Characterization of TAP Ambr 250 disposable bioreactors,as a reliable scale‐down model for biologics process development

Demands for development of biological therapies is rapidly increasing, as is the drive to reduce time to patient. In order to speed up development, the disposable Automated Microscale Bioreactor (Ambr 250) system is increasingly gaining interest due to its advantages, including highly automated control, high throughput capacity, and short turnaround time. Traditional early stage upstream process development conducted in 2 ‐ 5 L bench‐top bioreactors requires high foot‐print, and running cost. The establishment of the Ambr 250 as a scale‐down model leads to many benefits in process development. In this study, a comprehensive characterization of mass transfer coefficient (k_La) in the Ambr 250 was conducted to define optimal operational conditions. Scale‐down approaches, including dimensionless volumetric flow rate (vvm), power per unit volume (P/V) and k_La have been evaluated using different cell lines. This study demonstrates that the Ambr 250 generated comparable profiles of cell growth and protein production, as seen at 5‐L and 1000‐L bioreactor scales, when using k_La as a scale‐down parameter. In addition to mimicking processes at large scales, the suitability of the Ambr 250 as a tool for clone selection, which is traditionally conducted in bench‐top bioreactors, was investigated. Data show that cell growth, productivity, metabolite profiles, and product qualities of material generated using the Ambr 250 were comparable to those from 5‐L bioreactors. Therefore, Ambr 250 can be used for clone selection and process development as a replacement for traditional bench‐top bioreactors minimizing resource utilization during the early stages of development in the biopharmaceutical industry. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:478–489, 2017     

Available nitrogen: A time-based study of manipulated resource islands

Spatial and temporal heterogeneity of available nitrogen are critical determinants of the distribution and abundance of plants and animals in ecosystems. Evidence for the resource island theory suggests that soils below tree and shrub canopies contain higher amounts of resources, including available nitrogen, than are present in interspace areas. Disturbances, such as prescribed fire and tree removal, are common management practices in shrub-woodland ecosystems, but it is not known if these practices affect resource islands. We examined temporal variation in resource islands of available nitrogen and their retention after fire and woody plant removal. From August 1997 to October 1998, soil nitrate (NO₃^–) and ammonium (NH₄⁺) were measured monthly from canopy and interspace plots within four juniper-sagebrush sites along a precipitation gradient in central Oregon, USA. At each site, soil samples were collected from untreated plots, plots in which woody plants were removed, and those treated with prescribed fire in fall 1997. In burned treatments, canopy concentrations were significantly higher than interspace concentrations throughout the measurement period. Canopy NO₃^– and NH₄⁺ concentrations were significantly higher on burned vs. unburned treatments for four months after fire. After woody plant removal, NO₃^– and NH₄⁺ concentrations did not differ from the controls. Untreated control areas had higher NO₃^– and NH₄⁺ concentrations under juniper canopies for nearly all months. Wetter sites had smaller differences between canopy and interspace concentrations through time than did the two drier sites. In relation to NO₃^– and NH₄⁺ in this ecosystem, resource islands appear to be more ephemeral in wetter sites, and more pronounced following fire disturbances than in controls or those treated by woody plant removal.     

Structural and biophysical characterization of the EphB4*ephrinB2 protein-protein interaction and receptor specificity

Increasing evidence implicates the interaction of the EphB4 receptor with its preferred ligand, ephrinB2, in pathological forms of angiogenesis and in tumorigenesis. To identify the molecular determinants of the unique specificity of EphB4 for ephrinB2, we determined the crystal structure of the ligand binding domain of EphB4 in complex with the extracellular domain of ephrinB2. This structural analysis suggested that one amino acid, Leu-95, plays a particularly important role in defining the structural features that confer the ligand selectivity of EphB4. Indeed, all other Eph receptors, which promiscuously bind many ephrins, have a conserved arginine at the position corresponding to Leu-95 of EphB4. We have also found that amino acid changes in the EphB4 ligand binding cavity, designed based on comparison with the crystal structure of the more promiscuous EphB2 receptor, yield EphB4 variants with altered binding affinity for ephrinB2 and an antagonistic peptide. Isothermal titration calorimetry experiments with an EphB4 Leu-95 to arginine mutant confirmed the importance of this amino acid in conferring high affinity binding to both ephrinB2 and the antagonistic peptide ligand. Isothermal titration calorimetry measurements also revealed an interesting thermodynamic discrepancy between ephrinB2 binding, which is an entropically driven process, and peptide binding, which is an enthalpically driven process. These results provide critical information on the EphB4*ephrinB2 protein interfaces and their mode of interaction, which will facilitate development of small molecule compounds inhibiting the EphB4*ephrinB2 interaction as novel cancer therapeutics.     

