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Manufacturing has been the key factor limiting rollout of vaccination during the COVID-19 pandemic, requiring rapid development and large-scale implementation of novel manufacturing technologies. ChAdOx1 nCoV-19 (AZD1222, Vaxzevria) is an efficacious vaccine against SARS-CoV-2, based upon an adenovirus vector. We describe the development of a process for the production of this vaccine and others based upon the same platform, including novel features to facilitate very large-scale production. We discuss the process economics and the "distributed manufacturing" approach we have taken to provide the vaccine at globally-relevant scale and with international security of supply. Together, these approaches have enabled the largest viral vector manufacturing campaign to date, providing a substantial proportion of global COVID-19 vaccine supply at low cost.
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Vacinas contra COVID-19 , COVID-19/prevenção & controle , ChAdOx1 nCoV-19 , Indústria Farmacêutica/métodos , Desenvolvimento de Vacinas , Animais , Escherichia coli , Geografia , Células HEK293 , Humanos , Pan troglodytes , SARS-CoV-2 , Tecnologia Farmacêutica , Vacinação/instrumentaçãoRESUMO
INTRODUCTION: Mammalian cells like Chinese hamster ovary (CHO) cells are routinely used for production of recombinant therapeutic proteins. Cells require a continuous supply of energy and nutrients to sustain high cell densities whilst expressing high titres of recombinant proteins. Cultured mammalian cells are primarily dependent on glucose and glutamine metabolism for energy production. OBJECTIVES: The TCA cycle is the main source of energy production and its continuous flow is essential for cell survival. Modulated regulation of TCA cycle can affect ATP production and influence CHO cell productivity. METHODS: To determine the key metabolic reactions of the cycle associated with cell growth in CHO cells, we transiently silenced each gene of the TCA cycle using RNAi. RESULTS: Silencing of at least four TCA cycle genes was detrimental to CHO cell growth. With an exception of mitochondrial aconitase (or Aco2), all other genes were associated with ATP production reactions of the TCA cycle and their resulting substrates can be supplied by other anaplerotic and cataplerotic reactions. This study is the first of its kind to have established key role of aconitase gene in CHO cells. We further investigated the temporal effects of aconitase silencing on energy production, CHO cell metabolism, oxidative stress and recombinant protein production. CONCLUSION: Transient silencing of mitochondrial aconitase inhibited cell growth, reduced ATP production, increased production of reactive oxygen species and reduced cell specific productivity of a recombinant CHO cell line by at least twofold.
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Aconitato Hidratase/metabolismo , Mitocôndrias/enzimologia , Trifosfato de Adenosina/biossíntese , Animais , Células CHO , Proliferação de Células , CricetulusRESUMO
Aliphatic crystallites, characteristic of the eicosane and docosane components of naturally occurring lipids, were found to form microtextures that were structured by specific interactions with ordered graphite (HOPG) used as the underlying substratum, as confirmed by scanning electron microscopy (SEM) and fast Fourier transform (FFT) analysis. Confocal scanning laser microscopy (CLSM) showed highly directed bacterial alignment for two bacterial species (spherical and rod-shaped), reflecting the preferential orientation of the crystallite-air-water interfaces to give linear and triangular bacterial patterning. The mechanisms of bacterial attachment are demonstrated in terms of the balance between effective radial adhesional forces and the capillary forces resulting from the water contact angle of the bacteria at the three-phase line (TPL) of the lipid surface. It is suggested that these microtextured surfaces, which exhibit the ability to limit bacterial adhesion to a precise patterning at the lipid TPL, could be used as a means of controlling bacterial colonization.
