Visualization of SARS-CoV-2 particles in naso/oropharyngeal swabs by thin section electron microscopy

Background SARS-CoV-2 replicates efficiently in the upper airways of humans and produces high loads of virus RNA and, at least in the initial phase after infection, many infectious virus particles. Studying virus ultrastructure, such as particle integrity or presence of spike proteins, and effects on their host cells in patient samples is important to understand the pathogenicity of SARS-CoV-2. Methods Suspensions from swab samples with a high load of virus RNA (Ct < 20) were sedimented by desktop ultracentrifugation and prepared for thin section electron microscopy using a novel method which is described in detail. Embedding was performed in Epon or in LR White resin using standard or rapid protocols. Thin sections were examined using transmission electron microscopy. Results Virus particles could be regularly detected in the extracellular space, embedded in a background of heterogenous material (e.g. vesicles and needle-like crystals), and within ciliated cells. Morphology (i.e. shape, size, spike density) of virus particles in the swab samples was very similar to particle morphology in cell culture. However, in some of the samples the virus particles hardly revealed spikes. Infected ciliated cells occasionally showed replication organelles, such as double-membrane vesicles. The most common cells in all samples were keratinocytes from the mucosa and bacteria. Conclusions The new method allows the ultrastructural visualization and analysis of coronavirus particles and of infected host cells from easy to collect naso/oropharyngeal patient swab samples.Peer Reviewe

Morphometry of SARS-CoV and SARS-CoV-2 particles in ultrathin plastic sections of infected Vero cell cultures

SARS-CoV-2 is the causative of the COVID-19 disease, which has spread pandemically around the globe within a few months. It is therefore necessary to collect fundamental information about the disease, its epidemiology and treatment, as well as about the virus itself. While the virus has been identified rapidly, detailed ultrastructural analysis of virus cell biology and architecture is still in its infancy. We therefore studied the virus morphology and morphometry of SARS-CoV-2 in comparison to SARS-CoV as it appears in Vero cell cultures by using conventional thin section electron microscopy and electron tomography. Both virus isolates, SARS-CoV Frankfurt 1 and SARS-CoV-2 Italy-INMI1, were virtually identical at the ultrastructural level and revealed a very similar particle size distribution with a median of about 100 nm without spikes. Maximal spike length of both viruses was 23 nm. The number of spikes per virus particle was about 30% higher in the SARS-CoV than in the SARS-CoV-2 isolate. This result complements a previous qualitative finding, which was related to a lower productivity of SARS-CoV-2 in cell culture in comparison to SARS-CoV.Peer Reviewe

Tax Burden of Russian Oil Companies after Tax Consolidation

This article takes studying the impact of tax consolidation on oil companies' tax burden as its focus. Oil companies are crucial for the Russian economy while oil and gas money are as well important for the Russian budget. Besides, a lot of oil companies being major taxpayers have had the opportunity to consolidate corporate profit tax since 2012. The article's goal is to analyze the results of creating consolidated groups of taxpayers for tax burden in terms of corporate profit tax exemplified by oil companies in order to assess the importance of profit tax consolidation as a way of reducing corporate profit tax. Upon analyzing the data provided by the Federal Tax Service of Russia and 2010-2015 oil companies' financial reports, it is possible to conclude that the largest tax burden falls on fossil fuel industry in comparison with other industries what can be explained first of all by the mineral extraction tax. Corporate profit tax burden for most oil companies in 2014 accounted for 5% of revenue. At the same time, profit tax burdens on consolidated groups of taxpayers producing oil vary a lot; in 2012, the general trend was falling, but then it started to grow again. Thus, it is impossible to state that tax obligations and corporate profit tax burden have significantly decreased as a result of creating consolidated groups of taxpayers for oil producers. Quantitative analysis of money spent for paying the profit tax might be interestingfor describing consequences of creating consolidated groups of taxpayers

Lack of Effects of Extended Sessions of Transcranial Direct Current Stimulation (tDCS) Over Dorsolateral Prefrontal Cortex on Craving and Relapses in Crack-Cocaine Users

