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

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

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

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

Infrared spectroscopic studies of hydrogen bonding in substituted nitrophenols: substituent and solvent effects

A detailed infrared spectroscopic study of the substituted phenols 2-cyano-4,6-dinitrophenol and 4-cyano-2,6-dinitrophenol has been carried out (in several different solvents) in order to investigate the substituent and solvent effects on their intra- and inter-molecular hydrogen bonding properties. In benzene or dichloromethane it is found that both isomers form strong intramolecular hydrogen bonds with the 2-cyano (2CN) isomer having a stronger intramolecular interaction (in accordance with the higher pKa). The 4-cyano (4CN) isomer shows two distinct NO2 groups and exchange between the two possible hydrogen bonding sites is probably slow on the infrared time-scale. In protic solvents such as methanol the intramolecular hydrogen bonds are broken (more easily for the 4CN isomer) by intermolecular hydrogen bonding to the solvent. The differential “reactivity” towards methanol may be associated with steric congestion in the 4CN isomer leading to the forcing of at least one of the NO2 groups out of the aromatic plane. The use of mixed solvents (benzene-methanol) has established that the two hydrogen bonded species are observed together and that a high concentration of methanol is required to drive the equilibrium towards the intermolecular hydrogen bonded species. In dimethyl sulphoxide the behaviour of the two isomers is even more interesting. The 4CN isomer is ionised to produce the corresponding phenolate. However the 2CN isomer remains neutral (but highly solvated). We attribute this difference to the requirement for the 4CN isomer to allow the 2- and 6-NO2 groups to recover planarity with the aromatic ring. The energy compensation involved in this process is clearly sufficient to break a stronger intramolecular hydrogen bond.info:eu-repo/semantics/publishedVersio

The basicity of alkali metal methoxides in methanol. The effects of ion association on methoxide additions to activated anisoles

The formation of adducts with 1 :2 and 1:3 stoichiometry by methoxide addition to nitro-activated anisoles has been examined spectrophotometrically. For these equilibria the ‘basicity’ of sodium methoxide solutions in methanol is appreciably greater than that of corresponding potassium methoxide solutions. This is in contrast with other measures of basicity and is attributed to the association of the multi-charged adducts with cations which is stronger with sodium than with potassium ions.info:eu-repo/semantics/publishedVersio

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

Levantamento das etapas do processo de rastreabilidade bovina em propriedades dos municípios de Dom Pedrito e Bagé.

CONGREGA 2012