pinecone/core-2020-05-10-deduplication huggingface dataset:
Effect of the DnaK chaperone on the conformational quality of JCV VP1 virus-like particles produced in Escherichia coli

Altres ajuts: CIBER de Bioingeniería, Biomateriales y Nanomedicina ; VI National R&D&i Plan 2008–2011 ; Iniciativa Ingenio 2010 ; Consolider Program ; CIBER Actions ; Instituto de Salud Carlos III ; European Regional Development FundProtein nanoparticles such as virus-like particles (VLPs) can be obtained by recombinant protein production of viral capsid proteins and spontaneous self-assembling in cell factories. Contrarily to infective viral particles, VLPs lack infective viral genome while retaining important viral properties like cellular tropism and intracellular delivery of internalized molecules. These properties make VLPs promising and fully biocompatible nanovehicles for drug delivery. VLPs of human JC virus (hJCV) VP1 capsid protein produced in Escherichia coli elicit variable hemagglutination properties when incubated at different NaCl concentrations and pH conditions, being optimal at 200 mM NaCl and at pH range between 5.8 and 7.5. In addition, the presence or absence of chaperone DnaK in E. coli cells influence the solubility of recombinant VP1 and the conformational quality of this protein in the VLPs. The hemagglutination ability of hJCV VP1 VLPs contained in E. coli cell extracts can be modulated by buffer composition in the hemagglutination assay. It has been also determined that the production of recombinant hJCV VP1 in E. coli is favored by the absence of chaperone DnaK as observed by Western Blot analysis in different E. coli genetic backgrounds, indicating a proteolysis targeting role for DnaK. However, solubility is highly compromised in a DnaK(-) E. coli strain suggesting an important role of this chaperone in reduction of protein aggregates. Finally, hemagglutination efficiency of recombinant VP1 is directly related to the presence of DnaK in the producing cells
Enhancement of the antimicrobial properties of bacteriophage-K via stabilisation using oil-in-water nano-emulsions

Bacteriophage therapy is a promising new treatment that may help overcome the threat posed by antibiotic-resistant pathogenic bacteria, which are increasingly identified in hospitalised patients. The development of biocompatible and sustainable vehicles for incorporation of viable bacterial viruses into a wound dressing is a promising alternative. This paper evaluates the anti-microbial efficacy of Bacteriophage K against Staphylococcus aureus over time, when stabilised and delivered via an oil-in-water nano-emulsion. Nano-emulsions were formulated via thermal phase inversion emulsification, and then bacterial growth was challenged with either native emulsion, or emulsion combined with Bacteriophage K. Bacteriophage infectivity, and the influence of storage time of the preparation, were assessed by turbidity measurements of bacterial samples. Newly prepared Bacteriophage K / nano-emulsion formulations have greater anti-microbial activity than freely suspended bacteriophage. The phage loaded emulsions caused rapid and complete bacterial death of three different strains of Staphylococcus aureus. The same effect was observed for preparations that were either stored at room temperature (18 - 20°C), or chilled at 4°C, for up to 10 days of storage. A response surface design of experiments was used to gain insight on the relative effects of the emulsion formulation on bacterial growth and phage lytic activity. More diluted emulsions had a less significant effect on bacterial growth, and diluted bacteriophage-emulsion preparations yielded greater antibacterial activity. The enhancement of bacteriophage activity when delivered via nano-emulsions has yet to be reported. This prompts further investigation into the use of these formulations for the development of novel anti-microbial wound management strategies
Immobilization of lipase on woolen fabrics:enhanced effectiveness in stain removal

