Clone selection and process lever optimization using an AMBR® 15 system for conversion of a roller bottle process to a suspension, perfusion bioreactor platform

Due to the high capital costs for a new roller bottle facility, a new suspension bioreactor perfusion platform was pursued as a potential option to improve the supply network for an existing commercial roller bottle cell culture process. The first step in developing the bioreactor process was the adaptation of the current commercial working cell bank to serum-free, suspension conditions. Subcloning of this serum-free, suspension adapted pool was performed to reduce the pool to the top 50 clones based on titer and activity. In deep well plates and shake flask cultures, the top 50 clones were further screened to yield the top 10 clones based on yield, activity, and important product quality attributes including sialylation and Mannose-6-Phosphate (M6P) content, which were determined by high throughput analytical methods specifically designed for this molecule. In parallel with these activities, an ambr® 15 perfusion scale down model was developed to evaluate multiple process levers (e.g. medium osmolarity, target viable cell concentration, cell specific perfusion rate, etc.) for the serum-free, suspension adapted pool using definitive screening designs. After ambr® 15 scale down model development, the Top 10 clones identified in subcloning were evaluated in the top conditions identified from the process lever optimization study. Several of the best combinations of clones / bioreactor conditions were then repeated in 10L bench scale bioreactors to ensure reproducible cell culture performance. Furthermore, the 10L cultures were harvested and purified to mock drug substance to confirm significant product quality attributes were consistent between the ambr® 15 scale down model and bench scale bioreactors and within desired commercial specification ranges. These results suggest that the ambr® 15 perfusion scale down model can be deployed for clone selection, process optimization, and process characterization activities for the current suspension, perfusion bioreactor process, as well as development of future perfusion processes, to ensure successful launch at large scale

Gadd45β and Gadd45γ are critical for regulating autoimmunity

The number of effector T cells is controlled by proliferation and programmed cell death. Loss of these controls on self-destructive effector T cells may precipitate autoimmunity. Here, we show that two members of the growth arrest and DNA damage-inducible (Gadd45) family, β and γ, are critical in the development of pathogenic effector T cells. CD4+ T cells lacking Gadd45β can rapidly expand and invade the central nervous system in response to myelin immunization, provoking an exacerbated and prolonged autoimmune encephalomyelitis in mice. Importantly, mice with compound deficiency in Gadd45β and Gadd45γ spontaneously developed signs of autoimmune lymphoproliferative syndrome and systemic lupus erythematosus. Our findings therefore identify the Gadd45β/Gadd45γ-mediated control of effector autoimmune lymphocytes as an attractive novel target for autoimmune disease therapy

In vitro PKA phosphorylation-mediated human PDE4A4 activation

AbstractThe PDE4 catalytic machinery comprises, in part, two divalent cations in a binuclear motif. Here we report that PDE4A4 expressed in Sf9 cells exhibits a biphasic Mg2+ dose–response (EC50 of ∼0.15 and >10 mM) in catalyzing cAMP hydrolysis. In vitro phosphorylation of PDE4A4 by the PKA-catalytic subunit increases the enzyme’s sensitivity to Mg2+, leading to 4-fold increased cAMP hydrolysis without affecting its Km. The phosphorylation also increases the potencies of (R)- and (S)-rolipram without affecting CDP-840 and SB-207499. The results support that modulating the cofactor binding affinity of PDE4 represents a mechanism for regulating its activity

Guiding kinetic trajectories between jammed and unjammed states in 2D colloidal nanocrystal-polymer assemblies with zwitterionic ligands

Mesostructured matter composed of colloidal nanocrystals in solidified architectures abounds with broadly tunable catalytic, magnetic, optoelectronic, and energy storing properties. Less common are liquid-like assemblies of colloidal nanocrystals in a condensed phase, which may have different energy transduction behaviors owing to their dynamic character. Limiting investigations into dynamic colloidal nanocrystal architectures is the lack of schemes to control or redirect the tendency of the system to solidify. We show how to solidify and subsequently reconfigure colloidal nanocrystal assemblies dimensionally confined to a liquid-liquid interface. Our success in this regard hinged on the development of competitive chemistries anchoring or releasing the nanocrystals to or from the interface. With these chemistries, it was possible to control the kinetic trajectory between quasi–two-dimensional jammed (solid-like) and unjammed (liquid-like) states. In future schemes, it may be possible to leverage this control to direct the formation or destruction of explicit physical pathways for energy carriers to migrate in the system in response to an external field

Wildlife Underpass Use and Environmental Impact Assessment: A Southern California Case Study

Environmental planners often rely on transportation structures (i.e., underpasses, bridges) to provide connectivity for animals across developed landscapes. Environmental assessments of predicted environmental impacts from proposed developments often rely on literature reviews or other indirect measures to establish the importance of wildlife crossings. Literature-based evaluations of wildlife crossings may not be accurate, and result in under-estimation of impacts or establishment of inappropriate mitigation measures. To investigate the adequacy of literature-based evaluations, we monitored wildlife use of a freeway underpass that had been identified as critically important to wildlife connectivity, and which was evaluated in an environmental review document. Photographs were obtained from a network of trail cameras over 3 years. Six mid- to large-sized native mammal species used the underpass and two other mammal species were photographed near the underpass but not using it. American badger (Taxidea taxus) was photographed at a higher rate in the underpass than in the surrounding area. Gray fox (Urocyon cinereoargenteus) was rarely detected in the underpass relative to surrounding habitats, whereas the absence of mule deer (Odocoileus hemionus) in the underpass was unexpected, given relatively frequent detection in adjacent habitats. These results differed from the environmental assessment in that American badger was listed as potentially present while mule deer were expected to use the underpass. Results underscore importance of gathering data to document wildlife use of corridors, because some species do not or rarely take advantage of apparently suitable corridors, while others may be present when assumed to be absent

Facilitating Healthy Eating in Latin American Restaurants: Examining Acceptability and Barriers Among Restaurant Owners and Staff

Objectives: Examine the acceptability and potential barriers for the implementation of healthy eating promoting (HEP) strategies in independently-owned Latin American restaurants (LAR), including the influence of the COVID-19 pandemic

Supporting stimulation needs in dementia care through wall-sized displays

Beside reminiscing, the increasing cognitive decline in dementia can also be addressed through sensory stimulation allowing the immediate, nonverbal engagement with the world through one’s senses. Much HCI work has prioritized cognitive stimulation for reminiscing or personhood often on small screens, while less research has explored sensory stimulation like the one enabled by large displays. We describe a year-long deployment in a residential care home of a wall-sized display, and explored its domestication through 24 contextual interviews. Findings indicate strong engagement and attachment to the display which has inspired four psychosocial interventions using online generic content. We discuss the value of these findings for personhood through residents’ exercise of choices, the tension between generic/personal content and its public/private use, the importance of participatory research approach to domestication, and the infrastructure-based prototype, illustrated by the DementiaWall and its generative quality

Interleukin-22 signaling attenuates necrotizing enterocolitis by promoting epithelial cell regeneration

Necrotizing enterocolitis (NEC) is a deadly intestinal inflammatory disorder that primarily affects premature infants and lacks adequate therapeutics. Interleukin (IL)-22 plays a critical role in gut barrier maintenance, promoting epithelial regeneration, and controlling intestinal inflammation in adult animal models. However, the importance of IL-22 signaling in neonates during NEC remains unknown. We investigated the role of IL-22 in the neonatal intestine under homeostatic and inflammatory conditions by using a mouse model of NEC. Our data reveal tha