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Inefficient knock-in of transgene cargos limits the potential of cell-based medicines. In this study, we used a CRISPR nuclease that targets a site within an exon of an essential gene and designed a cargo template so that correct knock-in would retain essential gene function while also integrating the transgene(s) of interest. Cells with non-productive insertions and deletions would undergo negative selection. This technology, called SLEEK (SeLection by Essential-gene Exon Knock-in), achieved knock-in efficiencies of more than 90% in clinically relevant cell types without impacting long-term viability or expansion. SLEEK knock-in rates in T cells are more efficient than state-of-the-art TRAC knock-in with AAV6 and surpass more than 90% efficiency even with non-viral DNA cargos. As a clinical application, natural killer cells generated from induced pluripotent stem cells containing SLEEK knock-in of CD16 and mbIL-15 show substantially improved tumor killing and persistence in vivo. Transgene knock-ins into housekeeping genes lead to high efficiency of cell selection.

Maternal immune activation (MIA) disrupts the central innate immune system during a critical neurodevelopmental period. Microglia are primary innate immune cells in the brain although their direct influence on the MIA phenotype is largely unknown. Here we show that MIA alters microglial gene expression with upregulation of cellular protrusion/neuritogenic pathways, concurrently causing repetitive behavior, social deficits, and synaptic dysfunction to layer V intrinsically bursting pyramidal neurons in the prefrontal cortex of mice. MIA increases plastic dendritic spines of the intrinsically bursting neurons and their interaction with hyper-ramified microglia. Treating MIA offspring by colony stimulating factor 1 receptor inhibitors induces depletion and repopulation of microglia, and corrects protein expression of the newly identified MIA-associated neuritogenic molecules in microglia, which coalesces with correction of MIA-associated synaptic, neurophysiological, and behavioral abnormalities. Our study demonstrates that maternal immune insults perturb microglial phenotypes and influence neuronal functions throughout adulthood, and reveals a potent effect of colony stimulating factor 1 receptor inhibitors on the correction of MIA-associated microglial, synaptic, and neurobehavioral dysfunctions.

The role of Arc in synaptic plasticity and memory consolidation has been investigated for many years with recent evidence that defects in the expression or activity of this immediate-early gene may also contribute to the pathophysiology of brain disorders including schizophrenia and fragile X syndrome. These results bring forward the concept that reversing Arc abnormalities could provide an avenue to improve cognitive or neurological impairments in different disease contexts, but how to achieve this therapeutic objective has remained elusive. Here, we present results from a chemogenomic screen that probed a mechanistically diverse library of small molecules for modulators of BDNF-induced Arc expression in primary cortical neurons. This effort identified compounds with a range of influences on Arc, including promoting its acetylation—a previously uncharacterized post-translational modification of this protein. Together, our data provide insights into the control of Arc that could be targeted to harness neuroplasticity for clinical applications. The activity-regulated cytoskeleton-associated protein (Arc) has been implicated in synaptic plasticity and memory consolidation. Here the authors show that Arc acetylation regulates its stability and identify small molecules that modulate Arc expression.

Healthy bone homeostasis hinges upon a delicate balance and regulation of multiple processes that contribute to bone development and metabolism. While examining hematopoietic regulation by Tle4 , we have uncovered a previously unappreciated role of Tle4 on bone calcification using a novel Tle4 null mouse model. Given the significance of osteoblasts in both hematopoiesis and bone development, this study investigated how loss of Tle4 affects osteoblast function. We used dynamic bone formation parameters and microCT to characterize the adverse effects of Tle4 loss on bone development. We further demonstrated loss of Tle4 impacts expression of several key osteoblastogenic genes, including Runx2 , Oc , and Ap , pointing toward a potential novel mechanism for Tle4 -dependent regulation of mammalian bone development in collaboration with the RUNX family members.

The UK is required to reduce its greenhouse gas emissions by 80 per cent from 1990 levels, by 2050. Greenhouse gas emissions attributed to the UK higher education sector have increased by 34.5 per cent from 1990 to 2005. Higher education institutions have a unique role in the UK greenhouse gas emissions inventory, beyond management of their own estates and compliance with policy and legislation, higher education institutions have responsibilities as innovators and educators, inspiring students and employees through example and best practice. This study sought to understand acceptability of greenhouse gas emissions reduction policies among employees of a higher education institution. The value-belief-norm theory was used in a questionnaire to understand individual attitudinal factors thought to influence policy acceptability (N=405). Recognising that an employee's attitudinal factors may be influenced by their work colleagues, this study used social network analysis to understand the social context within which individual attitudinal factors sit. Support was found for higher education institutions to reduce their greenhouse gas emissions. Employees found policies that encouraged desired behaviours, such as assistance with train travel costs and working from home, to be more acceptable than policies that discouraged undesired behaviours, such as doubling the price of a car-parking permit. Support was found for the structure and content of the value-belief-norm theory, but logistic regression suggested that it provided a weak explanation of employee policy acceptability, indicating that other factors may have a greater role. Analysis of workplace social networks suggested that employees have small social groups (x̄=8) and do not select to be close to colleagues that reflect their own perspectives. Practitioners and policymakers should seek to address this void in environmental social norms through recruitment of more environmental champions to deliver strong and persuasive pro-environmental messages.

