LentiPro stable producer cells: Delivering scalable and reliable lentiviral vector manufacturing

Lentiviral vectors are one of the most currently used viral vectors for gene and cell therapies. Their use in clinical protocols has significantly increased in the past 5 years with the approval of several gene therapeutic products relying on lentiviral vector gene delivery. Capable of transducing non-dividing cells and presenting safer integration profiles as self-inactivating vectors, lentiviral vectors have progressively undertaken gammaretroviral vector use in gene therapies. However the knowledge on lentiviral vector manufacture is far more immature than that of gammaretroviral vectors. While the production of gammaretrovirus rely on stable producer cell lines and perfusion systems, enabling high cell density and longer term productions, most of the bioprocesses for lentiviral bioproducts rely on transient transfections and short term batch productions. At the upstream process, many of the challenges lentiviral bioproducts present in their manufacturing are related to the apoptosis leading cytotoxicity of some of the vector components. Supported on our long track experience and enabling tools developed for gammaretrovirus manufacturing, we carried out the challenge of establishing a constitutive stable lentiviral producer cell line. To surpass the challenges we proposed to eliminate or reduce the cytotoxicity of the lentiviral vector expression components1. Several strategic novelties were introduced in the development of the cell line namely: (i) the use of a modified gag-pro-pol, (ii) introduction of all the third generation lentiviral expression cassettes by chemical transfection instead of viral transduction and (iii) performing only one clone screening step (enabling the use on the ‘Single step cloning screening’ protocol developed by our group2). After establishing a stable producer cell line the culture conditions were developed with the main aim of extending bioreaction culture time and viral vector total yields. A lentiviral producer cell line constitutively producing infective titers above 106 TU.mL-1.day-1 was established. Moreover the new protocol to generate the cell line enabled its development in less than six months. The cell line showed to be stable, consistently maintaining vector productivity over one month in the absence of antibiotics. At the bioreaction process it was possible to maintain the cells continuously producing over 10 days1. These results validate the transition to continuous or perfusion large-scale production systems qualifying the strengths and advantages of the strategies followed. This work to be presented will discuss the challenges on the manufacture and scale-up of lentiviral vectors as well the strategies and novel technologies to be adopted to enable effective upstream processes. 1Tomás et al. (2018) ‘LentiPro26: novel stable cell lines for constitutive lentiviral vector production\u27 Sci Rep. 8(1):5271 2 Rodrigues et al. (2015) ‘Single step cloning-screening method: a new tool for developing and studying high-titer viral vector producer cells\u27 Gene Ther. 22(9):6

FEM applied to building physics: modeling solar radiation and heat transfer of PCM enhanced test cells

In passive solar buildings, energy can be stored using either sensible heat materials or latent heat materials. Phase change materials (PCM) can contribute to temperature control in passive solar buildings when melting occurs near to comfort temperature required for building’s interior spaces. The use of finite element method (FEM) as a numerical methodology for solving the thermal problem associated with heat transfer in current building materials and PCMs make sense, as it is a well-known technique, generalized and dominated, however, still little applied to the domain of building physics. In this work, a solar model was developed and applied in order to simulate numerically the effect of solar radiation incidence on each face of the test cells (with different solar exposures) without neglecting the main objective of the recommended numerical simulation: the study of the action of PCM. During the experimental campaign, two test cells with distinct inner layers were used to evaluate the effect of solar radiation: (i) REFM test cell (without PCM) with a reference mortar; (ii) PCMM test cell (with PCM) with a PCM mortar. The temperatures monitored inside the REFM and PCMM test cells were compared with the values resulting from the numerical simulation, using FEM with 3D discretization and the explicit modeling of the solar radiation, and the obtained results revealed a significant coherence of values

Catalytic performance of bulk and colloidal Co/Al layered double hydroxide with Au nanoparticles in aerobic olefin oxidation

