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     

Gene expression in autumn leaves

Two cDNA libraries were prepared, one from leaves of a field-grown aspen (Populus tremula) tree, harvested just before any visible sign of leaf senescence in the autumn, and one from young but fully expanded leaves of greenhouse-grown aspen (Populus tremula x tremuloides). Expressed sequence tags (ESTs; 5,128 and 4,841, respectively) were obtained from the two libraries. A semiautomatic method of annotation and functional classification of the ESTs, according to a modified Munich Institute of Protein Sequences classification scheme, was developed, utilizing information from three different databases. The patterns of gene expression in the two libraries were strikingly different. In the autumn leaf library, ESTs encoding metallothionein, early light-inducible proteins, and cysteine proteases were most abundant. Clones encoding other proteases and proteins involved in respiration and breakdown of lipids and pigments, as well as stress-related genes, were also well represented. We identified homologs to many known senescence-associated genes, as well as seven different genes encoding cysteine proteases, two encoding aspartic proteases, five encoding metallothioneins, and 35 additional genes that were up-regulated in autumn leaves. We also indirectly estimated the rate of plastid protein synthesis in the autumn leaves to be less that 10% of that in young leaves.     

SpecNet: A Spatial Network Algorithm that Generates a Wide Range of Specific Structures

Network measures are used to predict the behavior of different systems. To be able to investigate how various structures behave and interact we need a wide range of theoretical networks to explore. Both spatial and non-spatial methods exist for generating networks but they are limited in the ability of producing wide range of network structures. We extend an earlier version of a spatial spectral network algorithm to generate a large variety of networks across almost all the theoretical spectra of the following network measures: average clustering coefficient, degree assortativity, fragmentation index, and mean degree. We compare this extended spatial spectral network-generating algorithm with a non-spatial algorithm regarding their ability to create networks with different structures and network measures. The spatial spectral network-generating algorithm can generate networks over a much broader scale than the non-spatial and other known network algorithms. To exemplify the ability to regenerate real networks, we regenerate networks with structures similar to two real Swedish swine transport networks. Results show that the spatial algorithm is an appropriate model with correlation coefficients at 0.99. This novel algorithm can even create negative assortativity and managed to achieve assortativity values that spans over almost the entire theoretical range.     

Mitigation of acidified salmon rivers – effects of liming on young brown trout Salmo trutta

In southern Norway, 22 acidified rivers supporting anadromous salmonids were mitigated with lime to improve water quality and restore fish populations. In 13 of these rivers, effects on Salmo trutta and Salmo salar densities were monitored over 10–12 years, grouped into age 0 and age ≥ 1 year fish. These rivers had a mean annual discharge of between 4·9 and 85·5 m³ s^?1, and six of them were regulated for hydro‐power production. Salmo salar were lost in six of these rivers prior to liming, and highly reduced in the remaining seven rivers. Post‐liming, S. salar became re‐established in all six rivers with lost populations, and recovered in the seven other rivers. Salmo trutta occurred in all 13 study rivers prior to liming. Despite the improved water quality, both age 0 and age ≥ 1 year S. trutta densities decreased as S. salar density increased, with an average reduction of >50% after 10 years of liming. For age 0 year S. trutta this effect was less strong in rivers where S. salar were present prior to liming. In contrast, densities of S. trutta increased in unlimed streams above the anadromous stretches in two of the rivers following improved water quality due to natural recovery. Density increases of both age 0 and age ≥ 1 year S. salar showed a positive effect of river discharge. The results suggest that the decline in S. trutta density after liming is related to interspecific resource competition due to the recovery of S. salar. Thus, improved water quality through liming may not only sustain susceptible species, but can have a negative effect on species that are more tolerant prior to the treatment, such as S. trutta.     

KERA: analysis tool for multi-process,multi-state single-molecule data

Molecular machines within cells dynamically assemble, disassemble and reorganize. Molecular interactions between their components can be observed at the single-molecule level and quantified using colocalization single-molecule spectroscopy, in which individual labeled molecules are seen transiently associating with a surface-tethered partner, or other total internal reflection fluorescence microscopy approaches in which the interactions elicit changes in fluorescence in the labeled surface-tethered partner. When multiple interacting partners can form ternary, quaternary and higher order complexes, the types of spatial and temporal organization of these complexes can be deduced from the order of appearance and reorganization of the components. Time evolution of complex architectures can be followed by changes in the fluorescence behavior in multiple channels. Here, we describe the kinetic event resolving algorithm (KERA), a software tool for organizing and sorting the discretized fluorescent trajectories from a range of single-molecule experiments. KERA organizes the data in groups by transition patterns, and displays exhaustive dwell time data for each interaction sequence. Enumerating and quantifying sequences of molecular interactions provides important information regarding the underlying mechanism of the assembly, dynamics and architecture of the macromolecular complexes. We demonstrate KERA’s utility by analyzing conformational dynamics of two DNA binding proteins: replication protein A and xeroderma pigmentosum complementation group D helicase.     

Influence of substrate structure on disintegration activity of Moloney murine leukemia virus integrase.