Background: Non-invasive brain stimulation such as transcranial direct current stimulation (tDCS) has been investigated as additional therapeutic tool for drug use disorder. In a previous study, we showed that five sessions of tDCS applied bilaterally over the dorsolateral prefrontal cortex (dlPFC) reduced craving to the use of crack-cocaine in inpatients from a specialized clinic. In the present study, we examine if an extended number of sessions of the same intervention would reduce craving even further and affect also relapses to crack-cocaine use.Methods: A randomized, double-blind, sham-controlled, clinical trial with parallel arms was conducted (https://clinicaltrials.gov/ct2/show/NCT02091167). Crack-cocaine patients from two private and one public clinics for treatment of drug use disorder were randomly allocated to two groups: real tDCS (5 cm × 7 cm, 2 mA, for 20 min, cathodal over the left dlPFC and anodal over the right dlPFC, n = 19) and sham-tDCS (n = 16). Real or sham-tDCS was applied once a day, every other day, in a total of 10 sessions. Craving was monitored by a 5-item obsessive compulsive drinking scale once a week (one time before, three times during and once after brain stimulation) over about 5 weeks and relapse was monitored after their discharge from clinics for up to 60 days.Results: Craving scores progressively decreased over five measurements in both sham- and real tDCS groups. Corrected Hedges’ within-group (initial and final) effect sizes of craving scores were of 0.77 for the sham-tDCS and of 0.97 for the real tDCS group. The between-groups effect size was of 0.34, in favor of the real tDCS group over sham-tDCS group. Relapse rates were high and quite similar between groups in the 30- and 60-days follow-up after discharge from the hospital.Conclusion: Extended repetitive bilateral tDCS over the dlPFC had no add-on effects over regular treatment when considering craving and relapses to the crack-cocaine use in a sample of crack-cocaine patients with severe use disorder. Different tDCS montages targeting other cortical regions and perhaps additional extension of sessions need to be investigated to reach more efficiency in managing craving and relapses to crack-cocaine use

SARS-CoV-2 Omicron variant is attenuated for replication in a polarized human lung epithelial cell model

SARS-CoV-2 and its emerging variants of concern remain a major threat for global health. Here we introduce an infection model based upon polarized human Alveolar Epithelial Lentivirus immortalized (hAELVi) cells grown at the air–liquid interface to estimate replication and epidemic potential of respiratory viruses in the human lower respiratory tract. hAELVI cultures are highly permissive for different human coronaviruses and seasonal influenza A virus and upregulate various mediators following virus infection. Our analysis revealed a significantly reduced capacity of SARS-CoV-2 Omicron BA.1 and BA.2 variants to propagate in this human model compared to earlier D614G and Delta variants, which extends early risk assessments from epidemiological and animal studies suggesting a reduced pathogenicity of Omicron.Peer Reviewe

Impact of age on tDCS effects on pain threshold and working memory : results of a proof of concept cross-over randomized controlled study

Background: Age is an important factor that impacts the variability of tDCS effects. Objective/Hypothesis: To compare effects of anodal (a)-tDCS over the left dorsolateral prefrontal cortex (DLPFC), and primary motor cortex (M1) in adolescents, adults, and elderly on heat pain threshold (HPT; primary outcome) and the working memory (WM; secondary outcome). We hypothesized that the effect of tDCS on HPT and WM performance would be the largest in adolescents because their pre-frontal cortex is more prone to neuroplasticity. Methods: We included 30 healthy women within the age ranges of 15–16 (adolescents, n = 10), 30–40 (adults, n = 10), and 60–70 (elderly, n = 10) years. In this crossover single-blinded study, participants received three interventions applied over the DLPF and M1. The active stimulation intensity was two mA for 30 min. From 20 min of stimulation onset, the tDCS session was coupled with an online n-back task. The a-tDCS and sham were applied in a random sequence, with a washout time of a minimum 7 days between each trial. HPT was evaluated before and after stimulation. The WM performance with an n-back task was assessed after the tDCS session. Results: A Generalized Estimating Equation (GEE) model revealed a significant effect of the a-tDCS over the left DLPFC to reduce the HPT in adolescents compared with sham. It increased the pain perception significantly [a large effect size (ES) of 1.09)]. In the adults, a-tDCS over M1 enhanced the HPT significantly (a large ES of 1.25) compared to sham. No significant effect for HPT was found in the elderly. Response time for hits was reduced for a-tDCS over the DLPFC in adolescents, as compared to the other two age groups. Conclusions: These findings suggest that a-tDCS modulates pain perception and WM differentially according to age and target area of stimulation. In adolescents, anodal stimulation over the DLPFC increased the pain perception, while in adults, the stimulation over the M1 increased the pain threshold. Thus, they elucidate the impact of tDCS for different age groups and can help to define what is the appropriate intervention according to age in further clinical trials

Development of a Genus-Specific Antigen Capture ELISA for Orthopoxviruses – Target Selection and Optimized Screening

