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     

Conservation genomics: disequilibrium mapping of domestic cattle chromosomal segments in North American bison populations

Introgressive hybridization is one of the major threats to species conservation, and is often induced by human influence on the natural habitat of wildlife species. The ability to accurately identify introgression is critical to understanding its importance in evolution and effective conservation management of species. Hybridization between North American bison (Bison bison) and domestic cattle (Bos taurus) as a result of human activities has been recorded for over 100 years, and domestic cattle mitochondrial DNA was previously detected in bison populations. In this study, linked microsatellite markers were used to identify domestic cattle chromosomal segments in 14 genomic regions from 14 bison populations. Cattle nuclear introgression was identified in five populations, with an average frequency per population ranging from 0.56% to 1.80%. This study represents the first use of linked molecular markers to examine introgression between mammalian species and the first demonstration of domestic cattle nuclear introgression in bison. To date, six public bison populations have been identified with no evidence of mitochondrial or nuclear domestic cattle introgression, providing information critical to the future management of bison genetic resources. The ability to identify even low levels of introgression resulting from historic hybridization events suggests that the use of linked molecular markers to identify introgression is a significant development in the study of introgressive hybridization across a broad range of taxa.     

ACC synthase expression regulates leaf performance and drought tolerance in maize

Ethylene regulates entry into several types of plant developmental cell death and senescence programs besides mediating plant responses to biotic and abiotic stress. The response of cereals to conditions of drought includes loss of leaf function and premature onset of senescence in older leaves. In this study, ACC synthase ( ACS ) mutants, affecting the first step in ethylene biosynthesis, were isolated in maize and their effect on leaf function examined. Loss of ZmACS6 expression resulted in delayed leaf senescence under normal growth conditions and inhibited drought-induced senescence. Zmacs6 leaves continued to be photosynthetically active under both conditions indicating that leaf function was maintained. The delayed senescence phenotype associated with loss of ZmACS6 expression was complemented by exogenous ACC. Surprisingly, elevated levels of foliar chlorophyll, Rubisco, and soluble protein as well as improved leaf performance was observed for all Zmasc6 leaves, including young and fully expanded leaves which were far from initiating senescence. These observations suggest that ethylene may serve to regulate leaf performance throughout its lifespan as well as to determine the onset of natural senescence and mediate drought-induced senescence.     

Structure of botulinum neurotoxin type D light chain at 1.65 A resolution: repercussions for VAMP-2 substrate specificity

The seven serotypes (A-G) of botulinum neurotoxins (BoNTs) function through their proteolytic cleavage of one of three proteins (SNAP-25, Syntaxin, and VAMP) that form the SNARE complex required for synaptic vesicle fusion. The different BoNTs have very specific protease recognition requirements, between 15 and 50 amino acids in length depending on the serotype. However, the structural details involved in substrate recognition remain largely unknown. Here is reported the 1.65 A resolution crystal structure of the catalytic domain of BoNT serotype D (BoNT/D-LC), providing insight into the protein-protein binding interaction and final proteolysis of VAMP-2. Structural analysis has identified a hydrophobic pocket potentially involved in substrate recognition of the P1' VAMP residue (Leu 60) and a second remote site for recognition of the V1 SNARE motif that is critical for activity. A structural comparison of BoNT/D-LC with BoNT/F-LC that also recognizes VAMP-2 one residue away from the BoNT/D-LC site provides additional molecular details about the unique serotype specific activities. In particular, BoNT/D prefers a hydrophobic interaction for the V1 motif of VAMP-2, while BoNT/F adopts a more hydrophilic strategy for recognition of the same V1 motif.     

Alternative mating strategies in a desert grasshopper: evidence of density-dependence

Field observations revealed that, on a given day, male mating behaviour in a population of the grasshopper Ligurotettix coquilletti ranged from little or no stridulation (inactive) to relatively persistent singing (active signalling). Inactive males were usually located in the territories of active signallers. Actively signalling male achieved more matings, and also more frequently approached and mounted females in incidents terminating in the male departing the female without copulating. Individuals switched between inactive and actively-signalling behaviour during their adult lives, and males that were usually active signallers achieved greater lifetime mating success. Thus, we obtained no evidence to support the hypothesis that variation in mating behaviour was maintained by negative frequency-dependent selection. By comparing the behaviour of males in two field plots maintained at different population densities, however, we found that high density was directly related to a higher incidence of inactive behaviour. Males perched on creosote (Larrea tridentata) bushes (their host plant), and certain bushes. Mating behaviour was independent of body size and, while young (<7 days old) males tended to be inactive, neither age nor the time of adult maturation during the season could fully account for the incidence of inactive behaviour among males. Instead, we suggest that the adoption of inactive behaviour resulted partly from aggressive encounters between males.     

dSPRINT: predicting DNA,RNA, ion,peptide and small molecule interaction sites within protein domains

Domains are instrumental in facilitating protein interactions with DNA, RNA, small molecules, ions and peptides. Identifying ligand-binding domains within sequences is a critical step in protein function annotation, and the ligand-binding properties of proteins are frequently analyzed based upon whether they contain one of these domains. To date, however, knowledge of whether and how protein domains interact with ligands has been limited to domains that have been observed in co-crystal structures; this leaves approximately two-thirds of human protein domain families uncharacterized with respect to whether and how they bind DNA, RNA, small molecules, ions and peptides. To fill this gap, we introduce dSPRINT, a novel ensemble machine learning method for predicting whether a domain binds DNA, RNA, small molecules, ions or peptides, along with the positions within it that participate in these types of interactions. In stringent cross-validation testing, we demonstrate that dSPRINT has an excellent performance in uncovering ligand-binding positions and domains. We also apply dSPRINT to newly characterize the molecular functions of domains of unknown function. dSPRINT’s predictions can be transferred from domains to sequences, enabling predictions about the ligand-binding properties of 95% of human genes. The dSPRINT framework and its predictions for 6503 human protein domains are freely available at http://protdomain.princeton.edu/dsprint.     

HIV protease as a target for retrovirus vector-mediated gene therapy

The dimeric aspartyl protease of HIV has been the subject of intense research for almost a decade. Knowledge of the substrate specificity and catalytic mechanism of this enzyme initially guided the development of several potent peptidomimetic small molecule inhibitors. More recently, the solution of the HIV protease structure led to the structure-based design of improved peptidomimetic and non-peptidomimetic antiviral compounds. Despite the qualified success of these inhibitors, the high mutation rate associated with RNA viruses continues to hamper the long-term clinical efficacy of HIV protease inhibitors. The dimeric nature of the viral protease has been conducive to the investigation of dominant-negative inhibitors of the enzyme. Some of these inhibitors are defective protease monomers that interact with functional monomers to form inactive protease heterodimers. An advantage of macromolecular inhibitors as compared to small-molecule inhibitors is the increased surface area of interaction between the inhibitor and the target gene product. Point mutations that preserve enzyme activity but confer resistance to small-molecule inhibitors are less likely to have an adverse effect on macromolecular interactions. The use of efficient retrovirus vectors has facilitated the delivery of these macromolecular inhibitors to primary human lymphocytes. The vector-transduced cells were less susceptible to HIV infection in vitro, and showed similar levels of protection compared to other macromolecular inhibitors of HIV replication, such as RevM10. These preliminary results encourage the further development of dominant-negative HIV protease inhibitors as a gene therapy-based antiviral strategy.