Роль физической культуры и спорта в жизни воцерковленного человека

В данной статье автор рассматривает отношение Православной Церкви к физической культуре и спорту, значение физической культуры и спорта в духовном воспитании человека; анализирует взаимодействие физического и духовного в жизни верующего человека.У данiй статтi автор розглядає вiдношення Православної Церкви до фiзичної культури i спорту, значення фiзичної культури i спорту в духовному вихованнi людини; аналізує взаємодію фiзичного i духовного в життi вiруючої людини.In this article an author examines attitude of the Orthodox Church toward a physical culture and sport. A value of physical culture and sport is in spiritual education of man. Interaction of physical and spiritual in life believing man

Virus-like particles (VLPs) as a platform for the development of yellow fever and Zika virus vaccine candidate

Flaviviruses are arboviruses that have been posing serious challenges to global health since 2015. Zika virus (ZIKV) emerged in 2015 in Brazil and quickly spread to over 60 countries in Africa, Asia and the Americas, causing Guillain-Barré syndrome in adults and serious congenital malformations in fetuses of infected mothers. Besides mosquito-borne transmission, zika virus can persist for months in sexual fluids and thus poses risk also to non-endemic countries due to sexual transmission of returning travelers. Although the number of cases decreased significantly due to herd immunity in affected countries, development of a vaccine for ZIKV is of great importance to avoid future resurgence of the virus in endemic areas or future spread to currently non-endemic regions. Yellow fever (YF) is a “historically devastating disease” (Paules and Fauci, 2017), which in past centuries killed approximately 10% of the population of cities like Philadelphia and Barcelona. Although a very effective vaccine exists for YFV, it can cause fatal adverse effects in a small proportion of vaccinees, and recent outbreaks have shown that due to its limited production in embryonated eggs the risk of serious vaccine shortages is high. Fractionating the vaccine dosis (1/5) was the emergency solution introduced by the WHO in 2016 to stop an outbreak in Africa, and is currently being adopted for mass vaccination in Brazil to try to stop the serious outbreak ongoing since 2017. The potential risk of YFV spreading to highly populated areas with no vaccination coverage, where the mosquito vector is present, such as Asia, makes urgent the development of new YFV vaccines. In this context, virus-like particles (VLPs) can be a promising platform for developing safe and effective vaccines for YFV, ZIKV and other flaviviruses. In this work, we developed stable recombinant cell lines constitutively expressing the structural prM (pre-membrane) and E (envelope) proteins of ZIKV and YFV. Sucrose cushion ultracentrifugation and TEM images have confirmed that VLPs resembling in both size and shape the respective native viruses are formed. In order to optimize expression, cell transfection protocol was optimized using different transfection reagents, media and host cell lines, including CHO, HEK293, BHK, MDCK and Vero cells. Stable cell lines derived from CHO-K1 and HEK293-3F6 gave the most promising results and were followed for up to 20 weeks post-transfection in the presence and absence of the selection marker, showing that cells grow to high densities with high viabilities and keeping the expression of VLPs. The use of FACS to sort for high producer cells allowed obtaining enriched cell pools producing significantly higher amounts of VLPs and confirmed the hypothesis that secreted VLPs can be transiently detected on the cell membrane surface. Kinetic studies to evaluate different culture media and cultivation conditions under batch, pseudoperfusion and perfusion mode were carried out with the final aim of increasing productivity and reducing production costs. Ongoing studies are focusing, on one hand, to purify the ZIKV and YFV VLPs for immunogenicity studies and, on the other hand, to express VLPs of other flaviviruses circulating in the Americas, as preparedness measure for future threats. VLPs of DENV 1, 2, 3 and 4, as well as of Saint Louis encephalitis (SLEV), Ilheus (ILHV) and Cacipacore (CPCV) viruses have already been successfully expressed by transient transfection. Acknowledgements: The authors wish to gratefully acknowledge the Vaccine Research Center of NIAID/NIH (USA) for transferring the DENV, SLEV, ILHV and CPCV gene constructs developed during a sabbatical work of L. R. Castilho and used in this work. References: Paules CI, Fauci AS (2017), Yellow Fever - Once Again on the Radar Screen in the Americas, N Engl J Med 376:1397-1399, doi: 10.1056/NEJMp1702172