Orthopoxvirus species like cowpox, vaccinia and monkeypox virus cause zoonotic infections in humans worldwide. Infections often occur in rural areas lacking proper diagnostic infrastructure as exemplified by monkeypox, which is endemic in Western and Central Africa. While PCR detection requires demanding equipment and is restricted to genome detection, the evidence of virus particles can complement or replace PCR. Therefore, an easily distributable and manageable antigen capture enzyme-linked immunosorbent assay (ELISA) for the detection of orthopoxviruses was developed to facilitate particle detection. By comparing the virus particle binding properties of polyclonal antibodies developed against surface-exposed attachment or fusion proteins, the surface protein A27 was found to be a well-bound, highly immunogenic and exposed target for antibodies aiming at virus particle detection. Subsequently, eight monoclonal anti-A27 antibodies were generated and characterized by peptide epitope mapping and surface plasmon resonance measurements. All antibodies were found to bind with high affinity to two epitopes at the heparin binding site of A27, toward either the N- or C-terminal of the crucial KKEP-segment of A27. Two antibodies recognizing different epitopes were implemented in an antigen capture ELISA. Validation showed robust detection of virus particles from 11 different orthopoxvirus isolates pathogenic to humans, with the exception of MVA, which is apathogenic to humans. Most orthopoxviruses could be detected reliably for viral loads above 1 × 103 PFU/mL. To our knowledge, this is the first solely monoclonal and therefore reproducible antibody-based antigen capture ELISA able to detect all human pathogenic orthopoxviruses including monkeypox virus, except variola virus which was not included. Therefore, the newly developed antibody-based assay represents important progress towards feasible particle detection of this important genus of viruses

Discrimination of SARS-CoV-2 Infections From Other Viral Respiratory Infections by Scent Detection Dogs

Background: Testing of possibly infected individuals remains cornerstone of containing the spread of SARS-CoV-2. Detection dogs could contribute to mass screening. Previous research demonstrated canines' ability to detect SARS-CoV-2-infections but has not investigated if dogs can differentiate between COVID-19 and other virus infections. Methods: Twelve dogs were trained to detect SARS-CoV-2 positive samples. Three test scenarios were performed to evaluate their ability to discriminate SARS-CoV-2-infections from viral infections of a different aetiology. Naso- and oropharyngeal swab samples from individuals and samples from cell culture both infected with one of 15 viruses that may cause COVID-19-like symptoms were presented as distractors in a randomised, double-blind study. Dogs were either trained with SARS-CoV-2 positive saliva samples (test scenario I and II) or with supernatant from cell cultures (test scenario III). Results: When using swab samples from individuals infected with viruses other than SARS-CoV-2 as distractors (test scenario I), dogs detected swab samples from SARS-CoV-2-infected individuals with a mean diagnostic sensitivity of 73.8% (95% CI: 66.0–81.7%) and a specificity of 95.1% (95% CI: 92.6–97.7%). In test scenario II and III cell culture supernatant from cells infected with SARS-CoV-2, cells infected with other coronaviruses and non-infected cells were presented. Dogs achieved mean diagnostic sensitivities of 61.2% (95% CI: 50.7–71.6%, test scenario II) and 75.8% (95% CI: 53.0–98.5%, test scenario III), respectively. The diagnostic specificities were 90.9% (95% CI: 87.3–94.6%, test scenario II) and 90.2% (95% CI: 81.1–99.4%, test scenario III), respectively. Conclusion: In all three test scenarios the mean specificities were above 90% which indicates that dogs can distinguish SARS-CoV-2-infections from other viral infections. However, compared to earlier studies our scent dogs achieved lower diagnostic sensitivities. To deploy COVID-19 detection dogs as a reliable screening method it is therefore mandatory to include a variety of samples from different viral respiratory tract infections in dog training to ensure a successful discrimination process.Peer Reviewe

Голодомор 1932 –– 1933 рр. в Україні як геноцид

Given its fundamental role in development and cancer, the Wnt-beta-catenin signaling pathway is tightly controlled at multiple levels. RING finger protein 43 (RNF43) is an E3 ubiquitin ligase originally found in stem cells and proposed to inhibit Wnt signaling by interacting with the Wnt receptors of the Frizzled family. We detected endogenous RNF43 in the nucleus of human intestinal crypt and colon cancer cells. We found that RNF43 physically interacted with T cell factor 4 (TCF4) in cells and tethered TCF4 to the nuclear membrane, thus silencing TCF4 transcriptional activity even in the presence of constitutively active mutants of beta-catenin. This inhibitory mechanism was disrupted by the expression of RNF43 bearing mutations found in human gastrointestinal tumors, and transactivation of the Wnt pathway was observed in various cells and in Xenopus embryos when the RING domain of RNF43 was mutated. Our findings indicate that RNF43 inhibits the Wnt pathway downstream of oncogenic mutations that activate the pathway. Mimicking or enhancing this inhibitory activity of RNF43 may be useful to treat cancers arising from aberrant activation of the Wnt pathwa

Analysis of the African coelacanth genome sheds light on tetrapod evolution

It was a zoological sensation when a living specimen of the coelacanth was first discovered in 1938, as this lineage of lobe-finned fish was thought to have gone extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features . Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain, and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues demonstrate the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution