining 195 people were randomized into 3 groups (high-intensity intervention, n=73, low-intensity, n=73; standard care group, n=49). Conclusions: The trial protocol has been designed so that the methodology can be adapted for use in Russia.

Cul4A and DDB1 associate with Skp2 to target p27Kip1 for proteolysis involving the COP9 signalosome.

DDB1, a subunit of the damaged-DNA binding protein DDB, has been shown to function also as an adaptor for Cul4A, a member of the cullin family of E3 ubiquitin ligase. The Cul4A-DDB1 complex remains associated with the COP9 signalosome, and that interaction is conserved from fission yeast to human. Studies with fission yeast suggested a role of the Pcu4-Ddb1-signalosome complex in the proteolysis of the replication inhibitor Spd1. Here we provide evidence that the function of replication inhibitor proteolysis is conserved in the mammalian DDB1-Cul4A-signalosome complex. We show that small interfering RNA-mediated knockdown of DDB1, CSN1 (a subunit of the signalosome), and Cul4A in mammalian cells causes an accumulation of p27Kip1. Moreover, expression of DDB1 reduces the level of p27Kip1 by increasing its decay rate. The DDB1-induced proteolysis of p27Kip1 requires signalosome and Cul4A, because DDB1 failed to increase the decay rate of p27Kip1 in cells deficient in CSN1 or Cul4A. Surprisingly, the DDB1-induced proteolysis of p27Kip1 also involves Skp2, an F-box protein that allows targeting of p27Kip1 for ubiquitination by the Skp1-Cul1-F-box complex. Moreover, we provide evidence for a physical association between Cul4A, DDB1, and Skp2. We speculate that the F-box protein Skp2, in addition to utilizing Cul1-Skp1, utilizes Cul4A-DDB1 to induce proteolysis of p27Kip1.

Probabilistic modeling of the flows and environmental risks of nano-silica.

Nano-silica, the engineered nanomaterial with one of the largest production volumes, has a wide range of applications in consumer products and industry. This study aimed to quantify the exposure of nano-silica to the environment and to assess its risk to surface waters. Concentrations were calculated for four environmental (air, soil, surface water, sediments) and two technical compartments (wastewater, solid waste) for the EU and Switzerland using probabilistic material flow modeling. The corresponding median concentration in surface water is predicted to be 0.12 µg/l in the EU (0.053-3.3 µg/l, 15/85% quantiles). The concentrations in sediments in the complete sedimentation scenario were found to be the largest among all environmental compartments, with a median annual increase of 0.43 mg/kg · y in the EU (0.19-12 mg/kg · y, 15/85% quantiles). Moreover, probabilistic species sensitivity distributions (PSSD) were computed and the risk of nano-silica in surface waters was quantified by comparing the predicted environmental concentration (PEC) with the predicted no-effect concentration (PNEC) distribution, which was derived from the cumulative PSSD. This assessment suggests that nano-silica currently poses no risk to aquatic organisms in surface waters. Further investigations are needed to assess the risk of nano-silica in other environmental compartments, which is currently not possible due to a lack of ecotoxicological data.

Economic Burden of Cardiovascular Diseases in the Russian Federation.

OBJECTIVES: In the Russian Federation, cardiovascular disease (CVD) is the primary cause of death and premature death; however, to date, there have been no systematic cost-of-illness studies to assess the economic impact of CVD. METHODS: The economic burden of CVD was estimated from statistic data on morbidity, mortality, and health care resource use. Health care costs were estimated on the basis of expenditure on primary, outpatient, emergency, and inpatient care, as well as medications. Non-health care costs included economic losses due to morbidity and premature death in the working age. RESULTS: CVD was estimated to cost Russia RUR 836.1 billion (€24,517.8 million) in 2006 and RUR 1076 billion (€24,400.4 million) in 2009. Of the total costs of CVD, 14.5% in 2006 and 21.3% in 2009 were due to health care, with 85.5% and 78.7%, respectively, due to non-health care costs. CONCLUSIONS: CVD is a leading public health problem. We first assessed the economic burden of CVD in Russia. Our results can be used for planning investments in prevention programs and measures for improving care for patients with CVD. Regular monitoring of the economic burden of CVD in the future at the federal, regional, and municipal levels will allow assessment of the dynamics of economic burden, as well as the effectiveness of investments in the economy in primary and secondary prevention. Because data are relatively unavailable, there are important limitations to this study, which highlight the need for more accurate CVD-specific information.