A Co/Al layered double hydroxide material was synthesized in both bulk and exfoliated (colloidal) forms. Anion exchange with methionine allowed immobilization of Au nanoparticles previously prepared by a biomimetic method using an anti-oxidant tea aqueous extract to reduce the Au salt solution. The catalytic performance of bulk and exfoliated clays Au-hybrid materials was assessed in aerobic olefin epoxidation. Both catalysts were very active towards the epoxide products and with very interesting substrate conversion levels after 80 h reaction time. The Au-exfoliated material, where the nanosheets work as large ligands, yielded higher product stereoselectivity in the case of limonene epoxidation. This arises from a confined environment around the Au nanoparticles wrapped by the clay nanosheets modulating access to the catalytic active centres by reagents. Mechanistic assessment was also accomplished for styrene oxidation by DFT methodspublishe

Effect of sonic agitation of a binary mixture of solvents on filling remnants removal as an alternative to apical enlargement — a micro-CT study

Background: This work aimed to evaluate the efficacy of sonic agitation of a binary mixture of solvents (methyl ethyl ketone/tetrachloroethylene) on filling remnants removal and compare the effects of solvent agitation with the enlargement to the next instrument size. Methods: Twenty-four mandibular incisors were prepared with ProTaper Next (X1, X2) and obturated with the single-cone technique and AH Plus sealer. The teeth were retreated with ProTaper Universal Retreatment and ProTaper Next and divided into two groups (n = 12) according to the final instrument (X3 or X4). All canals were submitted to a supplementary procedure consisting of a mixture of solvents―methyl ethyl ketone/tetrachloroethylene, agitated with EndoActivator. The volume of filling remnants was assessed through micro-computed tomography in the apical 5 mm. Statistical analysis was performed with a significance level of 5%. Results: The supplementary procedure of agitation of the solvent mixture was beneficial in both groups (p p > 0.05). Conclusions: An additional step with a two-solvent solution potentiated by EndoActivator showed to be very effective for the removal of gutta-percha and resinous sealer remnants from apical root canals of mandibular incisors, avoiding further enlargement.This article was supported by National Funds through FCT-Fundacao para a Ciencia e a Tecnologia, I.P., within CINTESIS, R&D Unit (reference UIDB/4255/2020)

The impact of CdSe/ZnS Quantum Dots in cells of Medicago sativa in suspension culture

<p>Abstract</p> <p>Background</p> <p>Nanotechnology has the potential to provide agriculture with new tools that may be used in the rapid detection and molecular treatment of diseases and enhancement of plant ability to absorb nutrients, among others. Data on nanoparticle toxicity in plants is largely heterogeneous with a diversity of physicochemical parameters reported, which difficult generalizations. Here a cell biology approach was used to evaluate the impact of Quantum Dots (QDs) nanocrystals on plant cells, including their effect on cell growth, cell viability, oxidative stress and ROS accumulation, besides their cytomobility.</p> <p>Results</p> <p>A plant cell suspension culture of <it>Medicago sativa </it>was settled for the assessment of the impact of the addition of mercaptopropanoic acid coated CdSe/ZnS QDs. Cell growth was significantly reduced when 100 mM of mercaptopropanoic acid -QDs was added during the exponential growth phase, with less than 50% of the cells viable 72 hours after mercaptopropanoic acid -QDs addition. They were up taken by <it>Medicago sativa </it>cells and accumulated in the cytoplasm and nucleus as revealed by optical thin confocal imaging. As part of the cellular response to internalization, <it>Medicago sativa </it>cells were found to increase the production of Reactive Oxygen Species (ROS) in a dose and time dependent manner. Using the fluorescent dye H₂DCFDA it was observable that mercaptopropanoic acid-QDs concentrations between 5-180 nM led to a progressive and linear increase of ROS accumulation.</p> <p>Conclusions</p> <p>Our results showed that the extent of mercaptopropanoic acid coated CdSe/ZnS QDs cytotoxicity in plant cells is dependent upon a number of factors including QDs properties, dose and the environmental conditions of administration and that, for <it>Medicago sativa </it>cells, a safe range of 1-5 nM should not be exceeded for biological applications.</p

