Acordo de Livre Comércio com a UE: a vulnerabilidade dos produtos industriais produzidos pelo Mercosul à competição européia [Free Trade Agreement - EU and Mercosur: vulnerability of Mercosur products to European competition]

The UE and Mercosur are currently negotiating a free trade agreement. The offer made by Mercosur reflects its production and protection structure and is more "generous" to agriculture than to industry. In fact, the main competitive fragilities of Mercosur are concentrated in manufactured goods while European competitive fragilities are concentrated in the agriculture sector. As the UE has an important market share in the Mercosur countries, it poses a major threat to the domestic producers. Our paper seeks to identify, at a disaggregated level, the Mercosur manufactured goods for which the growth of the European manufacture exports can represent a threat. We consider that trade growth depends on the existence of complementarity between the two regions and the existence of a margin for liberalization in the destination market, and that the greater the trade among Mercosur countries the greater the threat from European goods to local produces.free trade agreement, manufactured goods, Mercosur, European Union

Purification of flavivirus VLPs by a two-step chromatographic process

Flaviviruses are enveloped viruses with positive-sense, single-stranded RNA, which are most commonly transmitted by infected mosquitoes. Besides for example dengue viruses (DENV), which have been already for decades posing challenges to public health worldwide, zika virus (ZIKV) and yellow fever virus (YFV) are flaviviruses that have caused significant outbreaks in the last few years. Thus, based on our experience of expressing virus-like particles (VLPs) of several different flaviviruses in recombinant mammalian cells, this work focuses on the development of efficient chromatographic purification processes for zika and yellow-fever VLPs. ZIKV has been discovered in 1947, and since 2007 it has caused isolated outbreaks in Pacific Islands. However, in 2015 it was identified for the first time in Brazil and then quickly spread to over 60 countries between 2015 and 2016. Although most zika patients are asymptomatic, in a small proportion of adults ZIKV infection can cause Guillain-Barré syndrome, and in fetuses of infected women it frequently causes serious congenital malformations, especially in the central nervous system. Since it can be transmitted also by the sexual route and can persist for very long periods in body fluids (including sperm), the development of a vaccine is needed to prevent the spread of the virus to non-endemic countries and to prevent outbreaks to periodically occur in regions where the virus is already circulating. Yellow fever virus is a highly lethal virus, which causes death in about 6-10% of non-vaccinated individuals. In past centuries, before the introduction of the current live-attenuated vaccine, 10% of the population of cities like Philadelphia (USA) and Barcelona (Spain) died in YF outbreaks. The current vaccine is very safe and provides life-long protection from a single dose. However, it can also cause fatal adverse effects in a small proportion of vaccines, and the egg-based production is limited in capacity. This latter fact led to worldwide vaccine shortages during an outbreak in Africa in 2016 and in Brazil in 2017-2018. Although the WHO introduced during the African outbreak in 2016 the use of a fractional (1/5) dose as an emergency measure to control outbreaks, even if using fractional doses of the current vaccine, shortage would be an issue if YF outbreaks spread and especially if it gets to be locally transmitted in Asia, where the mosquito vector is widespread. In this work, a two-step chromatographic process was developed for the purification of zika and yellow fever VLPs from CHO- and HEK293-derived cell culture supernatant, building on previous experience acquired on the purification of yellow fever whole virus from Vero cell culture (Pato et al., 2014, doi: 10.1016/j.vaccine.2014.02.036). The initial clarification of the cell culture suspension was performed by centrifugation and/or filtration, followed by anion exchange chromatography and then a multimodal chromatographic step. The anion exchanger used was a Q membrane adsorber, due to its easy scalability, simplicity to handle, absence of diffusional limitations, and good performance at high flow rates for the capture of large molecules such as VLPs. This capture step allowed a high degree of concentration and an efficient DNA removal. In order to enhance HCP removal, a CaptoCore 700 multimodal column was used in a flow-through mode, allowing contaminants to be adsorbed while VLPs were excluded by size. Samples from all steps of the process were characterized by immunoassays, total protein determination, SDS-PAGE and Western blot. The promising results obtained for zika and yellow fever VLPs indicate that this process could be potentially applied also to other flavivirus VLPs that we have been expressing in our lab, such as DENV1-4, SLEV, CPCV and ILHV. Overall, the presented downstream process could potentially represent a simple, robust and economic platform technology for the production of cell culture-derived recombinant flavivirus vaccines. Acknowledgements: T. P. Pato (Biomanguinhos, FIOCRUZ, Brazil) for fruitful discussions, and B. S. Graham (VRC, NIH, USA) for fruitful discussions and sharing reagents

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

Detailed Analysis of Nearby Bulgelike Dwarf Stars II. Lithium Abundances

Li abundances are derived for a sample of bulgelike stars with isochronal ages of 10-11 Gyr. These stars have orbits with pericentric distances, Rp, as small as 2-3 kpc and Zmax < 1 kpc. The sample comprises G and K dwarf stars in the metallicity range -0.80<[Fe/H]< +0.40. Few data of Li abundances in old turn-off stars (> 4.5 Gyr) within the present metallicity range are available. M67 (4.7 Gyr) and NGC 188 (6 Gyr) are the oldest studied metal-rich open clusters with late-type stars. Li abundances have also been studied for few samples of old metal-rich field stars. In the present work a high dispersion in Li abundances is found for bulgelike stars for all the metallicity range, comparable with values in M67. The role of metallicity and age on a Li depletion pattern is discussed. The possible connection between Li depletion and oxygen abundance due to atmospheric opacity effects is investigated.Comment: 9 pages, 7 figure