Destabilization of Rb by human papillomavirus E7 is cell cycle dependent: E2-25K is involved in the proteolysis.

The HPV oncoprotein E7 promotes proteasomal degradation of the tumor suppressor protein Rb. In this study, we analyzed the regulation of E7-induced Rb proteolysis in HPV-containing Caski cervical cancer cells. We show that the Rb proteolysis is cell cycle dependent; in S phase Rb is stable while in post-mitotic early G1 phase cells and in differentiated cells, Rb is unstable. Similarly, the in vivo Rb/E7 interaction is not detected in S-phase cells, but is readily detected in differentiating Caski cells. The ubiquitinating enzymes involved in Rb proteolysis have not been identified. We find that the E3 ligase MDM2 is not involved in the Rb proteolysis in Caski cells. An in vivo analysis using multiple catalytic site mutant dominant negative E2 enzymes show that the C92A E2-25K most effectively blocks E7-induced Rb proteolysis. Taken together, these results show that E7 induces Rb proteolysis in growth-arrested cells and E2-25K is involved in the proteolysis.

Micropropagation of ornamental Prunus spp. and GF305 peach, a Prunus viral indicator.

A micropropagation approach was developed for nine ornamental Prunus species, P. americana, P. cistena, P. glandulosa, P. serrulata 'Kwanzan', P. laurocerasus, P. sargentii, P. tomentosa, P. triloba, P. virginiana 'Schubert', commercially important in North America, and GF305 peach, commonly used for Prunus virus indexing. The micropropagation cycle based on proliferation of vegetative tissues includes establishment of tissue culture through introduction of shoot meristems in vitro, shoot proliferation, root induction and plant acclimatization steps and can be completed in 5 months. A meristem sterilization protocol minimized bacterial and fungal contamination. Multiple shoot formation in ornamental Prunus was obtained through the use of 1 mg l(-1) 6-benzyladenine. For GF305 peach, alteration in the sugar composition, fructose instead of sucrose, and addition of 1 mg l(-1 )ferulic acid had a significant impact on the shoot proliferation rate and maintenance of long-term in vitro culture. Rooting and plant acclimatization conditions were improved using a two-step protocol with a 4-day root induction in indole-3-butiric acid (IBA)-containing media with consequent 3-week root elongation in IBA-free media. One-month incubation of rooted shoots in a vermiculite-based medium resulted in additional shoot and root growth and provided better acclimatization and plant recovery. The micropropagation approach can be used for maintenance of the clonal properties for Prunus spp. as well as a protocol to support meristem therapy against viral infection.

Deregulation of eIF4E: 4E-BP1 in differentiated human papillomavirus-containing cells leads to high levels of expression of the E7 oncoprotein.

Infections with high-risk human papillomaviruses (HPVs) are linked to more than 95% of cervical cancers. HPVs replicate exclusively in differentiated cells and the function of the HPV E7 oncoprotein is essential for viral replication. In this study, we investigated the mechanism that regulates E7 expression in differentiated cells. The level of E7 protein was strongly induced in HPV-containing Caski, HOK-16B, and BaP-T cells during growth in methylcellulose-containing medium, a condition that induces differentiation. Enhanced expression of E7 was observed between 4 and 8 h of culturing in methylcellulose and was maintained for up to 24 h. The increase was not due to altered stability of the E7 protein or an increase in the steady-state level of the E7 mRNA. Instead, the translation of the E7 mRNA was enhanced during differentiation. More than 70 to 80% of the E7 mRNA was found in the polysome fractions in the differentiated cells. Consistent with this observation, higher levels of the phosphorylated translator inhibitor 4E-BP1 were observed in differentiated HPV-containing cells but not in differentiated non-HPV tumor cells or primary keratinocytes. The mTOR kinase inhibitor rapamycin blocked phosphorylation of 4E-BP1 and significantly decreased the level of E7 protein in Caski cells, suggesting that phosphorylation of 4E-BP1 is linked to E7 expression. Prevailing models for the molecular mechanisms underlying E7 expression have focused largely on transcriptional regulation. The results presented in this study demonstrate a significant role of the cellular translation machinery to maintain a high level of E7 protein in differentiated cells.