Both neuroinflammation, and an increase in microglial cells, have been associated with Autism Spectrum Disorder (ASD) through observation in human subjects as well as in mouse models. A mother having an infection early in pregnancy increases the chances for autism in her child. (Atladottir et al., 2012). This process is known as Maternal Immune Activation (MIA), and the proposed mechanism is that inflammatory signals cross from the mother to child; then in response to increased pro-inflammatory cytokines, microglia within the brain are activated to combat the infection. Microglia are essential to healthy synaptogenesis and neuronal growth, and a change in their signaling early in development has been shown to alter behavior in mouse models that replicate MIA. We use microglial depletion as a therapy to counteract the potentially harmful pro-inflammatory response in the developing mouse brain. Four experimental groups—control, MIA, microglial depleted, and a therapy group (MIA plus microglial depletion)—were run through a comprehensive series of behavioral and electrophysiological assessments. Layer 5 pyramidal cells (L5PNs) were targeted for recording in medial frontal cortex—a mouse cortical area important for cognition and social behavior. L5PNs are a heterogeneous population with cortical and subcortical targeting. Subcortical targeting neurons are thick tufted morphologically, and have an intrinsically bursting spike pattern. Analysis of the intrinsically bursting neurons revealed significant differences between the maternal inflammation and the microglial depletion groups across multiple physiological properties. Therefore, the therapy group had electrophysiological characteristics more consistent with the microglial depleted model than the autism model.

Again in 2 Timothy 4:12, [Paul] has him running as a trouble- shooter to Ephesus for him. Not mentioned in Colossians, but in 2 Timothy we find Titus was off and running for Paul too, as his job was that of a trouble-shooter for the Apostle. Also in the Colossians letter we find Onesimus mentioned. He is called a \"faithful and beloved brother.\" In fact, Onesimus was the runaway slave of Philemon who had found Paul and gotten his life straightened out. Onesimus was now a slave of Jesus Christ and a brother to both Paul and Philemon. Next Paul mentions Aristarchus. He and Gaius, who is not mentioned here, ran into Paul in Ephesus. They were seized by a mob after the hassle over the drop in sales of the silver shrines of the Ephesians' goddess Diana. Aristarchus and Gaius were dragged into the coliseum and beaten before the wild mob was quieted. Here, in Colossians, we find Aristarchus standing with Paul again when he's in hot water, as Paul calls him \"my fellow prisoner.\"

SCSI is a peripheral bus based on the ANSI X3.131-1986 standard. SCSI is capable of connecting eight peripheral devices on the bus at data rates up to 4 Mbytes/second. The design process includes the selection of appropriate VLSI devices and necessary support logic. The logic devices are connected for complete functionality of the bridge and schematics are generated to show proper connection. The design is verified using worst case timing analysis based on manufacturer specifications.

Diel vertical migration is a widespread behavioral phenomenon where organisms migrate through the water column and may modify behavior relative to changing environmental conditions based on physiological tolerances. Here, we combined a novel suite of biologging technologies to examine the thermal physiology (intramuscular temperature), fine-scale swimming behavior and activity (overall dynamic body acceleration as a proxy for energy expenditure) of bluntnose sixgill sharks (Hexanchus griseus) in response to environmental changes (depth, water temperature, dissolved oxygen) experienced during diel vertical migrations. In the subtropical waters off Hawai'i, sixgill sharks undertook pronounced diel vertical migrations and spent considerable amounts of time in cold (5-7°C), low oxygen conditions (10-25% saturation) during their deeper daytime distribution. Further, sixgill sharks spent the majority of their deeper daytime distribution with intramuscular temperatures warmer than ambient water temperatures, thereby providing them with a significant thermal advantage over non-vertically migrating and smaller-sized prey. Sixgill sharks exhibited relatively high rates of activity during both shallow (night) and deep (day) phases and contrary to our predictions, did not reduce activity levels during their deeper daytime distribution while experiencing low temperature and dissolved oxygen levels. This demonstrates an ability to tolerate the low oxygen conditions occurring within the local oxygen minimum zone. The novel combination of biologging technologies used here enabled innovative in situ deep-sea natural experiments and provided significant insight into the behavioral and physiological ecology of an ecologically important deepwater species.