The aim of this research was to examine the effectiveness of an enzyme in enhancing the cleaning effectiveness of woolen fabric without addition of any detergent. As a model enzyme, lipase from Pseudomonas fluoresces was immobilized onto a woolen cloth using a unique protocol that involved: chlorination of the wool, adsorbing a polyethyleneimine (PEI) spacer, adsorbing, and cross-linking with glutaraldehyde (GA) followed by adsorption of the lipase. It was determined that for this protocol, the immobilized activity was dependent on the GA solution pH and not on its concentration. The cloth exhibited excellent oily stain removal ability: after being stained with olive oil and stored for 1 day in air at room temperature, the oily stain could be easily removed by 0.05 M pH 8.5 Tris buffer without any detergent addition. This enhanced cleaning was stable also over a period of one month. The activity of the cloth (based on activity assay) dropped considerably over just 15 days storage in air. This therefore likely indicates that the enhanced cleaning seen over an extended storage period may not require as high an enzyme activity. The activity of the immobilized lipase was also very stable when stored under near ideal conditions: when the immobilized cloth was stored in 0.05 M Tris buffer (pH 8.5) for more than 80 days in a refrigerator, more than 80% of the lipase activity remained. Overall, results indicate that this immobilization protocol is a promising step towards producing a woolen fabric with enhanced cleaning properties
Measurement of cooling and warming rates in vitrification-based plant cryopreservation protocols

Cryopreservation protocols include the use of additives and pre-treatments aimed to reduce the probability of ice nucleation at all temperatures, mainly through micro-viscosity increase. Still, there is a risk of ice formation in the temperature region comprised between the equilibrium freezing (Tf) and the glass transition (TG) temperatures. Consequently, fast cooling and warming, especially in this region, is a must to avoid ice-derived damage. Vitrification and droplet-vitrification techniques, frequently used cryopreservation protocols based in fast cooling, were studied, alongside with the corresponding warming procedures. A very fast data acquisition system, able to read very low temperatures, down to that of liquid nitrogen, was employed. Cooling rates, measured between -20 and -120 ºC, ranged from ca. 5 ºC s-1 to 400 ºC s-1, while warming rates spanned from ca. 2 ºC s-1 to 280 ºC s-1, for the different protocols and conditions studied. A wider measuring window (0 ºC to -150 ºC) produced lower rates for all cases. The cooling and warming rates were also related to the survival observed after the different procedures. Those protocols with the faster rates yielded the highest survival percentages.Fil: Schneider Teixeira, Aline. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Centro de Investigaciones En Criotecnología de Alimentos (i); Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas; Argentina. Instituto de Ciencia y Tecnología de Alimentos y Nutrición; EspañaFil: Gonzalez Benito, M. Elena. Universidad Politecnica de Madrid; EspañaFil: Molina García, Antonio D.. Consejo Superior de Investigaciones Científicas. Instituto de Ciencia y Tecnologia de Alimentos y Nutrición; Españ
Nanotubular structures developed from whey-based α-lactalbumin fractions for food applications

Whey proteins have high nutritional value providing use in dietary purposes and improvement of technological properties in processed foods. Functionality of the whey-based α-lactalbumin (α-La) may be increased when assembled in the form of nanotubes, promising novel potential applications subject to investigation. The purpose of this study was to extract highly pure α-La from whey protein isolate (WPI) and whey powder (WP) and to construct protein nanotubes from them for industrial applications. For protein fractionation, WPI was directly fed to chromatography, however, WP was first subjected to membrane filtration and the retentate fraction, whey protein concentrate (WPC), was obtained and then used for chromatographic separation. α-La and, additionally β-Lg, were purified at the same batches with the purities in the range of 95%-99%. After enzymatic hydrolysis, WPI-based α-La produced chain-like and long nanotubules with ∼20 nm width while WPC-based α-La produced thinner, miscellaneous, and fibril-like nanostructures by self-assembly. Raman and FT-IR spectroscopies revealed that α-La fractions, obtained from both sources and the nanostructures, developed using both fractions have some structural differences due to conformation of secondary structure elements. Nanotube formation induced gelation and nanotubular gel network entrapped a colorant uniformly with a transparent appearance. Dairy-based α-La protein nanotubules could be served as alternative gelling agents and the carriers of natural colorants in various food processes.Scientific and Technological Research Council of Turkey(109O866); Izmir Institute of Technology (IYTE03
Monitoring the ex-vivo expansion of human mesenchymal stem/stromal cells in xeno-free microcarrier-based reactor systems by MIR spectroscopy