Quantitative profiling of drug-associated proteomic alterations by combined 2-nitrobenzenesulfenyl chloride (NBS) isotope labeling and 2DE/MS identification

The identification of drug-responsive biomarkers in complex protein mixtures is an important goal of quantitative proteomics. Here, we describe a novel approach for identifying such drug-induced protein alterations, which combines 2-nitrobenzenesulfenyl chloride (NBS) tryptophan labeling with two-dimensional gel electrophoresis (2DE)/mass spectrometry (MS). Lysates from drug-treated and control samples are labeled with light or heavy NBS moiety and separated on a common 2DE gel, and protein alterations are identified by MS through the differential intensity of paired NBS peptide peaks. Using NBS/2DE/MS, we profiled the proteomic alterations induced by tamoxifen (TAM) in the estrogen receptor (ER) positive MCF-7 breast cancer cell line. Of 88 protein spots that significantly changed upon TAM treatment, 44 spots representing 23 distinct protein species were successfully identified with NBS-paired peptides. Of these 23 TAM-altered proteins, 16 (70%) have not been previously associated with TAM or ER activity. We found the NBS labeling procedure to be both technically and biologically reproducible, and the NBS/2DE/MS alterations exhibited good concordance with conventional 2DE differential protein quantitation, with discrepancies largely due to the comigration of distinct proteins in the regular 2DE gels. To validate the NBS/2DE/MS results, we used immunoblotting to confirm GRP78, CK19, and PA2G4 as bona fide TAM-regulated proteins. Furthermore, we demonstrate that PA2G4 expression can serve as a novel prognostic factor for disease-free survival in two independent breast cancer patient cohorts. To our knowledge, this is the first report describing the proteomic changes in breast cancer cells induced by TAM, the most commonly used selective estrogen receptor modulator (SERM). Our results indicate that NBS/2DE/MS may represent a more reliable approach for cellular protein quantitation than conventional 2DE approaches.     

Analysis of Immune Response Markers in Jorge Lobo's Disease Lesions Suggests the Occurrence of Mixed T Helper Responses with the Dominance of Regulatory T Cell Activity

Jorge Lobo’s disease (JLD) is a chronic infection that affects the skin and subcutaneous tissues. Its etiologic agent is the fungus Lacazia loboi. Lesions are classified as localized, multifocal, or disseminated, depending on their location. Early diagnosis and the surgical removal of lesions are the best therapeutic options currently available for JLD. The few studies that evaluate the immunological response of JLD patients show a predominance of Th2 response, as well as a high frequency of TGF-β and IL-10 positive cells in the lesions; however, the overall immunological status of the lesions in terms of their T cell phenotype has yet to be determined. Therefore, the objective of this study was to evaluate the pattern of Th1, Th2, Th17 and regulatory T cell (Treg) markers mRNA in JLD patients by means of real-time PCR. Biopsies of JLD lesions (N = 102) were classified according to their clinical and histopathological features and then analyzed using real-time PCR in order to determine the expression levels of TGF-β1, FoxP3, CTLA4, IKZF2, IL-10, T-bet, IFN-γ, GATA3, IL-4, IL-5, IL-13, IL-33, RORC, IL-17A, IL-17F, and IL-22 and to compare these levels to those of healthy control skin (N = 12). The results showed an increased expression of FoxP3, CTLA4, TGF-β1, IL-10, T-bet, IL-17F, and IL-17A in lesions, while GATA3 and IL-4 levels were found to be lower in diseased skin than in the control group. When the clinical forms were compared, TGF-β1 was found to be highly expressed in patients with a single localized lesion while IL-5 and IL-17A levels were higher in patients with multiple/disseminated lesions. These results demonstrate the occurrence of mixed T helper responses and suggest the dominance of regulatory T cell activity, which could inhibit Th-dependent protective responses to intracellular fungi such as L. loboi. Therefore, Tregs may play a key role in JLD pathogenesis.     