Axillary meristem development in the branchless Zu-0 ecotype of Arabidopsis thaliana.

Axillary meristems form in the leaf axils during post-embryonic development. In order to initiate the genetic dissection of axillary meristem development, we have characterized the late-flowering branchless ecotype of Arabidopsis thaliana (L.) Heynh., Zu-0. The first-formed rosette leaves of Zu-0 plants all initiate axillary meristems, but later-formed leaves of the rosette remain branchless. Alteration in the meristem development is axillary meristem-specific because the shoot apical and floral meristems develop normally. Scanning electron microscopy, histology and RNA in situ analysis with SHOOTMERISTEMLESS ( STM), a marker for meristematic tissues, show that a mound of cells form and STM mRNA accumulates in barren leaf axils, indicating that axillary meristems initiate but arrest in their development prior to organizing a meristem proper. Expression and retention of the STM RNA in barren leaf axils further suggests that STM expression is not sufficient for the establishment of the axillary meristem proper.

The papillomavirus E7 oncoprotein is ubiquitinated by UbcH7 and Cullin 1- and Skp2-containing E3 ligase.

Recurrent infections with high-risk human papillomaviruses (HPVs) are associated with human cervical cancers. All HPV-associated cancer tissues express the viral oncoproteins E6 and E7, which stimulate cell growth. The expression of E7 is crucial for both the initiation and the maintenance of HPV-associated cancer. Recent studies showed that the level of E7 in cancer cells is regulated by ubiquitin-dependent proteolysis through the 26S proteasome. In this study, we characterized the enzymes involved in the ubiquitin-dependent proteolysis of E7. We show that UbcH7, an E2 ubiquitin-conjugating enzyme, is specifically involved in the ubiquitination of E7. Furthermore, we show that E7 interacts with the SCF (Skp-Cullin-F box) ubiquitin ligase complex containing Cullin 1 (Cul1) and Skp2 and can be ubiquitinated by the Cul1-containing ubiquitin ligase in vitro. Coimmunoprecipitation analyses revealed that E7 interacts with Skp2 and Cul1 in vivo. Finally, the half-life of E7 was found to be significantly longer in Skp2(-/-) mouse embryo fibroblasts (MEFs) than in wild-type MEFs. Taken together, these results suggest that the Cul1- and Skp2-containing ubiquitin ligase plays a role in the ubiquitination and proteolysis of E7. In HPV type 16-containing cervical carcinoma cell line Caski, E7 localizes to both the cytoplasm and the nucleus. Brief treatment of Caski cells with MG132 (a proteasome inhibitor) causes the accumulation of E7 in discrete nuclear bodies. These nuclear bodies are detergent insoluble and contain polyubiquitinated E7. We suggest that E7 relocates to specific nuclear bodies for proteolysis in HPV-containing epithelial cells.

COPD: can prevention be improved? Proposal for an integrated intervention strategy.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a worldwide public health problem with increasing morbidity and mortality. The aim of this proposal is to contribute to the improvement of COPD prevention by identifying groups at risk for COPD and targeting them for preventive interventions. METHODS: Based on the existing organizational structures for COPD detection, detailed analysis of the determinants of COPD will allow to identify groups at high risk to develop COPD. The Stepwise Target Group-Oriented Prevention (STOP) model developed during this study proposes an integrated identification and intervention strategy for high-risk groups. RESULTS: Apart from smoking, other environmental determinants and host factors contribute to further lung function's rapid decline. Combined with smoking, these factors increase the risk for COPD. Target groups for early disease detection and appropriate interventions can be identified by the presence of one or more known risk factors and by identification of high-risk groups. CONCLUSION: The Stepwise Target Group-Oriented Prevention (STOP) strategy is a step toward improvement in COPD prevention, by shifting the focus from the group of a focus symptomatic smokers aged 45+ years to much earlier and preventable stages of the disease, that is, from disease treatment to risk management.