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Alcanos/química , Aderência Bacteriana , Lipídeos/química , Análise de Fourier , Teste de Materiais , Microscopia de Força Atômica , Microscopia Confocal , Microscopia Eletrônica de Varredura , Pseudomonas aeruginosa/crescimento & desenvolvimento , Análise Espectral Raman , Staphylococcus aureus/crescimento & desenvolvimento , Propriedades de Superfície , MolhabilidadeRESUMO
We describe for the first time the creation of a library of 140 synthetic promoters specifically designed to regulate the expression of recombinant genes in CHO cells. Initially, 10 common viral promoter sequences known to be active in CHO cells were analyzed using bioinformatic sequence analysis programs to determine the identity and relative abundance of transcription factor regulatory elements (TFREs; or transcription factor binding sites) they contained. Based on this, 28 synthetic reporters were constructed that each harbored seven repeats of a discrete TFRE sequence upstream of a minimal CMV core promoter element and secreted alkaline phosphatase (SEAP) reporter gene. After evaluation of the relative activity of TFREs by transient expression in CHO-S cells, we constructed a first generation library of 96 synthetic promoters derived from random ligation of six active TFREs inserted into the same reporter construct backbone. Comparison of the sequence and relative activity of first generation promoters revealed that individual TFRE blocks were either relatively abundant in active promoters (NFκB, E-box), equally distributed across promoters of varying activity (C/EBPα, GC-box) or relatively abundant in low activity promoters (E4F1, CRE). These data were utilized to create a second generation of 44 synthetic promoters based on random ligation of a fixed ratio of 4 TFREs (NFκB 5: E-box 3: C/EBPα 1: GC-box 1). Comparison of the sequence and relative activity of second generation promoters revealed that the most active promoters contained relatively high numbers of both NFκB and E-box TFREs in approximately equal proportion, with a correspondingly low number of GC-box and C/EBPα blocks. The most active second generation promoters achieved approximately twice the activity of a control construct harboring the human cytomegalovirus (CMV) promoter. Lastly, we evaluated the function of a subset of synthetic promoters exhibiting a broad range of activity in different CHO cell host cell lines (CHO-S, CHO-K1, and CHO-DG44) and across extended fed-batch transient expression in CHO-S cells. In general, the different synthetic promoters both maintained their relative activity and the most active promoters consistently and significantly exceeded the activity of the CMV control promoter. For advanced cell engineering strategies our synthetic promoter libraries offer precise control of recombinant transcriptional activity in CHO cells spanning over two orders of magnitude.
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Células CHO/metabolismo , Engenharia Celular/métodos , Regiões Promotoras Genéticas , Ativação Transcricional , Animais , Sequência de Bases , Cricetulus , Genes Reporter , Engenharia Genética/métodos , Humanos , Dados de Sequência Molecular , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
Transcription-factor decoys are short synthetic oligodeoxynucleotides that sequester cognate transcription factors and prevent their binding at target promoters. Current methods of decoy formation have primarily been optimized for potential therapeutic applications. However, they are not ideally suited to in vitro investigations into multi-transcription factor-mediated processes that may require multiple regulatory elements to be inhibited in varying combinations. In this study we describe a novel method for chimeric decoy formation in which blocks containing discrete transcription factor binding sites are combined into circular molecules. Unlike currently available methods, block decoys allow rapid construction of chimeric decoys targeting multiple regulatory elements. Further, they enable fine-tuning of binding-site copy ratios within chimeras, allowing sophisticated control of the cellular transcriptional landscape. We show that block decoys are exonuclease-resistant and specifically inhibit expression from target binding sites. The potential of block decoys to inhibit multiple elements simultaneously was demonstrated using a chimeric decoy containing molar optimized ratios of three regulatory elements, NF-κB-RE, CRE, and E-box. The chimeric decoy inhibited expression from all three elements simultaneously at equivalent levels. The primary intended use of block decoys is in vitro gene regulation studies in which bespoke chimeras can be rapidly constructed and utilized to determine a promoter's functional regulation.