Use of a biphasic perfusion process based on mild hypothermia for recombinant glucocerebrosidase (GBA) production

The main goal of this study was to develop an innovative CHO-based process for the production of glucocerebrosidase (GBA), an enzyme used for the replacement therapy of Type 1 Gaucher disease. The focus of the present study was on the development of a perfusion process, combining strategies that are commonly used for process optimization: temperature reduction, and supplementation of the culture medium with productivity enhancers, such as short chain fatty acids. The effects of mild hypothermic conditions combined with valeric acid supplementation were first studied in batch shake flasks for two clones (CHO-GBA-36K and CHO-GBA-65P), developed previously using as host the cell lines CHO.K1 (ATCC CCL-61) and CHO.PRO5 (a glycosylation mutant developed by Stanley et al. Cell 6:121, 1975), respectively. A DOE approach was used (Table 1) to select the most promising cultivation conditions to be further applied to a perfusion process. The best performance regarding both cell growth and GBA production was obtained for the CHO-GBA-65P clone under condition [1], at 31ºC with no valeric acid (Table 1). Under this condition, CHO-GBA-65P achieved a maximum qP of 58.4 mU/106 cells/d, which is 4.2 fold higher than qP at the control condition [2] and 2.7 fold higher than the maximum qP obtained for the CHO-GBA-36K clone, which was achieved at 31ºC with 2 mM valeric acid supplementation (condition [3]). Please click Additional Files below to see the full abstract

High Susceptibility Of Activated Lymphocytes To Oxidative Stress-induced Cell Death.

The present study provides evidence that activated spleen lymphocytes from Walker 256 tumor bearing rats are more susceptible than controls to tert-butyl hydroperoxide (t-BOOH)-induced necrotic cell death in vitro. The iron chelator and antioxidant deferoxamine, the intracellular Ca2+ chelator BAPTA, the L-type Ca2+ channel antagonist nifedipine or the mitochondrial permeability transition inhibitor cyclosporin A, but not the calcineurin inhibitor FK-506, render control and activated lymphocytes equally resistant to the toxic effects of t-BOOH. Incubation of activated lymphocytes in the presence of t-BOOH resulted in a cyclosporin A-sensitive decrease in mitochondrial membrane potential. These results indicate that the higher cytosolic Ca2+ level in activated lymphocytes increases their susceptibility to oxidative stress-induced cell death in a mechanism involving the participation of mitochondrial permeability transition.80137-4

Association or Causation? Exploring the Oral Microbiome and Cancer Links

Several epidemiological investigations have found associations between poor oral health and different types of cancer, including colorectal, lung, pancreatic, and oral malignancies. The oral health parameters underlying these relationships include deficient oral hygiene, gingival bleeding, and bone and tooth loss. These parameters are related to periodontal diseases, which are directly and indirectly mediated by oral bacteria. Given the increased accessibility of microbial sequencing platforms, many recent studies have investigated the link between the oral microbiome and these cancers. Overall, it seems that oral dysbiotic states can contribute to tumorigenesis in the oral cavity as well as in distant body sites. Further, it appears that certain oral bacterial species can contribute to carcinogenesis, in particular, Fusobacterium nucleatum and Porphyromonas gingivalis, based on results from epidemiological as well as mechanistic studies. Yet, the strength of the findings from these investigations is hampered by the heterogeneity of the methods used to measure oral diseases, the treatment of confounding factors, the study design, the platforms employed for microbial analysis, and types of samples analyzed. Despite these limitations, there is an overall indication that the presence of oral dysbiosis that leads to oral diseases may directly and/or indirectly contribute to carcinogenesis. Proper methodological standardized approaches should be implemented in future epidemiological studies as well as in the mechanistic investigations carried out to explore these results. © International & American Associations for Dental Research 202

Multimodal chromatography combining steric exclusion and cation exchange as an intermediate downstream step to purify yellow fever virus-like particles

Yellow fever (YF) is an hemorrhagic viral disease transmitted by infected mosquitoes, which is endemic in many African and Central/South American countries. The severe symptoms and the high mortality rate of the disease can have devastating effects in case an outbreak occurs in an area where the population is non-vaccinated. Before the current YF vaccine became available, outbreaks in cities like Barcelona (Spain) and Philadelphia (USA) led to the death of approximately 10% of the population. Recent outbreaks have shown that YF continues to be a major public health threat due to production capability issues and shortage of vaccine stockpiles, which even led to the use of an emergency fractional (1/5) dose in Africa in 2016 and in Brazil in 2018. Yellow fever virus-like particles (VLPs) represent an interesting alternative to develop a new YF vaccine. With the aim of developing an efficient and affordable process to purifiy yellow fever VLPs, in this work we developed a multimodal strategy combining cation exchange (CEX) and steric exclusion chromatography (SXC) under conditions where the product of interest does not bind to the CEX adsorber, whereas many contaminants do. In this way, the product of interest is retained just due to steric exclusion by the polyethylene glycol (PEG) added to the mobile phase. Product desorption can be achieved by decreasing PEG concentration, while contaminants remain bound to the adsorber and are eluted in the regeneration step. To the best of our knowledge, the application of such a multimodal strategy has not been published before. Please click Download on the upper right corner to see the full abstract