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Genome-edited human induced pluripotent stem cells (iPSCs) hold great promise for therapeutic applications. However, low editing efficiency has hampered the applications of CRISPR-Cas9 technology in creating knockout and homology-directed repair (HDR)-edited iPSC lines, particularly for silent genes. This is partially due to chromatin compaction, inevitably limiting Cas9 access to the target DNA. Among the six HDAC inhibitors we examined, vorinostat, or suberoylanilide hydroxamic acid (SAHA), led to the highest HDR efficiency at both open and closed loci, with acceptable toxicity. HDAC inhibitors equally increased non-homologous end joining (NHEJ) editing efficiencies (∼50%) at both open and closed loci, due to the considerable HDAC inhibitor-mediated increase in Cas9 and sgRNA expression. However, we observed more substantial HDR efficiency improvement at closed loci relative to open chromatin (2.8 vs. 1.7-fold change). These studies provide a new strategy for HDR-editing of silent genes in iPSCs.

We present an end-to-end architecture for embodied exploration inspired by two biological computations: predictive coding and uncertainty minimization. The architecture can be applied to any exploration setting in a task-independent and intrinsically driven manner. We first demonstrate our approach in a maze navigation task and show that it can discover the underlying transition distributions and spatial features of the environment. Second, we apply our model to a more complex active vision task, whereby an agent actively samples its visual environment to gather information. We show that our model builds unsupervised representations through exploration that allow it to efficiently categorize visual scenes. We further show that using these representations for downstream classification leads to superior data efficiency and learning speed compared to other baselines while maintaining lower parameter complexity. Finally, the modular structure of our model facilitates interpretability, allowing us to probe its internal mechanisms and representations during exploration.

The anatomy of the mammalian visual system, from the retina to the neocortex, is organized hierarchically1. However, direct observation of cellular-level functional interactions across this hierarchy is lacking due to the challenge of simultaneously recording activity across numerous regions. Here we describe a large, open dataset—part of the Allen Brain Observatory2—that surveys spiking from tens of thousands of units in six cortical and two thalamic regions in the brains of mice responding to a battery of visual stimuli. Using cross-correlation analysis, we reveal that the organization of inter-area functional connectivity during visual stimulation mirrors the anatomical hierarchy from the Allen Mouse Brain Connectivity Atlas3. We find that four classical hierarchical measures—response latency, receptive-field size, phase-locking to drifting gratings and response decay timescale—are all correlated with the hierarchy. Moreover, recordings obtained during a visual task reveal that the correlation between neural activity and behavioural choice also increases along the hierarchy. Our study provides a foundation for understanding coding and signal propagation across hierarchically organized cortical and thalamic visual areas.

The mammalian cortex is a laminar structure containing many areas and cell types that are densely interconnected in complex ways, and for which generalizable principles of organization remain mostly unknown. Here we describe a major expansion of the Allen Mouse Brain Connectivity Atlas resource1, involving around a thousand new tracer experiments in the cortex and its main satellite structure, the thalamus. We used Cre driver lines (mice expressing Cre recombinase) to comprehensively and selectively label brain-wide connections by layer and class of projection neuron. Through observations of axon termination patterns, we have derived a set of generalized anatomical rules to describe corticocortical, thalamocortical and corticothalamic projections. We have built a model to assign connection patterns between areas as either feedforward or feedback, and generated testable predictions of hierarchical positions for individual cortical and thalamic areas and for cortical network modules. Our results show that cell-class-specific connections are organized in a shallow hierarchy within the mouse corticothalamic network.

Ruling out the presence of cirrhosis is important for the management of chronic hepatitis B. We aimed to study and optimise the performance of two non-invasive indices for ruling out cirrhosis: the aspartate aminotransferase-platelet ratio index (APRI) and fibrosis score based on four factors (FIB-4).