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Regulação da Expressão Gênica , NF-kappa B/metabolismo , Oligodesoxirribonucleotídeos/metabolismo , Sequências Reguladoras de Ácido Nucleico , Animais , Sequência de Bases , Sítios de Ligação , Células CHO , Cricetulus , Elementos E-Box , Genes Reporter , Oligodesoxirribonucleotídeos/química , Regiões Promotoras GenéticasRESUMO
It has become increasingly recognized that polymer particle size can have a profound effect on the interactions of particle-based vaccines with antigen presenting cells (APCs) thereby influencing and modulating ensuing immune responses. With the aim of developing chitosan particle-based immunocontraceptive vaccines, we have compared the use of chitosan-based nanoparticles and chitosan-based microparticles as vaccine delivery vehicles for vaccine candidates based on luteinizing hormone-releasing hormone (LHRH). Particles, functionalized with chloroacetyl groups, which allows the covalent attachment of thiol-containing antigens, were able to adsorb ~60-70% of their weight of peptide-based antigen and 10-20% of their weight of protein-based antigen. Quantitation by amino acid analysis of antigen associated with particles demonstrated a correlation between associated antigen and the degree of chloracetylation of particles. Visualization of fluorescently labeled antigen-loaded particles by confocal microscopy indicated that the majority of antigen was localized at the particle surface with a smaller amount located in the interior. We also found that uptake of both fluoresceinated nanoparticles and microparticles by dendritic cells occurred in a manner dependent on particle concentration. Nanoparticles trafficked from the injection site to draining lymph nodes faster than microparticles; high numbers of nanoparticle-bearing cells appeared in draining lymph nodes on day 3 and microparticles on day 4. This difference in trafficking rate did not, however, appear to have any significant impact on the ensuing immune response because inoculation with both peptide-conjugated and protein-conjugated particles induced high levels of LHRH-specific antibodies. In the case of protein-conjugated particles, the levels of antibodies elicited were similar to those elicited following inoculation with antigen emulsified with complete Freund's adjuvant. The approach to vaccine design that we have described here could represent another useful method for inducing immune responses against microbial, viral and tumorigenic protein antigens.
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Quitosana/química , Portadores de Fármacos/administração & dosagem , Hormônio Liberador de Gonadotropina/administração & dosagem , Nanopartículas/química , Fragmentos de Peptídeos/administração & dosagem , Vacinas Anticoncepcionais/administração & dosagem , Acetatos/química , Acetilação/efeitos dos fármacos , Anidridos/química , Animais , Células Cultivadas , Quitosana/metabolismo , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Portadores de Fármacos/metabolismo , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/uso terapêutico , Composição de Medicamentos , Feminino , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Hormônio Liberador de Gonadotropina/farmacocinética , Hormônio Liberador de Gonadotropina/uso terapêutico , Halogenação/efeitos dos fármacos , Imunidade Ativa , Linfonodos/imunologia , Linfonodos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microesferas , Nanopartículas/ultraestrutura , Tamanho da Partícula , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/farmacocinética , Fragmentos de Peptídeos/uso terapêutico , Propriedades de Superfície , Distribuição Tecidual , Vacinas Anticoncepcionais/metabolismo , Vacinas Anticoncepcionais/farmacocinética , Vacinas Anticoncepcionais/uso terapêuticoRESUMO
In this work, we report on the incorporation of a siloxane copolymer additive, poly((2-phenylethyl) methylsiloxane)-co(1-phenylethyl) methylsiloxane)-co-dimethylsiloxane), which is fully soluble at room temperature, in a rapid-cure thermoset polyester coating formulation. The additive undergoes polymerization-induced phase segregation (PIPS) to self-assemble on the coating surface as discrete discoid nanofeatures during the resin cure process. Moreover, the copolymer facilitates surface co-segregation of titanium dioxide pigment microparticulate present in the coating. Depending on the composition, the coatings can display persistent superhydrophobicity and self-cleaning properties and, surprisingly, the titanium dioxide pigmented coatings that include the siloxane copolymer additive display high levels of antibacterial performance against Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria. This antibacterial performance is believed to be associated with the unique surface topology of these coatings, which comprise stimuli-responsive discoid nanofeatures. This paper provides details of the surface morphology of the coatings and how these relates to the antimicrobial properties of the coating.