Reproductive output of invasive versus native plants

Aim Propagule size and output are critical for the ability of a plant species to colonize new environments. If invasive species have a greater reproductive output than native species (via more and/or larger seeds), then they will have a greater dispersal and establishment ability. Previous comparisons within plant genera, families or environments have conflicted over the differences in reproductive traits between native and invasive species. We went beyond a genus‐, family‐ or habitat‐specific approach and analysed data for plant reproductive traits from the global literature, to investigate whether: (1) seed mass and production differ between the original and introduced ranges of invasive species; (2) seed mass and production differ between invasives and natives; and (3) invasives produce more seeds per unit seed mass than natives. Location Global. Methods We combined an existing data set of native plant reproductive data with a new data compilation for invasive species. We used t‐tests to compare original and introduced range populations, two‐way ANOVAs to compare natives and invasives, and an ANCOVA to examine the relationship between seed mass and production for natives and invasives. The ANCOVA was performed again incorporating phylogenetically independent contrasts to overcome any phylogenetic bias in the data sets. Results Neither seed mass nor seed production of invasive species differed between their introduced and original ranges. We found no significant difference in seed mass between invasives and natives after growth form had been accounted for. Seed production was greater for invasive species overall and within herb and woody growth forms. For a given seed mass, invasive species produced 6.7‐fold (all species), 6.9‐fold (herbs only) and 26.1‐fold (woody species only) more seeds per individual per year than native species. The phylogenetic ANCOVA verified that this trend did not appear to be influenced by phylogenetic bias within either data set. Main conclusions This study provides the first global examination of both seed mass and production traits in native and invasive species. Invasive species express a strategy of greater seed production both overall and per unit seed mass compared with natives. The consequent increased likelihood of establishment from long‐distance seed dispersal may significantly contribute to the invasiveness of many exotic species.     

Advances in approaches to seagrass restoration in Australia

Three case studies involving two temperate Australian seagrass species – Pondweed (Ruppia tuberosa) and Ribbon Weed (Posidonia australis) – highlight different approaches to their restoration. Seeds and rhizomes were used in three collaborative programmes to promote new approaches to scale up restoration outcomes.     

Mouse-adapted SARS-CoV-2 protects animals from lethal SARS-CoV challenge

The emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has resulted in a pandemic causing significant damage to public health and the economy. Efforts to understand the mechanisms of Coronavirus Disease 2019 (COVID-19) have been hampered by the lack of robust mouse models. To overcome this barrier, we used a reverse genetic system to generate a mouse-adapted strain of SARS-CoV-2. Incorporating key mutations found in SARS-CoV-2 variants, this model recapitulates critical elements of human infection including viral replication in the lung, immune cell infiltration, and significant in vivo disease. Importantly, mouse adaptation of SARS-CoV-2 does not impair replication in human airway cells and maintains antigenicity similar to human SARS-CoV-2 strains. Coupled with the incorporation of mutations found in variants of concern, CMA3p20 offers several advantages over other mouse-adapted SARS-CoV-2 strains. Using this model, we demonstrate that SARS-CoV-2–infected mice are protected from lethal challenge with the original Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), suggesting immunity from heterologous Coronavirus (CoV) strains. Together, the results highlight the use of this mouse model for further study of SARS-CoV-2 infection and disease.

Studying cross-protection from different coronaviruses is important to inform the research for a universal vaccine. This study uses a mouse-adapted SARS-CoV-2 strain to show that it confers protection from SARS-CoV challenge, suggesting possible immunity from heterologous challenge following natural infection.