One-dimensional (1-D) SnO2lines as a representative semiconducting oxide were formed by using electrohydrodynamic (EHD) jet-printing of a tin chloride pentahydrate and polyvinylpyrrolidone (PVP, 1,200k, Aldrich) solution ink. The 1-D polymer lines, including Sn precursors, were created by controlling the viscosity, that is, the polymer/tin precursor ratio, and by adjusting printing conditions such as the tip-to-substrate distance, the applied voltage, the flow rate of ink and its velocity. The printed lines were dried at 200 °C to get rid of solvent and were finally heat-treated at 600 °C to burn out PVP and form a tin oxide line. We found that the linearity and the shape of the aligned 1-D SnO2could be controlled by adjusting various parameters such as the viscosity of the precursor solution, the ratio of Sn to the PVP polymer in the solution, the shape of the cone, the size of a droplet, the applied voltage, the working distance, and the flow rate on glass slides and Si wafers with a SiO2layer. We found that the heat treatment for removal of the polymers should be tailored to produce continuous 1-D SnO2lines due to the drastic volume reduction (> 90%) of the aligned fibers during the annealing process. The electrical and the NO-gas-sensing properties of the 1-D SnO2aligned on Si wafers with Au electrode patterns were evaluated.

We compared ICU nonopioid analgesic use, opioid use, and pain before and after Pain, Agitation/Sedation, Delirium, Immobility, and Sleep guideline publication at one academic center among critically ill adults receiving an opioid infusion and greater than or equal to 24 hours of mechanical ventilation after major surgery. The 2017 (n= 77) and 2019 (n= 57) groups were similar at baseline. The 2019 (vs 2017) patients were more likely to receive scheduled IV/oral acetaminophen (84% vs 69%; p= 0.05), less likely to receive a lidocaine patch (33% vs 50%; p= 0.05), and just as likely to receive ketamine (4% vs 3%; p= 1.0), an nonsteroidal anti-inflammatory drug (7% vs 3%; p= 0.26), or gabapentin/pregabalin (16% vs 9%; p= 0.23). Daily average opioid exposure (in IV morphine milligram equivalent) was not different (70 [42–99] [2017] vs 78 mg [49–109 mg]; p= 0.94). The 2019 (vs 2017) group spent more ICU days with severe pain (p= 0.04). At our center, Pain, Agitation/Sedation, Delirium, Immobility, and Sleep guideline publication had little effect on nonopioid analgesic or opioid prescribing practices in critically ill surgical adults.

Background: Hemophilia A, a bleeding disorder resulting from F8mutations, can only be cured by gene therapy. A promising strategy is CRISPR-Cas9-mediated precise insertion of F8in hepatocytes at highly expressed gene loci, such as albumin (Alb). Unfortunately, the precise in vivo integration efficiency of a long insert is very low (~ 0.1%). Results: We report that the use of a double-cut donor leads to a 10- to 20-fold increase in liver editing efficiency, thereby completely reconstituting serum F8 activity in a mouse model of hemophilia A after hydrodynamic injection of Cas9-sgAlb and B domain-deleted (BDD) F8 donor plasmids. We find that the integration of a double-cut donor at the Alblocus in mouse liver is mainly through non-homologous end joining (NHEJ)-mediated knock-in. We then target BDDF8to multiple sites on introns 11 and 13 and find that NHEJ-mediated insertion of BDDF8restores hemostasis. Finally, using 3 AAV8 vectors to deliver genome editing components, including Cas9, sgRNA, and BDDF8donor, we observe the same therapeutic effects. A follow-up of 100 mice over 1 year shows no adverse effects. Conclusions: These findings lay the foundation for curing hemophilia A by NHEJ knock-in of BDDF8at Albintrons after AAV-mediated delivery of editing components.

Coats: Elf Zone, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees. Meng:Elf Zone, Inc.: Employment. Dovat:Elf Zone, Inc.: Membership on an entity's Board of Directors or advisory committees. Payne:Elf Zone, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Background. Approximately half of all Ph-like B cell acute lymphoblastic leukemia is characterized by overexpression of CRLF2 (CRLF2 B-ALL). CRLF2 B-ALL is associated with high rates of relapse and is more prevalent in Hispanic children with Native American ancestry. CRLF2, together with the IL-7 receptor alpha (IL-7Ra), comprises a receptor complex that is activated by the cytokine, TSLP. Receptor activation by TSLP induces JAK2/STAT5 and PI3/AKT/mTOR signals that promote survival and proliferation of leukemia cells. To study the role of TSLP in CRLF2 B-ALL, we developed a patient-derived xenograft (PDX) model of CRLF2 B-ALL that allows us to vary circulating levels of human TSLP (hTSLP) in a physiologic environment. We generated PDX from patients' CRLF2 B-ALL cells and compared leukemia burden in mice with varying levels of hTSLP. CRLF2 B-ALL cells grew robustly in PDX models with hTSLP levels at or below levels present in pediatric cancer patients (~10 pg/mL). In contrast, CRLF2 B-ALL cells were essentially eliminated in PDXs with hTSLP at high physiological levels (40-140 pg/mL). These data suggest that high physiologic levels of TSLP exert a potent anti-leukemia effect in Ph-like B-ALL with overexpression of CRLF2.