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The purpose of this study was to compare the effects of two practical precooling techniques (skin cooling vs. skin + core cooling) on cycling time trial performance in warm conditions. Six trained cyclists completed one maximal graded exercise test (VO2(peak) 71.4 +/- 3.2 ml x kg(-1) x min(-1)) and four approximately 40 min laboratory cycling time trials in a heat chamber (34.3 degrees C +/- 1.1 degrees C; 41.2% +/- 3.0% rh) using a fixed-power/variable-power format. Cyclists prepared for the time trial using three techniques administered in a randomised order prior to the warm-up: (1) no cooling (control), (2) cooling jacket for 40 min (jacket) or (3) 30-min water immersion followed by a cooling jacket application for 40 min (combined). Rectal temperature prior to the time trial was 37.8 degrees C +/- 0.1 degrees C in control, similar in jacket (37.8 degrees C +/- 0.3 degrees C) and lower in combined (37.1 degrees C +/- 0.2 degrees C, P < 0.01). Compared with the control trial, time trial performance was not different for jacket precooling (-16 +/- 36 s, -0.7%; P = 0.35) but was faster for combined precooling (-42 +/- 25 s, - .8%; P = 0.009). In conclusion, a practical combined precooling strategy that involves immersion in cool water followed by the use of a cooling jacket can produce decrease in rectal temperature that persist throughout a warm-up and improve laboratory cycling time trial performance in warm conditions.
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Temperatura Corporal , Exercício Físico/fisiologia , Adulto , Temperatura Corporal/fisiologia , Regulação da Temperatura Corporal/fisiologia , Temperatura Baixa , Estudos Cross-Over , Frequência Cardíaca , Humanos , Imersão , Masculino , Resistência Física/fisiologia , Análise e Desempenho de TarefasRESUMO
Wrinkled patterns, which possess an extensive surface area over a limited planar space, can provide surface features ranging across the nano- and microscale that have become an engineering material with the flexibility to be tuneable for a number of technologies. Here, we investigate the surface parameters that influence the attachment response of two model bacteria (P. aeruginosa and S. aureus) to wrinkled gold-coated polystyrene surfaces having topologies at the nano- and microscale. Together with flat gold films as the controls, surface feature heights spanned 2 orders of magnitude (15 nm, 200 nm, and 1 micron). The surface wrinkle topology was shown through confocal laser scanning microscopic, atomic force microscopic and scanning electron microscopic image analyses to consist of air-water interfacial areas unavailable for bacterial attachment, which were also shown to be stable by time-lapsed contact angle measurements. Imposition of the nanoscale wrinkles reduced P. aeruginosa attachment to 57% and S. aureus attachment to 20% of their flat equivalent surfaces whereas wrinkles at the microscale further reduced these attachments to 7.5% and 14.5%, respectively. The density of attachments indicated an inherent species specific selectivity that changed with feature dimension, attributable to the scale of the air-water interfaces in contact with the bacterial cell. Parameters influencing static bacterial attachment were the total projected surface areas minus the air-water interface areas and the scale of these respective air-water interfaces (area distribution) with respect to the cell morphology. The range of these controlling parameters may provide new design principles for the evolving suite of physical anti-biofouling materials not reliant on biocidal agents under development.