Interaction between cannabinoid type 1 and type 2 receptors in the modulation of subventricular zone and dentate Gyrus neurogenesis

Copyright © 2017 Rodrigues, Ribeiro, Ferreira, Vaz, Sebastião and Xapelli. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Neurogenesis in the adult mammalian brain occurs mainly in two neurogenic niches, the subventricular zone (SVZ) and the subgranular zone (SGZ) of the dentate gyrus (DG). Cannabinoid type 1 and 2 receptors (CB1R and CB2R) have been shown to differently modulate neurogenesis. However, low attention has been given to the interaction between CB1R and CB2R in modulating postnatal neurogenesis (proliferation, neuronal differentiation and maturation). We focused on a putative crosstalk between CB1R and CB2R to modulate neurogenesis and cultured SVZ and DG stem/progenitor cells from early postnatal (P1-3) Sprague-Dawley rats. Data showed that the non-selective cannabinoid receptor agonist WIN55,212-2 promotes DG cell proliferation (measured by BrdU staining), an effect blocked by either CB1R or CB2R selective antagonists. Experiments with selective agonists showed that facilitation of DG cell proliferation requires co-activation of both CB1R and CB2R. Cell proliferation in the SVZ was not affected by the non-selective receptor agonist, but it was enhanced by CB1R selective activation. However, either CB1R or CB2R selective antagonists abolished the effect of the CB1R agonist in SVZ cell proliferation. Neuronal differentiation (measured by immunocytochemistry against neuronal markers of different stages and calcium imaging) was facilitated by WIN55,212-2 at both SVZ and DG. This effect was mimicked by either CB1R or CB2R selective agonists and blocked by either CB1R or CB2R selective antagonists, cross-antagonism being evident. In summary, our findings indicate a tight interaction between CB1R and CB2R to modulate neurogenesis in the two major neurogenic niches, thus contributing to further unraveling the mechanisms behind the action of endocannabinoids in the brain.This work was supported by LISBOA-01-0145-FEDER-007391, project co-funded by FEDER through POR Lisboa 2020 (Programa Operacional Regional de Lisboa) from PORTUGAL 2020, and by Fundação para a Ciência e a Tecnologia (FCT). AS thanks the following supports: PTDC/DTP-FTO/3346/2014 from FCT and H2020 Twinning Action from EU (SynaNet 692340). SX is grateful for the support by the COST action BM1402. RR (IMM/BI/42-2016), FR (SFRH/BD/74662/2010), SV (SFRH/BPD/81627/2011), and SX (SFRH/BPD/76642/2011 and IF/01227/2015) were in receipt of a fellowship from FCT.info:eu-repo/semantics/publishedVersio

Variation in pea (Pisum Sativum L.) seed quality traits defined by physicochemical functional properties

Pea is one of the most produced and consumed pulse crops around the world. The study of genetic variability within pea germplasm is an important tool to identify outstanding accessions with optimal functional and nutritional qualities. In the present study, a collection of 105 pea accessions was analysed for physicochemical properties, pasting viscosity, and basic composition parameters. While pasting viscosities were negatively correlated to hydration capacity, cooking time, and basic composition, a positive correlation was found between the hydration capacity and the basic composition parameters. Basic composition (protein, fibre, fat, and resistant starch) parameters were further evaluated regarding seed trait morphology, namely, seed shape, colour, and surface. Allelic characterisation at the r and rb genetic loci was performed in a subgroup of 32 accessions (3 phenotyped as smooth and 29 as rough seeded), revealing that none of the initially classified rough-seeded accessions were rb mutants, 19 were r mutants, and 13 were neither r nor rb. Despite their initial phenotypic classification, the 13 accessions genetically classified as smooth behaved differently (p < 0.05) to the 19 r mutants in terms of physicochemical properties, pasting viscosity, and basic composition parameters. Using multivariate analysis of the most discriminatory parameters for the food-related traits studied, the best-performing accessions at functional and nutritional levels were identified for future plant breeding to improve field pea production and consumption.info:eu-repo/semantics/publishedVersio