Human mesenchymal stem/stromal cells (MSCs) have received considerable attention in the field of cell-based therapies due to their high differentiation potential and ability to modulate immune responses. However, since these cells can only be isolated in very low quantities, successful realization of these therapies requires MSCs ex-vivo expansion to achieve relevant cell doses. The metabolic activity is one of the parameters often monitored during MSCs cultivation by using expensive multi-analytical methods, some of them time-consuming. The present work evaluates the use of mid-infrared (MIR) spectroscopy, through rapid and economic high-throughput analyses associated to multivariate data analysis, to monitor three different MSCs cultivation runs conducted in spinner flasks, under xeno-free culture conditions, which differ in the type of microcarriers used and the culture feeding strategy applied. After evaluating diverse spectral preprocessing techniques, the optimized partial least square (PLS) regression models based on the MIR spectra to estimate the glucose, lactate and ammonia concentrations yielded high coefficients of determination (R2 ≥ 0.98, ≥0.98, and ≥0.94, respectively) and low prediction errors (RMSECV ≤ 4.7%, ≤4.4% and ≤5.7%, respectively). Besides PLS models valid for specific expansion protocols, a robust model simultaneously valid for the three processes was also built for predicting glucose, lactate and ammonia, yielding a R2 of 0.95, 0.97 and 0.86, and a RMSECV of 0.33, 0.57, and 0.09 mM, respectively. Therefore, MIR spectroscopy combined with multivariate data analysis represents a promising tool for both optimization and control of MSCs expansion processes
Nanoparticles for bone tissue engineering

Tissue engineering (TE) envisions the creation of functional substitutes for damaged tissues through integrated solutions, where medical, biological, and engineering principles are combined. Bone regeneration is one of the areas in which designing a model that mimics all tissue properties is still a challenge. The hierarchical structure and high vascularization of bone hampers a TE approach, especially in large bone defects. Nanotechnology can open up a new era for TE, allowing the creation of nanostructures that are comparable in size to those appearing in natural bone. Therefore, nanoengineered systems are now able to more closely mimic the structures observed in naturally occurring systems, and it is also possible to combine several approaches - such as drug delivery and cell labeling - within a single system. This review aims to cover the most recent developments on the use of different nanoparticles for bone TE, with emphasis on their application for scaffolds improvement; drug and gene delivery carriers, and labeling techniques.This study was funded by QREN (ON.2 - NORTE-01-0124-FEDER-000018), as well as the European Union’s FP7 Programme under grant agreement number REGPOTCT2012-316331-POLARIS.
Sılvia Vieira was awarded an FCT PhD scholarship (SFRH/BD/102710/2014). The FCT distinction attributed to J.M.O. under the Investigator FCT program (IF/00423/2012 and IF/01285/2015) is also greatly acknowledged.info:eu-repo/semantics/publishedVersio
Selection of best conditions of inoculum preparation for optimum performance of the pigment production process by Talaromyces spp. using the Taguchi method

Process optimisation techniques increasingly need to be used early on in research and development of processes for new ingredients. There are different approaches and this article illustrates the main issues at stake with a method that is an industry best practice, the Taguchi method, suggesting a procedure to assess the potential impact of its drawbacks. The Taguchi method has been widely used in various industrial sectors because it minimises the experimental requirements to define an optimum region of operation, which is particularly relevant when minimising variability is a target. However, it also has drawbacks, especially the intricate confoundings generated by the experimental designs used. This work reports a process optimisation of the synthesis of red pigments by a fungal strain, Talaromyces spp. using the Taguchi methodology and proposes an approach to assess from validation trials whether the conclusions can be accepted with confidence. The work focused on optimising the inoculum characteristics, and the studied factors were spore age and concentration, agitation speed and incubation time. It was concluded that spore age was the most important factor for both responses, with optimum results at 5 days old, with the best other conditions being spores concentration, 100,000 (spores/mL); agitation, 200 rpm; and incubation time, 84 h. The interactive effects can be considered negligible and therefore this is an example where a simple experimental design approach was successful in speedily indicating conditions able to increase pigment production by 63% compared to an average choice of settings
Technological progresses in monoclonal antibody production systems