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Aderência Bacteriana , Incrustação Biológica , Ouro , Poliestirenos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Staphylococcus aureus/crescimento & desenvolvimento , Propriedades de SuperfícieRESUMO
One of the major challenges faced by the biomedical industry is the development of robust synthetic surfaces that can resist bacterial colonization. Much inspiration has been drawn recently from naturally occurring mechano-bactericidal surfaces such as the wings of cicada (Psaltoda claripennis) and dragonfly (Diplacodes bipunctata) species in fabricating their synthetic analogs. However, the bactericidal activity of nanostructured surfaces is observed in a particular range of parameters reflecting the geometry of nanostructures and surface wettability. Here, several of the nanometer-scale characteristics of black silicon (bSi) surfaces including the density and height of the nanopillars that have the potential to influence the bactericidal efficiency of these nanostructured surfaces have been investigated. The results provide important evidence that minor variations in the nanoarchitecture of substrata can substantially alter their performance as bactericidal surfaces.
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A method for the evaluation of quantities that are experimentally inaccessible such as the surface tension at the solid-vacuum interface and the superficial tension of the fluid in contact with the solid is presented. The approach is based on consideration of an equilibrium of a fluid in solid capillary wherein a balance between surface and capillary forces has been replaced by conceptual alternative interfacial and centrifugal forces. This approach involves the simultaneous numerical solution one the special forms of the Gibbs equation for solid-fluid interface and a generalized Kelvin equation derived earlier. The latter equation takes into account interactions between the solid thick cylindrical wall and confined fluid, this body-body interaction potential has been primarily calculated using the Lennard-Jones (6-12) expression for the atom-atom pair potentials and expressed by hypergeometrical functions having good convergences. All numerical calculations shown here have been performed for the model graphite-argon system at 90 K. Finally, an analysis of the accuracy of the proposed method is considered.
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Managing the impact of anthropogenic and climate induced stress on plant growth remains a challenge. Here we show that polymeric hydrogels, which maintain their hydrous state, can be designed to exploit functional interactions with soil microorganisms. This microbial enhancement may mitigate biotic and abiotic stresses limiting productivity. The presence of mannan chains within synthetic polyacrylic acid (PAA) enhanced the dynamics and selectivity of bacterial ingress in model microbial systems and soil microcosms. Pseudomonas fluorescens exhibiting high mannan binding adhesins showed higher ingress and localised microcolonies throughout the polymeric network. In contrast, ingress of Bacillus subtilis, lacking adhesins, was unaltered by mannan showing motility comparable to bulk liquids. Incubation within microcosms of an agricultural soil yielded hydrogel populations significantly increased from the corresponding soil. Bacterial diversity was markedly higher in mannan containing hydrogels compared to both control polymer and soil, indicating enhanced selectivity towards microbial families that contain plant beneficial species. Here we propose functional polymers applied to the potential root zone which can positively influence rhizobacteria colonization and potentially plant growth as a new approach to stress tolerance.
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Bactérias/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , Polímeros/farmacologia , Bacillus subtilis/efeitos dos fármacos , Bactérias/efeitos dos fármacos , Biodiversidade , Contagem de Colônia Microbiana , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Raízes de Plantas/efeitos dos fármacos , Porosidade , Análise de Componente Principal , Microbiologia do SoloRESUMO
The wings of insects such as cicadas and dragonflies have been found to possess nanostructure arrays that are assembled from fatty acids. These arrays can physically interact with the bacterial cell membranes, leading to the death of the cell. Such mechanobactericidal surfaces are of significant interest, as they can kill bacteria without the need for antibacterial chemicals. Here, we report on the bactericidal effect of two of the main lipid components of the insect wing epicuticle, palmitic (C16) and stearic (C18) fatty acids. Films of these fatty acids were re-crystallised on the surface of highly ordered pyrolytic graphite. It appeared that the presence of two additional CH2 groups in the alkyl chain resulted in the formation of different surface structures. Scanning electron microscopy and atomic force microscopy showed that the palmitic acid microcrystallites were more asymmetric than those of the stearic acid, where the palmitic acid microcrystallites were observed to be an angular abutment in the scanning electron micrographs. The principal differences between the two types of long-chain saturated fatty acid crystallites were the larger density of peaks in the upper contact plane of the palmitic acid crystallites, as well as their greater proportion of asymmetrical shapes, in comparison to that of the stearic acid film. These two parameters might contribute to higher bactericidal activity on surfaces derived from palmitic acid. Both the palmitic and stearic acid crystallite surfaces displayed activity against Gram-negative, rod-shaped Pseudomonas aeruginosa and Gram-positive, spherical Staphylococcus aureus cells. These microcrystallite interfaces might be a useful tool in the fabrication of effective bactericidal nanocoatings. STATEMENT OF SIGNIFICANCE: Nanostructured cicada and dragonfly wing surfaces have been discovered to be able physically kill bacterial cells. Here, we report on the successful fabrication of bactericidal three-dimensional structures of two main lipid components of the epicuticle of insect wings, palmitic (C16) and stearic (C18) acids. After crystallisation onto highly ordered pyrolytic graphite, both the palmitic and stearic acid films displayed bactericidal activity against both Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus cells. The simplicity of the production of these microcrystallite interfaces suggests that a fabrication technique, based on solution deposition, could be an effective technique for the application of bactericidal nanocoatings.
