Directional, seamless, and restriction enzyme-free construction of random-primed complementary DNA libraries using phosphorothioate-modified primers

Directional cloning of complementary DNA (cDNA) primed by oligo(dT) is commonly achieved by appending a restriction site to the primer, whereas the second strand is synthesized through the combined action of RNase H and Escherichia coli DNA polymerase I (PolI). Although random primers provide more uniform and complete coverage, directional cloning with the same strategy is highly inefficient. We report that phosphorothioate linkages protect the tail sequence appended to random primers from the 5′ → 3′ exonuclease activity of PolI. We present a simple strategy for constructing a random-primed cDNA library using the efficient, size-independent, and seamless In-Fusion cloning method instead of restriction enzymes.     

Automated dynamic fed‐batch process and media optimization for high productivity cell culture process development

Current industry practices for large‐scale mammalian cell cultures typically employ a standard platform fed‐batch process with fixed volume bolus feeding. Although widely used, these processes are unable to respond to actual nutrient consumption demands from the culture, which can result in accumulation of by‐products and depletion of certain nutrients. This work demonstrates the application of a fully automated cell culture control, monitoring, and data processing system to achieve significant productivity improvement via dynamic feeding and media optimization. Two distinct feeding algorithms were used to dynamically alter feed rates. The first method is based upon on‐line capacitance measurements where cultures were fed based on growth and nutrient consumption rates estimated from integrated capacitance. The second method is based upon automated glucose measurements obtained from the Nova Bioprofile FLEX® autosampler where cultures were fed to maintain a target glucose level which in turn maintained other nutrients based on a stoichiometric ratio. All of the calculations were done automatically through in‐house integration with a Delta V process control system. Through both media and feed strategy optimization, a titer increase from the original platform titer of 5 to 6.3 g/L was achieved for cell line A, and a substantial titer increase of 4 to over 9 g/L was achieved for cell line B with comparable product quality. Glucose was found to be the best feed indicator, but not all cell lines benefited from dynamic feeding and optimized feed media was critical to process improvement. Our work demonstrated that dynamic feeding has the ability to automatically adjust feed rates according to culture behavior, and that the advantage can be best realized during early and rapid process development stages where different cell lines or large changes in culture conditions might lead to dramatically different nutrient demands. Biotechnol. Bioeng. 2013; 110: 191–205. © 2012 Wiley Periodicals, Inc.     

Evolution of the Influenza A Virus Genome during Development of Oseltamivir Resistance In Vitro

Influenza A virus (IAV) is a major cause of morbidity and mortality throughout the world. Current antiviral therapies include oseltamivir, a neuraminidase inhibitor that prevents the release of nascent viral particles from infected cells. However, the IAV genome can evolve rapidly, and oseltamivir resistance mutations have been detected in numerous clinical samples. Using an in vitro evolution platform and whole-genome population sequencing, we investigated the population genomics of IAV during the development of oseltamivir resistance. Strain A/Brisbane/59/2007 (H1N1) was grown in Madin-Darby canine kidney cells with or without escalating concentrations of oseltamivir over serial passages. Following drug treatment, the H274Y resistance mutation fixed reproducibly within the population. The presence of the H274Y mutation in the viral population, at either a low or a high frequency, led to measurable changes in the neuraminidase inhibition assay. Surprisingly, fixation of the resistance mutation was not accompanied by alterations of viral population diversity or differentiation, and oseltamivir did not alter the selective environment. While the neighboring K248E mutation was also a target of positive selection prior to H274Y fixation, H274Y was the primary beneficial mutation in the population. In addition, once evolved, the H274Y mutation persisted after the withdrawal of the drug, even when not fixed in viral populations. We conclude that only selection of H274Y is required for oseltamivir resistance and that H274Y is not deleterious in the absence of the drug. These collective results could offer an explanation for the recent reproducible rise in oseltamivir resistance in seasonal H1N1 IAV strains in humans.     

Embryonic Stem Cells Do Not Stiffen on Rigid Substrates

It has been previously established that living cells, including mesenchymal stem cells, stiffen in response to elevation of substrate stiffness. This stiffening is largely attributed to the elevation of the tractions at the cell base that is associated with increases in cell spreading on more-rigid substrates. We show here, surprisingly, that mouse embryonic stem cells (ESCs) do not stiffen when substrate stiffness increases. As shown recently, these cells do not increase spreading on more-rigid substrates either. However, these ESCs do increase their basal tractions as substrate stiffness increases. We conclude that these ESCs exhibit mechanical behaviors distinct from those of mesenchymal stem cells and of terminally differentiated cells, and decouple its apical cell stiffness from its basal tractional stresses during the substrate rigidity response.     