Production of Zika virus-like particles (VLPs) by perfusion processes

Zika virus (ZIKV) emerged as a major international public health concern in 2015 and rapidly spread to more than 80 countries in Africa, Asia and the Americas. ZIKV infection has been shown to cause Guillain-Barré syndrome in adults, as well as severe congenital malformations in fetuses from as much as 42% of infected mothers (Brasil et al., 2016, doi:10.1056/NEJMoa1602412). While no ZIKV vaccine becomes approved for human use, periodic outbreaks will continue to occur in endemic regions and the risk of spreading to non-endemic regions will continue to exist, especially because ZIKV persists in body fluids for very long time after infection and can be transmitted via the sexual route. Among many different vaccine platforms currently under study, virus-like particles (VLPs) are a promising alternative for the development of vaccines, since three-dimensional structures, constituted by recombinant structural proteins of the virus but lacking the viral genome, are able to display the antigen in a repetitive pattern, triggering a robust immune response. In this work, we investigated the production of Zika virus-like particles by both intermittent and continuous perfusion processes, using a recombinant HEK293 cell pool previously generated in our laboratory, which constitutively expresses the VLPs. In order to improve production levels, we first enriched the recombinant cell pool for high producers by means of fluorescence-activated cell sorting (FACS). Using this FACS-enriched cell pool, small-scale shake flask studies showed that intermittent perfusion (also known as pseudoperfusion) with daily medium exchange enhanced viable cell density by 3.5 fold and VLP titer by 4 fold when compared to batch cultures. Continuous perfusion in a controlled stirred-tank bioreactor was carried out using an ATF-2 unit as cell retention device. A steady-state viable cell concentration of 25-30 × 106 cells/mL was maintained at a cell-specific perfusion rate (CSPR) of 50-60 pL/cell/day. VLP titers inside the bioreactor were higher than in the harvest, evidencing product retention by the ATF hollow fiber, especially from day 14 of cultivation on. Our results show that the use of cell lines constitutively expressing zika VLPs, cultured in stirred-tank perfusion bioreactors, represents a promising system for the production of a VLP-based Zika vaccine candidate. This process could potentially be more cost-effective than traditional viral vaccine platforms based on batch production of whole viruses, especially considering that VLPs can be produced in lower biosafety level plants, and that perfusion systems are characterized by higher volumetric productivities, reduced bioreactor sizes, smaller plant footprint and lower investment costs when compared to batch processes

Perfusion process for the production of a new, VLP-based yellow fever vaccine candidate

Yellow fever (YF) is an acute viral hemorrhagic disease endemic in tropical areas of Africa, Central and South America, which is transmitted by the bite of infected mosquitoes. It is a “historically devastating disease” (Paules and Fauci, 2017) that killed during outbreaks in past centuries, before the introduction of the current vaccine, approximately 10% of the population of cities like Philadelphia (USA) and Barcelona (Spain). According to Garske et al. (2014), YF caused in 2013 78,000 deaths worldwide, which is a disease burden comparable to influenza. In the past few years, outbreaks in Angola (2016) and in Brazil (2017-2018) led to the depletion of the WHO vaccine stockpile and to the introduction of the emergency use of a fractional dose (1/5). Furthermore, the Angola outbreak in 2016 caused the first cases of YF ever to occur in Asia (11 imported cases to China), rising the concern about approximately 2 billion immunologically naïve people who would be at high risk in Asia in case local transmission of the virus starts to occur (Wilder-Smith et al., 2019). The urgent need for a new YF vaccine becomes evident from two major issues concerning the current vaccine, which consists of a live-attenuated virus propagated in chicken embryos: (i) vaccine shortage due to limitations in the manufacturing technology; (ii) rare, but fatal adverse effects. Therefore, this work focuses on the development of a safe, non-replicating YF vaccine, produced by a high-productivity perfusion process. Stable recombinant HEK293 cell lines constitutively expressing the structural proteins prM (pre-membrane) and E (envelope) of YFV were generated, enabling long-term production and secretion of recombinant virus-like particles (VLPs). FACS (fluorescence activated cell sorting) was used to sort the transfected population for high producer cells and allowed obtaining an enriched cell pool producing significantly higher amounts of VLPs. Small scale kinetic studies under intermittent perfusion (pseudoperfusion) were performed in order to investigate possible feeding strategies and to evaluate the use of short-chain fatty acids as productivity enhancers. Subsequently, perfusion runs were carried out in stirred-tank bioreactors in order to investigate optimal conditions for VLP production, as well as to evaluate different cell retention devices (e.g. inclined lamella settler and ATF-2). Partial retention of the VLPs in the perfusion bioreactor system occurred when the ATF-2 was used. VLPs produced by perfusion were purified by a two-step chromatographic process, and transmission electron microscopy (TEM) images confirmed the expected size and morphology of the VLPs, enabling their use in mouse immunogenicity studies. References: Garske T, Van Kerkhove MD, Yactayo S, Ronveaux O, Lewis RF, Staples JE, Perea W, Ferguson NM, Yellow Fever Expert Committee (2014). Yellow fever in Africa: estimating the burden of disease and impact of mass vaccination from outbreak and serological data. PLoS Medicine 11:e1001638. Paules CI, Fauci AS (2017), Yellow fever - once again on the radar screen in the Americas, N Engl J Med 376: 1397-1399. Wilder-Smith A, Lee V, Gubler DJ (2019), Yellow fever: is Asia prepared for an epidemic? The Lancet 19:241-242