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Antibacterianos , Grafite , Ácido Palmítico , Pseudomonas aeruginosa/crescimento & desenvolvimento , Staphylococcus aureus/crescimento & desenvolvimento , Ácidos Esteáricos , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Grafite/química , Grafite/farmacologia , Hemípteros/química , Odonatos/química , Ácido Palmítico/farmacologia , Ácidos Esteáricos/farmacologia , Propriedades de SuperfícieRESUMO
While insect wings are widely recognised as multi-functional, recent work showed that this extends to extensive bactericidal activity brought about by cell deformation and lysis on the wing nanotopology. We now quantitatively show that subtle changes to this topography result in substantial changes in bactericidal activity that are able to span an order of magnitude. Notably, the chemical composition of the lipid nanopillars was seen by XPS and synchrotron FTIR microspectroscopy to be similar across these activity differences. Modelling the interaction between bacterial cells and the wing surface lipids of 3 species of dragonflies, that inhabit similar environments, but with distinctly different behavioural repertoires, provided the relationship between surface structure and antibacterial functionality. In doing so, these principal behavioural patterns correlated with the demands for antimicrobial efficiency dictated by differences in their foraging strategies. This work now reveals a new feature in the design elegance of natural multi-functional surfaces as well providing insights into the bactericidal mechanism underlying inherently antimicrobial materials, while suggesting that nanotopology is related to the evolutionary development of a species through the demands of its behavioural repertoire. The underlying relationship between the processes of wetting, adhesion and capillarity of the lipid nanopillars and bactericidal efficiency suggests new prospects for purely mechano-responsive antibacterial surfaces.
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Nanotecnologia/métodos , Odonatos/classificação , Asas de Animais/fisiologia , Animais , Antibacterianos/química , Bacillus subtilis , Biomimética , Lipídeos/química , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Pseudomonas aeruginosa , Especificidade da Espécie , Espectroscopia de Infravermelho com Transformada de Fourier , Staphylococcus aureus , Propriedades de Superfície , Síncrotrons , MolhabilidadeRESUMO
With an aging population and the consequent increasing use of medical implants, managing the possible infections arising from implant surgery remains a global challenge. Here, we demonstrate for the first time that a precise nanotopology provides an effective intervention in bacterial cocolonization enabling the proliferation of eukaryotic cells on a substratum surface, preinfected by both live Gram-negative, Pseudomonas aeruginosa, and Gram-positive, Staphylococcus aureus, pathogenic bacteria. The topology of the model black silicon (bSi) substratum not only favors the proliferation of eukaryotic cells but is biocompatible, not triggering an inflammatory response in the host. The attachment behavior and development of filopodia when COS-7 fibroblast cells are placed in contact with the bSi surface are demonstrated in the dynamic study, which is based on the use of real-time sequential confocal imaging. Bactericidal nanotopology may enhance the prospect for further development of inherently responsive antibacterial nanomaterials for bionic applications such as prosthetics and implants.