Methodological considerations in assessing interlimb coordination on poststroke gait: a scoping review of biomechanical approaches and outcomes

To identify and summarize biomechanical assessment approaches in interlimb coordination on poststroke gait. Interlimb coordination involves complex neurophysiological mechanisms that can be expressed through the biomechanical output. The deepening of this concept would have a significant contribution in gait rehabilitation in patients with an asymmetric neurological impairment as poststroke adults. Poststroke adults (>19 years old), with assessment of interlimb coordination during gait, in an open context, according to the Population, Concept, Context framework. A literature search was performed in PubMed, Web of Science™, Scopus, and gray literature in Google Scholar™, according to the PRISMA-ScR recommendations. Studies written in Portuguese or English language and published between database inception and 14 November 2021 were included. Qualitative studies, conference proceedings, letters, and editorials were excluded. The main conceptual categories were “author/year”, “study design”, “participant’s characteristics”, “walking conditions”, “instruments” and “outcomes”. The search identified 827 potentially relevant studies, with a remaining seven fulfilling the established criteria. Interlimb coordination was assessed during walking in treadmill (n = 3), overground (n = 3) and both (n = 1). The instruments used monitored electromyography (n = 2), kinetics (n = 2), and kinematics (n = 4) to assess spatiotemporal parameters (n = 4), joint kinematics (n = 2), anteroposterior ground reaction forces (n = 2), and electromyography root mean square (n = 2) outcomes. These outcomes were mostly used to analyze symmetry indices or ratios, to calculate propulsive impulse and external mechanical power produced on the CoM, as well as antagonist coactivation. Assessment of interlimb coordination during gait is important for consideration of natural auto-selected overground walking, using kinematic, kinetic, and EMG instruments. These allow for the collection of the main biomechanical outcomes that could contribute to improve better knowledge of interlimb coordination assessment in poststroke patients.info:eu-repo/semantics/publishedVersio

Molecular characterization of Giardia lamblia in children less than 5 years of age with diarrhoea attending the Bengo General Hospital, Angola

Introduction - Giardia lamblia is a pathogenic intestinal protozoan with high prevalence in developing countries, especially among children. Molecular characterization has revealed the existence of eight assemblages, with A and B being more commonly described in human infections. Despite its importance, to our knowledge, this is the first published molecular analysis of G. lamblia assemblages in Angola. Methods - The present study aimed to identify the assemblages of G. lamblia in children with acute diarrhoea presenting at the Bengo General Hospital, Angola. A stool sample was collected and microscopy and immunochromatographic tests were used. DNA was extracted and assemblage determination was performed through amplification of the gene fragment ssu-rRNA (175 bp) and β-giardin (511 bp) through polymerase chain reaction and DNA sequencing. Results - Of the 16 stool samples screened, 12 were successfully sequenced. Eleven isolates were assigned to assemblage B and one to assemblage A. Subassemblage determination was not possible for assemblage B, while the single isolate assigned to assemblage A was identified as belonging to subassemblage A3. Conclusion - This study provides information about G. lamblia assemblages in Bengo Province, Angola and may contribute as a first step in understanding the molecular epidemiology of this protozoan in the country. GenBank accession numbers for the ssur-RNA gene: MF479750, MF479751, MF479752, MF479753, MF479754, MF479755, MF479756, MF479757, MF479758, MF479759, MF479760, MF479761. GenBank accession numbers for the β-giardin gene: MF565378, MF565379, MF565380, MF565381.info:eu-repo/semantics/publishedVersio