Monitoring of active but non-culturable bacterial cells by flow cytometry

Flow cytometric signatures (i.e., light scatter, red and green fluorescence) were obtained for the active but non-culturable (ABNC) cells of E. coli and a coliform isolate H03N1, in seawater microcosms using BacLight, a live-dead assay kit from Molecular Probes (Eugene/Portland, OR). Previous studies have reported that there are two major adaptations, which cells undergo during the formation of ABNC states: cell wall toughening and DNA condensation. Therefore, we hypothesized that the matured ABNC forms should be more resistant to extreme temperature treatments (i.e., by freezing in liquid nitrogen and thawing at room temperature) than the normal and transition populations. It was shown that the membrane-compromised cells (comprising of normal wild-type and dead cells which are less resistant to rapid freeze thaw) could be differentiated from the matured ABNC using BacLight staining and fluorescence detection by flow cytometry. The population of ABNC cells, which could not be cultured using m-FC media (for the enumeration of fecal coliforms), was resuscitated in phosphate buffer saline followed by growth in Luria broth. Flow cytometry was thus able to detect and differentiate the ABNC cells against a mixed population comprising of culturable cells, transition populations, and dead cells. The results also showed that the formation of ABNC is as early as 2 days in seawater microcosms. By directly comparing the coliform levels enumerated by the BacLight based flow cytometry assays and m-FC technique, it was shown that the presence of coliforms can be undetected by the membrane filtration method.     

Pre- and Post-Natal Maternal Depressive Symptoms in Relation with Infant Frontal Function,Connectivity, and Behaviors

This study investigated the relationships between pre- and early post-natal maternal depression and their changes with frontal electroencephalogram (EEG) activity and functional connectivity in 6- and 18-month olds, as well as externalizing and internalizing behaviors in 24-month olds (n = 258). Neither prenatal nor postnatal maternal depressive symptoms independently predicted neither the frontal EEG activity nor functional connectivity in 6- and 18-month infants. However, increasing maternal depressive symptoms from the prenatal to postnatal period predicted greater right frontal activity and relative right frontal asymmetry amongst 6-month infants but these finding were not observed amongst 18-month infants after adjusted for post-conceptual age on the EEG visit day. Subsequently increasing maternal depressive symptoms from the prenatal to postnatal period predicted lower right frontal connectivity within 18-month infants but not among 6-month infants after controlling for post-conceptual age on the EEG visit day. These findings were observed in the full sample and the female sample but not in the male sample. Moreover, both prenatal and early postnatal maternal depressive symptoms independently predicted children’s externalizing and internalizing behaviors at 24 months of age. This suggests that the altered frontal functional connectivity in infants born to mothers whose depressive symptomatology increases in the early postnatal period compared to that during pregnancy may reflect a neural basis for the familial transmission of phenotypes associated with mood disorders, particularly in girls.     

Plastic Compressed Collagen as a Novel Carrier for Expanded Human Corneal Endothelial Cells for Transplantation

Current treatments for reversible blindness caused by corneal endothelial cell failure involve replacing the failed endothelium with donor tissue using a one donor-one recipient strategy. Due to the increasing pressure of a worldwide donor cornea shortage there has been considerable interest in developing alternative strategies to treat endothelial disorders using expanded cell replacement therapy. Protocols have been developed which allow successful expansion of endothelial cells in vitro but this approach requires a supporting material that would allow easy transfer of cells to the recipient. We describe the first use of plastic compressed collagen as a highly effective, novel carrier for human corneal endothelial cells. A human corneal endothelial cell line and primary human corneal endothelial cells retained their characteristic cobblestone morphology and expression of tight junction protein ZO-1 and pump protein Na+/K+ ATPase α1 after culture on collagen constructs for up to 14 days. Additionally, ultrastructural analysis suggested a well-integrated endothelial layer with tightly opposed cells and apical microvilli. Plastic compressed collagen is a superior biomaterial in terms of its speed and ease of production and its ability to be manipulated in a clinically relevant manner without breakage. This method provides expanded endothelial cells with a substrate that could be suitable for transplantation allowing one donor cornea to potentially treat multiple patients.