Dual sites for SEC11 on the SNARE SYP121 implicate a binding exchange during secretory traffic

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins facilitate vesicle traffic through their assembly in a heteromeric complex that drives membrane fusion. Much of vesicle traffic at the Arabidopsis (Arabidopsis thaliana) plasma membrane is subject to the Sec1/Munc18 protein SEC11, which, along with plasma membrane K+ channels, selectively binds with the SNARE SYP121 to regulate its assembly in complex. How SEC11 binding is coordinated with the K+ channels is poorly understood, as both SEC11 and the channels are thought to compete for the same SNARE binding site. Here, we identify a second binding motif within the N terminus of SYP121 and demonstrate that this motif affects SEC11 binding independently of the F9xRF motif that is shared with the K+ channels. This second, previously unrecognized motif is centered on residues R20R21 of SYP121 and is essential for SEC11 interaction with SYP121. Mutation of the R20R21 motif blocked vesicle traffic without uncoupling the effects of SYP121 on solute and K+ uptake associated with the F9xRF motif; the mutation also mimicked the effects on traffic block observed on coexpression of the dominant-negative SEC11Δ149 fragment. We conclude that the R20R21 motif represents a secondary site of interaction for the Sec1/Munc18 protein during the transition of SYP121 from the occluded to the open conformation that leads to SNARE complex assembly

Towards updatable, multivalent Covid vaccines: A platform process to produce trimeric spike protein of SARS-COV-2 variants expressed in HEK293 stable cell clones

Within 2.5 years since SARS-COV-2 emergence, COVID-19 pandemic has caused more than 6.3 million registered deaths and 530 million registered cases. The quick development of safe and effective vaccines was very important to mitigate the sanitary crisis. However, the continuous emergence of virus variants with increasing transmissibility continues to cause periodic outbreaks worldwide. The original vaccines keep protecting from severe disease and death caused by variants, but not from transmission and mild disease. Thus, new and improved vaccines are necessary, and updatable, multivalent pan-variant vaccines might be one way to control SARS-COV-2. The spike (S) protein of SARS-COV-2, a highly glycosylated and very large protein (1380 amino acids), is a key target for diagnostics, therapeutics (e.g. monoclonal antibodies) and vaccines. Very early in the pandemic, Wrapp et al. (https://doi.org/10.1126/science.abb2507) produced the recombinant spike protein as a trimer stabilized in the prefusion conformation and determined its structure by cryoelectron microscopy, providing evidence that it binds to the ACE-2 receptor. The stabilized spike trimer forms the basis of most approved COVID vaccines, across vaccine platforms. In the present work, we studied the expression and purification of the trimeric prefusion-stabilized spike protein in HEK293 cell lines and developed a platform process applicable to SARS-COV-2 variants (current - and potentially future - ones). Parental HEK293 cells (NRC, Canada or Thermofisher, USA) were transfected by lipofection using Lipofectamine 3000 (Thermofisher Scientific, USA) with a plasmid containing the gene encoding the spike protein. At first, for the ancestral strain (Wuhan), we used a plasmid intended for transient expression kindly provided by VRC/NIAID/NIH (USA). However, for the variants, we ordered synthetic genes (Genscript, USA) that were subcloned in a plasmid intended for stable expression. After genetic modification, stably transfected cells were maintained in the presence of G418 sulfate selection agent. After 3-4 weeks, stable cell pools were obtained and submitted to single cell deposit (FACS Aria, BD Biosciences) in order to obtain clonally-derived cell lines. Documented research cell banks of selected clones were cryopreserved. Batch and fed-batch cultivations were investigated in shake flasks and bioreactors, using the chemically defined HEK TF culture medium and HEK FS feed solution (both Sartorius Xell, Germany). For protein purification, different chromatographic techniques were investigated using Akta Purifier and Akta Pilot systems. Detection of the spike protein secreted in cell culture supernatant was performed by immunoblot, whereas UV280 (Nanodrop, Thermofisher, USA) was used for protein quantitation in purified samples. After first expressing the spike protein in February 2020 by transient transfection, we developed a stable cell pool by co-transfecting the same transient expression plasmid and an empty stable expression plasmid. This stable cell pool allowed the generation of Wuhan protein that was used to develop serological tests and a hyperimmune equine serum (Cunha et al., doi: 10.1016/j.isci.2021.103315; Alvim et al., in press) and was donated so far to over 90 laboratories in Brazil for basic or applied research. Please click Download on the upper right corner to see the full abstract