The disintegration activity of Moloney murine leukemia virus (M-MuLV) integrase (IN) was investigated through structural and sequence modifications of a Y substrate that resembles an integration intermediate. The Y substrates, constructed from individual oligonucleotides, contain a single viral long terminal repeat (LTR) joined to a nicked target DNA. Truncation of the double-stranded LTR sequences distal to the conserved 5'-CA-3' dinucleotide progressively diminished disintegration activity. M-MuLV IN was also able to catalyze disintegration of a heterologous double-stranded LTR sequence. Significantly, the activity of M-MuLV IN on single-stranded LTR Y substrates was more dependent on the sequence and length of the LTR strand than that reported for human immunodeficiency virus type 1 (HIV-1) IN. Modifications introduced at the Y-substrate junction demonstrated that the 3'-hydroxyl group at the terminus of the target strand was necessary for efficient joining of the target DNA strands. The presence of a 2'-hydroxyl group at the 3' end of the target strand, as well as a single-nucleotide gap at the LTR-target junction, reduced disintegration activity. The absence of hydroxyl groups on the terminal nucleotide abolished joining of the target strands. The results presented here suggest that M-MuLV IN disintegration activity is dependent on substantially different LTR sequence requirements than those reported for HIV-1 IN and may be mediated primarily through a structural recognition event.     

Optimization of a histopathological biomarker for sphingomyelin accumulation in acid sphingomyelinase deficiency

Niemann-Pick disease (types A and B), or acid sphingomyelinase deficiency, is an inherited deficiency of acid sphingomyelinase, resulting in intralysosomal accumulation of sphingomyelin in cells throughout the body, particularly within those of the reticuloendothelial system. These cellular changes result in hepatosplenomegaly and pulmonary infiltrates in humans. A knockout mouse model mimics many elements of human ASMD and is useful for studying disease histopathology. However, traditional formalin-fixation and paraffin embedding of ASMD tissues dissolves sphingomyelin, resulting in tissues with a foamy cell appearance, making quantitative analysis of the substrate difficult. To optimize substrate fixation and staining, a modified osmium tetroxide and potassium dichromate postfixation method was developed to preserve sphingomyelin in epon-araldite embedded tissue and pulmonary cytology specimens. After processing, semi-thin sections were incubated with tannic acid solution followed by staining with toluidine blue/borax. This modified method provides excellent preservation and staining contrast of sphingomyelin with other cell structures. The resulting high-resolution light microscopy sections permit digital quantification of sphingomyelin in light microscopic fields. A lysenin affinity stain for sphingomyelin was also developed for use on these semi-thin epon sections. Finally, ultrathin serial sections can be cut from these same tissue blocks and stained for ultrastructural examination by electron microscopy.     

Lipid body formation plays a central role in cell fate determination during developmental differentiation of Myxococcus xanthus

Cell differentiation is widespread during the development of multicellular organisms, but rarely observed in prokaryotes. One example of prokaryotic differentiation is the Gram-negative bacterium Myxococcus xanthus . In response to starvation, this gliding bacterium initiates a complex developmental programme that results in the formation of spore-filled fruiting bodies. How the cells metabolically support the necessary complex cellular differentiation from rod-shaped vegetative cells into spherical spores is unknown. Here, we present evidence that intracellular lipid bodies provide the necessary metabolic fuel for the development of spores. Formed at the onset of starvation, these lipid bodies gradually disappear until they are completely used up by the time the cells have become mature spores. Moreover, it appears that lipid body formation in M. xanthus is an important initial step indicating cell fate during differentiation. Upon starvation, two subpopulations of cells occur: cells that form lipid bodies invariably develop into spores, while cells that do not form lipid bodies end up becoming peripheral rods, which are cells that lack signs of morphological differentiation and stay in a vegetative-like state. These data indicate that lipid bodies not only fuel cellular differentiation but that their formation represents the first known morphological sign indicating cell fate during differentiation.     

Individual wealth rank, community wealth inequality, and self-reported adult poor health: a test of hypotheses with panel data (2002-2006) from native Amazonians, Bolivia

Growing evidence suggests that economic inequality in a community harms the health of a person. Using panel data from a small-scale, preindustrial rural society, we test whether individual wealth rank and village wealth inequality affects self-reported poor health in a foraging-farming native Amazonian society. A person's wealth rank was negatively but weakly associated with self-reported morbidity. Each step up/year in the village wealth hierarchy reduced total self-reported days ill by 0.4 percent. The Gini coefficient of village wealth inequality bore a positive association with self-reported poor health that was large in size, but not statistically significant. We found small village wealth inequality, and evidence that individual economic rank did not change. The modest effects may have to do with having used subjective rather than objective measures of health, having small village wealth inequality, and with the possibly true modest effect of a person's wealth rank on health in a small-scale, kin-based society. Finally, we also found that an increase in mean individual wealth by village was related to worse self-reported health. As the Tsimane' integrate into the market economy, their possibilities of wealth accumulation rise, which may affect their well-being. Our work contributes to recent efforts in biocultural anthropology to link the study of social inequalities, human biology, and human-environment interactions.     

The biogeography of three Boeckella species (Copepoda: Calanoida) in New Zealand

The New Zealand distributions of three species of Boeckella (Copepoda, Calanoida), B. triarticulata, B. dilatata and B. hamata are mapped. B. triarticulata is primarily a pond dweller but is also found in reservoirs and shallow lakes. B. dilatata is mainly found in the deeper glacial lakes and ponds in the central region of the South Island and B. hamata has a more widespread distribution in lakes and ponds in the South Island and lower half of the North Island. Differences in temperature optima, food requirements and dispersal ability among the three Boeckella species are related to vicariant events to explain their distribution in New Zealand.