Automatic whole broth multi-fermentor sampling

Summary An efficient, aseptic method of obtaining whole broth fermentation samples was developed based on a piston-valve, a local sample loop, and an ability to drive the entire sample volume with sterile air through a sample line and into a remote tube. The configuration delivers 10-ml samples 10 m away with about 4 ml of broth wasted in the sampling process. An autosampler was enhanced and programmed to control acquisition into chilled tubes. The autosampler-based system represents a convenient way to provide frequent samples to profile intracellular and extracellular components for yeast and bacterial fermentations. A configuration to provide sampling from six fermentors with a multi-rack autosampler will be presented.     

Red-edge excitation fluorescence measurements of several two-tryptophan-containing proteins.

The dependence of the fluorescence emission maximum of the tryptophan residues in several two-tryptophan-containing proteins (horse liver alcohol dehydrogenase, yeast 3-phosphoglycerate kinase, Staphylococcus aureus metalloprotease and bee venom phospholipase A2) on the excitation wavelengths has been studied. Using fluorescence-resolved spectroscopy, we have dissected the contributions of particular tryptophan residues located in different parts of the protein molecule. The results demonstrate that dipolar structural relaxation can occur in the environment of tryptophan residues buried within protein molecules. The observed spectral shifts upon red-edge excitation of these residues can depend on temperature or ligand binding, as demonstrated in case of metalloprotease and alcohol dehydrogenase. No spectral shifts upon red-edge excitation have been observed for tryptophan residues totally exposed to the rapidly relaxing aqueous solvent.     

Species diversity and phylogeography of Cornus kousa (Asian dogwood) captured by genomic and genic microsatellites

Cornus kousa (Asian dogwood), an East Asia native tree, is the most economically important species of the dogwood genus, owing to its desirable horticultural traits and ability to hybridize with North America‐native dogwoods. To assess the species genetic diversity and to better inform the ongoing and future breeding efforts, we assembled an herbarium and arboretum collection of 131 noncultivated C. kousa specimens. Genotyping and capillary electrophoresis analyses of our C. kousa collection with the newly developed genic and published nuclear genomic microsatellites permitted assessment of genetic diversity and evolutionary history of the species. Regardless of the microsatellite type used, the study yielded generally similar insights into the C. kousa diversity with subtle differences deriving from and underlining the marker used. The accrued evidence pointed to the species distinct genetic pools related to the plant country of origin. This can be helpful in the development of the commercial cultivars for this important ornamental crop with increased pyramided utility traits. Analyses of the C. kousa evolutionary history using the accrued genotyping datasets pointed to an unsampled ancestor population, possibly now extinct, as per the phylogeography of the region. To our knowledge, there are few studies utilizing the same gDNA collection to compare performance of genomic and genic microsatellites. This is the first detailed report on C. kousa species diversity and evolutionary history inference.     

Effects of thermal and oxygen conditions during development on cell size in the common rough woodlice Porcellio scaber

During development, cells may adjust their size to balance between the tissue metabolic demand and the oxygen and resource supply: Small cells may effectively absorb oxygen and nutrients, but the relatively large area of the plasma membrane requires costly maintenance. Consequently, warm and hypoxic environments should favor ectotherms with small cells to meet increased metabolic demand by oxygen supply. To test these predictions, we compared cell size (hindgut epithelium, hepatopancreas B cells, ommatidia) in common rough woodlice (Porcellio scaber) that were developed under four developmental conditions designated by two temperatures (15 or 22°C) and two air O₂ concentrations (10% or 22%). To test whether small‐cell woodlice cope better under increased metabolic demand, the CO₂ production of each woodlouse was measured under cold, normoxic conditions and under warm, hypoxic conditions, and the magnitude of metabolic increase (MMI) was calculated. Cell sizes were highly intercorrelated, indicative of organism‐wide mechanisms of cell cycle control. Cell size differences among woodlice were largely linked with body size changes (larger cells in larger woodlice) and to a lesser degree with oxygen conditions (development of smaller cells under hypoxia), but not with temperature. Developmental conditions did not affect MMI, and contrary to predictions, large woodlice with large cells showed higher MMI than small woodlice with small cells. We also observed complex patterns of sexual difference in the size of hepatopancreatic cells and the size and number of ommatidia, which are indicative of sex differences in reproductive biology. We conclude that existing theories about the adaptiveness of cell size do not satisfactorily explain the patterns in cell size and metabolic performance observed here in P. scaber. Thus, future studies addressing physiological effects of cell size variance should simultaneously consider different organismal elements that can be involved in sustaining the metabolic demands of tissue, such as the characteristics of gas‐exchange organs and O₂‐binding proteins.     

Integration of high-throughput analytics and cell imaging enables direct early productivity and product quality assessment during Chinese Hamster ovary cell line development for a complex multi-subunit vaccine antigen

Mammalian cell line generation typically includes stable pool generation, single cell cloning and several rounds of clone selection based on cell growth, productivity and product quality criteria. Individual clone expansion and phenotype-based ranking is performed initially for hundreds or thousands of mini-scale cultures, representing the major operational challenge during cell line development. Automated cell culture and analytics systems have been developed to enable high complexity clone selection workflows; while ensuring traceability, safety, and quality of cell lines intended for biopharmaceutical applications. Here we show that comprehensive and quantitative assessment of cell growth, productivity, and product quality attributes are feasible at the 200–1,200 cell colony stage, within 14 days of the single cell cloning in static 96-well plate culture. The early cell line characterization performed prior to the clone expansion in suspension culture can be used for a single-step, direct selection of high quality clones. Such clones were comparable, both in terms of productivity and critical quality attributes (CQAs), to the top-ranked clones identified using an established iterative clone screening approach. Using a complex, multi-subunit antigen as a model protein, we observed stable CQA profiles independently of the cell culture format during the clonal expansion as well as in the batch and fed-batch processes. In conclusion, we propose an accelerated clone selection approach that can be readily incorporated into various cell line development workstreams, leading to significant reduction of the project timelines and resource requirements.     

Covid‐19.bioreproducibility.org: A web resource for SARS‐CoV‐2‐related structural models

The COVID‐19 pandemic has triggered numerous scientific activities aimed at understanding the SARS‐CoV‐2 virus and ultimately developing treatments. Structural biologists have already determined hundreds of experimental X‐ray, cryo‐EM, and NMR structures of proteins and nucleic acids related to this coronavirus, and this number is still growing. To help biomedical researchers, who may not necessarily be experts in structural biology, navigate through the flood of structural models, we have created an online resource, covid19.bioreproducibility.org, that aggregates expert‐verified information about SARS‐CoV‐2‐related macromolecular models. In this article, we describe this web resource along with the suite of tools and methodologies used for assessing the structures presented therein.