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Células Eucarióticas , Antibacterianos , Nanoestruturas , Pseudomonas aeruginosa , Staphylococcus aureus , Propriedades de SuperfícieRESUMO
Previous clinical studies on a subcutaneous injectable suspension of levodopa showed poor injectability into human tissue. When this formulation was rheologically characterised, a clinical shear thickening interval was observed at increased shear rates. The formulation parameters that contributed to this rheological behavior were systematically evaluated with the aim of removing this flow limitation while maintaining the concentration of 60% levodopa to retain the clinical applicability. The three suspension parameters examined were: levodopa volume fraction, concentration of the HPMC suspending vehicle, and particle size distribution. Shear thickening increased with the drug concentration and the critical shear rate was inversely dependent on the drug concentration. Increasing the vehicle concentration retarded the shear thickening but increased the overall suspension viscosity. There was an increase in shear thickening with increased average particle diameter. Combinations of micronized and non-micronized particles were used to prepare bimodal particle size distributions. The rheology of these bimodal distributions resulted in removal of shear thickening. This allowed the preparation of 60% levodopa formulations that showed a range of flow characteristics spanning near Newtonian flow or shear thinning at initial injectable viscosities of about 0.6 Pa.s and final viscosities in the range of 0.1 Pa.s, alleviating the shear thickening limitation of these levodopa formulations.
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Dopaminérgicos/química , Levodopa/química , Química Farmacêutica , Derivados da Hipromelose , Injeções Subcutâneas , Metilcelulose/análogos & derivados , Metilcelulose/química , Tamanho da Partícula , Veículos Farmacêuticos/química , Reologia , Estresse Mecânico , Suspensões , ViscosidadeRESUMO
Transient gene expression (TGE) in CHO cells is utilized to produce material for use in early stage drug development. These systems typically utilize the cytomegalovirus (CMV) promoter to drive recombinant gene transcription. In this study, we have mechanistically dissected CMV-mediated TGE in CHO cells in order to identify the key regulators of this process. An in silico analysis of the promoter composition of transcription factor regulatory elements (TFREs) and the CHO cell repertoire of transcription factors identified eight TFREs as likely effectors of CMV activity. We determined the regulatory function of these elements by preventing their cognate transcription factors from binding at the CMV promoter. This was achieved by both scrambling promoter binding site sequences and using decoy molecules to sequester intracellular transcription factors. We determined that the vast majority of CMV activity is mediated by just two discrete TFREs, showing that simultaneous inhibition of NF-κB and CRE-mediated transactivation reduced CMV-driven transient secreted alkaline phosphatase (SEAP) production by over 75%. Further, we identified a mechanism by which CMV-mediated TGE is negatively regulated in CHO cells, showing that inhibition of YY1-mediated transrepression increased SEAP production 1.5-fold. This work enables optimization and control of CMV-mediated TGE in CHO cells, in order to improve transient protein production yields.
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Fosfatase Alcalina/biossíntese , Células CHO , NF-kappa B/genética , Transcrição Gênica , Fator de Transcrição YY1/genética , Fosfatase Alcalina/genética , Animais , Sítios de Ligação , Cricetinae , Cricetulus , Citomegalovirus/genética , Descoberta de Drogas , Expressão Gênica , Integrases/genética , Regiões Promotoras Genéticas , Fator de Transcrição YY1/biossínteseRESUMO
Studies of microbial interactions during motility, micro-structuring and colonisation have predominately been limited to surface associated bacteria involving materials such as semi-solid biomolecular hydrogels and thin liquid films. Recently, these surfaces have been extended to synthetic polymers where they provide defined chemistries and structural properties. However, precise details of microbial ingress into the confined fluid volume of synthetic 3-D hydrogel networks and their subsequent microstructuring remain to be defined. Here, we show that Gram-positive and Gram-negative bacteria internally populate mesoporous polyacrylate hydrogels by quantifying: the dynamic advancing population front and the resultant spontaneous self-organisation into well-defined clusters and micro-colonies. Polymer chain conjugated fluorescent nanoparticles indicated that both bacterial clusters and micro-colonies associated directly with the polymer chains of the mesoporous hydrogel. Protonation of the K-polyacrylate made chains more hydrophobic and globular in conformation, reducing the swelling of the hydrogel by half. However, the bacterial population increased by 30% indicating the dominance of hydrophobic and viscoelastic interactions as well as the cation chemistry within the confined fluids of synthetic polymer hydrogels despite pore size reductions of 50%. Synthetic polymer hydrogels having a range of porosities when swollen together with controllable chemical and structural functionality can potentially offer well-defined microenvironments for bacterial populations in advancing biotechnologies such as inoculants and substrates in the production of therapeutic agents.