Monoclonal antibodies (mAbs) have become vitally important to modern medicine and are currently one of the major biopharmaceutical products in development. However, the high clinical dose requirements of mAbs demand a greater biomanufacturing capacity, leading to the development of new technologies for their large-scale production, with mammalian cell culture dominating the scenario. Although some companies have tried to meet these demands by creating bioreactors of increased capacity, the optimization of cell culture productivity in normal bioreactors appears as a better strategy. This review describes the main technological progresses made with this intent, presenting the advantages and limitations of each production system, as well as suggestions for improvements. New and upgraded bioreactors have emerged both for adherent and suspension cell culture, with disposable reactors attracting increased interest in the last years. Furthermore, the strategies and technologies used to control culture parameters are in constant evolution, aiming at the on-line multiparameter monitoring and considering now parameters not seen as relevant for process optimization in the past. All progresses being made have as primary goal the development of highly productive and economic mAb manufacturing processes that will allow the rapid introduction of the product in the biopharmaceutical market at more accessible prices. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 201
Functionalization of cellulose acetate fibers with engineered cutinases

In the present work, we describe for the first time the specific role of cutinase on surface modification of cellulose acetate fibers. Cutinase exhibits acetyl esterase activity on diacetate and triacetate of 0.010 U and 0.007 U, respectively. An increase on the hydroxyl groups at the fiber surface of 25% for diacetate and 317% for triacetate, after a 24 h treatment, is estimated by an indirect assay. Aiming at further improvement of cutinase affinity toward cellulose acetate, chimeric cutinases are genetically engineered by fusing the 3′-end coding sequence with a bacterial or a fungal carbohydrate-binding module and varying the linker DNA sequence. A comparative analysis of these genetic constructions is presented showing that, the superficial regeneration of cellulose hydrophilicity and reactivity on highly substituted cellulose acetates is achieved by chimeric cutinases.This work was supported by the Biosyntex Project, no. G5RD 2000-30110, from the European Community under the "Competitive and Sustainable Growth" Program, and by the PhD grant SFRH/BD/13423/2003, from Fundacao para a Ciencia e a Tecnologia
Thermal stability of carbonic anhydrase immobilized within polyurethane foam

Thermal stability of carbonic anhydrase (CA) immobilized within polyurethane (PU) foam was investigated. The catalytic activity of the enzyme was estimated by using p-nitrophenyl acetate (p-NPA) as the substrate in tris buffer containing 10% acetonitrile. The immobilized CA was stable during the repeatable washings and stability tests over 45 days stored in tris buffer at ambient conditions indicating that the CA was covalently attached to the polyurethane (PU) foam by crosslinking. The immobilized CA was found to be 98% stable below 50°C, whereas a drastic decrease was seen at temperatures between 50 and 60°C. The optimum temperature for the immobilized CA was found to be 45°C and it lost its activity completely at 60°C. Thermal deactivation energies for the free and immobilized CA were estimated to be 29 and 86 kcal/mol, respectively. The association of unfolded CA with the polymeric backbone chains of the PU foam was also addressed. It was concluded that the immobilized CA was highly stable at temperatures less than 50°C and could be used in biomimetic CO sequestration processes. © 2010 American Institute of Chemical EngineersIzmir Institute of Technology project numbers 2006IYTE29 and 2008IYTE15; Dow Chemicals HYPOL206
Protein hydrolysis by immobilized and stabilized trypsin