Ph-like B-cell acute lymphoblastic leukemia (B-ALL) is associated with poor outcomes and high relapse rates. Approximately 50% of Ph-like B-ALL cases occur due to genetic alterations leading to overexpression of the cytokine receptor, CRLF2. B-ALL with overexpression of CRLF2 (CRLF2 B-ALL) occurs 5 times more often in Hispanic children than others, is prevalent in adolescents and young adults, and makes up ~1/3 of adult B-ALL. CRLF2, together with the IL-7Rα, forms a receptor complex that can be activated by circulating TSLP cytokine in patients. The human CRLF2 receptor is not activated by mouse TSLP so classic patient-derived xenografts (PDX) do not model TSLP-induced CRLF2 signaling that occurs in patients. CRLF2-mediated JAK/STAT and PI3/AKT/mTOR signals are believed to contribute to survival and proliferation of leukemia cells. We developed a novel PDX model of CRLF2 B-ALL that allows us to vary circulating levels of human TSLP (+T PDX). Primary CRLF2 B-ALL cells injected into +T PDX with circulating human TSLP (hTSLP) levels similar to pediatric leukemia patients (~4-10 pg/ml), engrafted well and showed a gene expression pattern that was more similar to the original patient sample than when injected into classic PDX. To our surprise, when +T PDX expressed physiological, but elevated levels of hTSLP (> 40 pg/ml, upper end of range reported in healthy children), CRLF2 B-ALL cells were essentially eliminated, but grew robustly in PDX without hTSLP (-T PDX). These results have been observed in 4 independent experiments for a total of 17 +T PDX and 12 -T PDX mice produced using CRLF2 B-ALL cells from two different Hispanic pediatric patients with CRLF2 B-ALL. We hypothesize that anti-leukemia effects observed at hTSLP concentrations above 40 pg/ml are mediated via TSLP-induced upregulation of the Suppressor of Cytokine Signaling (SOCS) genes. SOCS genes encode a family of proteins that regulate cytokine signaling via negative feedback through multiple mechanisms including ubiquitin-mediated degradation of JAKs and cytokine receptor components. Using the CRLF2 B-ALL cell lines, MUTZ5 and CALL-4, we found that SOCS family proteins encoded by the SOCS1 and SOCS3 genes were upregulated following culture with 15 ng/ml hTSLP, as compared to controls without hTSLP. Similarly, primary CRLF2 B-ALL cells from Hispanic pediatric patients cultured with TSLP showed upregulation of SOCS1 and SOCS3 mRNA and proteins. Next, we determined whether the upregulation of SOCS proteins was accompanied by a loss of CRLF2-mediated signals. We found that CRLF2 B-ALL cell lines and primary cells cultured with hTSLP lost the ability to upregulate STAT5 and S6 phosphorylation following hTSLP stimulation. Further, cells cultured with TSLP showed a loss of cytokine receptors as well as STAT5 phosphorylation levels that were below the baseline observed in leukemia cells never exposed to hTSLP. These data suggest that TSLP exerts anti-leukemia effects by shutting down CRLF2-mediated signals and that this shut down may occur through SOCS-mediated degradation of cytokine receptor components and/or degradation of the mutant JAK2 that provides constitutively low level STAT5 activation in these CRLF2 B-ALL cell lines and in a majority of patients with this disease. In summary, these results provide evidence that elevated hTSLP exerts a therapeutic effect on CRLF2 B-ALL and suggest that this occurs via SOCS-mediated suppression of the CRLF2 signaling pathway. These studies identify the human TSLP cytokine as a potential biologic therapy to treat CRLF2 B-ALL and reduce cancer health disparities for Hispanic children with CRLF2 B-ALL. Supported by 1R01CA209829.