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The impact of non- and poorly wetting soils has become increasingly important, due to its direct influence on the water-limited potential yield of rain-fed grain crops at a time of enhanced global competition for fresh water. This study investigates the physical and compositional mechanisms underlying the influence of soil organic matter (SOM) on the wetting processes of model systems. These model systems are directly related to two sandy wheat-producing soils that have contrasting hydrophobicities. Atomic force microscopy (AFM), contact angle and Raman micro-spectroscopy measurements on model planar and particulate SOM-containing surfaces demonstrated the role of the hierarchical surface structure on the wetting dynamics of packed particulate beds. It was found that a nanoscale surface topology is superimposed over the microscale roughness of the packed particles, and this controls the extent of water ingress into particulate packed beds of these particles. Using two of the dominant component organic species found in the SOM of the two soils used in this study, it was found that the specific interactions taking place between the SOM components, rather than their absolute quantities, dictated the formation of highly hydrophobic surface nanotopologies. This hydrophobicity was demonstrated, using micro-Raman imaging, to arise from the surface being in a composite Cassie-Baxter wetting state. Raman imaging demonstrated that the particle surface nanotopography influenced the degree of air entrapment in the interstices within the particle bed. The influence of a conventional surfactant on the wetting kinetics of both the model planar surfaces and packed particulate beds was quantified in terms of their respective advancing contact angles and the capillary wetting force vector. The information obtained for all of the planar and particulate surfaces, together with that obtained for the two soils, allowed linear relationships to be obtained in plots of the contact angle data as a function of the wetting liquid surface tensions. These linear relationships were found to reflect the mechanisms underlying the surface energy parameter requirements for wetting.
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In this study, we describe a biodegradable vaccine depot which persists in vivo for at least 4-months, provides synergistic adjuvant effects and also allows dose sparing of both antigen and adjuvant. A single administration results in immediate release of a priming dose of vaccine, by a process of syneresis, which is then followed by release of remaining vaccine which maintains robust antibody levels that last for more than a year. The platform technology comprises two aqueous components; one contains chitosan and hydroxyapatite, in which the vaccine is incorporated, and the other consists of a crosslinking agent, tripolyphosphate (TPP) and chondroitin sulphate. When co-injected into tissue, they spontaneously crosslink forming a firm yet compliant vaccine-containing depot. Whole body imaging of animals inoculated with the material show that the depot persists in situ for up to 19 weeks. Vaccination of mice with depot formulations containing ovalbumin (OVA) emulsified in Montanide ISA 61 adjuvant results in the induction of robust antibody responses using doses of adjuvant 40-fold less than those recommended by the manufacturer. Dose sparing effects were also apparent with antigen when delivered in the depot. Similar dose sparing effects were observed with Montanide ISA 50, complete and incomplete Freund's adjuvants but not with aluminium hydroxide nor Quil A. Antibody titres, induced by a single dose of antigen/adjuvant formulation incorporated in the depot, persisted at high levels for at least 55 weeks following a single dose of vaccine.