El pdf del artículo es la versión pre-print.-- et al.The preparation of novel immobilized and stabilized derivatives of trypsin is reported here. The new derivatives preserved 80% of the initial catalytic activity toward synthetic substrates [benzoyl-arginine p-nitroanilide (BAPNA)] and were 50,000-fold more thermally stable than the diluted soluble enzyme in the absence of autolysis. Trypsin was immobilized on highly activated glyoxyl-Sepharose following a two-step immobilization strategy: (a) first, a multipoint covalent immobilization at pH 8.5 that only involves low pKa amino groups (e.g., those derived from the activation of trypsin from trypsinogen) is performed and (b) next, an additional alkaline incubation at pH 10 is performed to favor an intense, additional multipoint immobilization between the high concentration of proximate aldehyde groups on the support surface and the high pKa amino groups at the enzyme surface region that participated in the first immobilization step. Interestingly, the new, highly stable trypsin derivatives were also much more active in the proteolysis of high molecular weight proteins when compared with a nonstabilized derivative prepared on CNBr-activated Sepharose. In fact, all the proteins contained a cheese whey extract had been completely proteolyzed after 6 h at pH 9 and 50°C, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Under these experimental conditions, the immobilized biocatalysts preserve more than 90% of their initial activity after 20 days. Analysis of the three-dimensional (3D) structure of the best immobilized trypsin derivative showed a surface region containing two amino terminal groups and five lysine (Lys) residues that may be responsible for this novel and interesting immobilization and stabilization. Moreover, this region is relatively far from the active site of the enzyme, which could explain the good results obtained for the hydrolysis of high-molecular weight proteins. © 2011 American Institute of Chemical Engineers (AIChE).Peer Reviewe
Polymer-based microparticles in tissue engineering and regenerative medicine

Different types of biomaterials, processed into different shapes, have been proposed as temporary support for cells in tissue engineering (TE) strategies. The manufacturing methods used in the production of particles in drug delivery strategies have been adapted for the development of microparticles in the fields of TE and regenerative medicine (RM). Microparticles have been applied as building blocks and matrices for the delivery of soluble factors, aiming for the construction of TE scaffolds, either by fusion giving rise to porous scaffolds or as injectable systems for in situ scaffold formation, avoiding complicated surgery procedures. More recently, organ printing strategies have been developed by the fusion of hydrogel particles with encapsulated cells, aiming the production of organs in in vitro conditions. Mesoscale self-assembly of hydrogel microblocks and the use of leachable particles in three-dimensional (3D) layer-by-layer (LbL) techniques have been suggested as well in recent works. Along with innovative applications, new perspectives are open for the use of these versatile structures, and different directions can still be followed to use all the potential that such systems can bring. This review focuses on polymeric microparticle processing techniques and overviews several examples and general concepts related to the use of these systems in TE and RE applications. The use of materials in the development of microparticles from research to clinical applications is also discussed
Large-scale compatible methods for the preservation of human embryonic stem cells: current perspectives

This article was published in the journal, Biotechnology Progress [© American Institute of Chemical Engineers (AIChE)] and the definitive version is available at: http://dx.doi.org/10.1002/btpr.680Human embryonic stem cells (hESCs) and hESC-derived cells are of great interest, not only because of their therapeutic potential but also their prospective uses in in vitro drug and toxicity screening. The ability to preserve these cells is critical, allowing for the generation of quality-controlled stocks of cells, transport of cells between sites, and avoiding the need for expensive and time-consuming continuous culture. Current methodologies, namely conventional slow freezing and vitrification, can successfully preserve hESCs and their differentiated progeny, retaining the key characteristics of the cells. However, there is a significant gap between the number of cells potentially needed to either treat patients or run a high-throughput drug screen and how many cells can be preserved using these techniques. Therefore, this review focuses on the scalability of slow freezing and vitrification, identifying key barriers to success and whether they can be overcome. Given the precedent with other mammalian cells in using slow freezing to successfully preserve large quantities of cells and its compatibility with current and emerging culture methods for hESCs, it is likely to become the method of choice for cryopreserving these cells at scale. However, issues other than scale still exist; therefore, alternatives to cryopreservation should also be explored. Here, the potential to lyophilize hESCs for long-term storage is considered as one such alternative