Synergy Among Exocyst and SNARE Interactions Identifies a Functional Hierarchy in Secretion during Vegetative Growth

Vesicle exocytosis underpins signaling and development in plants and is vital for wall remodeling during cell expansion. Vesicle tethering and fusion are thought to occur sequentially, tethering mediated by the exocyst and fusion driven by assembly of SNARE protein complexes. Interactions between these two protein complexes are known, although insights into their functional consequences are largely unexplored. We now identify a clear hierarchy of interactions leading to secretion in Arabidopsis. Mating-based split-ubiquitin screens and in vivo FRET analyses showed that exocyst EXO70 subunits bind preferentially with cognate plasma membrane SNAREs, notably SYP121 and VAMP721. The exo70A1 mutant affected SNARE distributions and suppressed vesicle traffic like the dominant-negative SYP121deltaC, consistent with the epistasis of exo70A1 over the exo70A1syp121 double mutant. However, the exo70A1vamp721 mutant showed a strong synergistic suppression of growth. These data are best explained by a spatiotemporal hierarchy of exocyst recruitment with the R-SNARE to the plasma membrane, plausibly with the VAMP721 longin domain as a nexus for binding

Membrane voltage as a dynamic platform for spatio-temporal signalling, physiological and developmental regulation

Membrane voltage arises from the transport of ions through ion-translocating ATPases, ion-coupled transport of solutes, and ion channels, and is an integral part of the bioenergetic ′currency′ of the membrane. The dynamics of membrane voltage - so-called action, systemic, and variation potentials - have also led to a recognition of their contributions to signal transduction, both within cells and across tissues. Here we review the origins of our understanding of membrane voltage and its place as a central element in regulating transport and signal transmission. We stress the importance of understanding voltage as a common intermediate that acts both as a driving force for transport - an electrical ′substrate′ - and as a product of charge flux across the membrane, thereby interconnecting all charge-carrying transport across the membrane. The voltage interconnection is vital to signalling via second messengers that rely on ion flux, including cytosolic free Ca2+, H+, and the synthesis of reactive oxygen species generated by integral membrane, respiratory burst oxidases. These characteristics inform on the ways in which long-distance voltage signals and voltage oscillations give rise to unique gene expression patterns and influence physiological, developmental, and adaptive responses such as systemic acquired resistance to pathogens and to insect herbivory

Simplification of State Transition Diagrams in Average Unavailability Analysis by Using Generalized Perturbation Theory

Safety analysis studies in nuclear engineering, more specifically system reliability, usually handle a great number of components, so that computational difficulties may arise. To face the problem of many component systems a method for simplifying the state transition diagram in Markovian reliability analyses has been proposed, using the edges which can be cut, since these latter have a smaller influence on system failure probability. In order to extend the application of GPT (Generalized Perturbation Theory), this work uses GPT formalism to reduce the number of states in a transition diagram, not considering the state probability as the integral quantity of interest, but the mean system unavailability instead. Therefore, after simplifying the original diagram, the mean unavailability for the new system was calculated and the results were very close to those of the original diagram integral quantity (giving a relative error of about 2%), showing that the proposed simplification is quite reasonable and simple to apply

Association between C-reactive protein with all-cause mortality in ELSA-Brasil cohort

Background: High-sensitive C-reactive protein (hsCRP) has been proposed as a marker of incident cardiovascular disease and vascular mortality, and it may also be a marker of non-vascular mortality. However, most evidence comes from either North American or European cohorts. The present proposal aims to investigate the association of high-sensitive C-reactive protein with the risk of all-cause mortality in a multi-ethnic Brazilian population Methods: Cohort data from baseline (2008–2010) of 14 792 subjects participating in the Brazilian Longitudinal Study of Adult Health were used. HsCRP was assayed with Immunochemistry. The association of baseline covariates with all-cause mortality was calculated by Cox regression for univariate model and adjusted for different confounders after mean follow-up of 8.0 ± 1.1 years. The final model was adjusted for age, sex, self-rated race/ethnicity, schooling, health behaviours and prevalent chronic disease. Results: The risk of death increased steadily by quartiles of hsCRP from 1.45 (95% Confidence Interval: 1.05, 2.01) in Quartile 2 to 1.95 (1.42, 2.69) in Quartile 4 compared to Quartile 1. Furthermore, the persistence of a significant graded association after the exclusion of deaths in the first year of follow-up suggests that these results are unlikely to be due to reverse causality. Finally, the hazard ratios were unaffected by the exclusion of participants that had self-reported past medical history for diabetes, cancer and chronic obstructive pulmonary disease. Conclusions: Our study shows that hsCRP levels is associated with mortality in a highly admixed population, independently of a large set of lifestyle and clinical variables