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Chronic stimulation can cause T cell dysfunction and limit the efficacy of cellular immunotherapies. Improved methods are required to compare large numbers of synthetic knockin (KI) sequences to reprogram cell functions. Here, we developed modular pooled KI screening (ModPoKI), an adaptable platform for modular construction of DNA KI libraries using barcoded multicistronic adaptors. We built two ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors (SRs). Over 30 ModPoKI screens across human TCR- and CAR-T cells in diverse conditions identified a transcription factor AP4 (TFAP4) construct that enhanced fitness of chronically stimulated CAR-T cells and anti-cancer function in vitro and in vivo. ModPoKI's modularity allowed us to generate an ∼10,000-member library of TF combinations. Non-viral KI of a combined BATF-TFAP4 polycistronic construct enhanced fitness. Overexpressed BATF and TFAP4 co-occupy and regulate key gene targets to reprogram T cell function. ModPoKI facilitates the discovery of complex gene constructs to program cellular functions.

This investigation examined whether the mode of locomotion matters in how 8-, 10-, 12-, and 14-year-old children (N = 91) judge dynamic affordances in a complex perception-action task with significant safety risks. The primarily European American children in the sample came from the area of Iowa City, Iowa and were balanced for gender. The same children crossed a single lane of continuous traffic on foot and on bike (order counterbalanced) in identical immersive virtual environments. We found that although 8-year-olds chose significantly larger gaps when crossing on bike than on foot, these gaps were not large enough to compensate for their delay in entering the gap and their slowness in crossing the road. As a result, they ended up with less time to spare when exiting the roadway on bike than on foot. In contrast, 14-year-olds exhibited no difference in their gap choices on bike than on foot, nor did they exhibit a difference in their timing of entry into the gap. However, they crossed the road much more quickly on bike, resulting in significantly more time to spare when crossing on bike than on foot. The 10- and 12-year-olds' performance fit neatly between that of the 8- and 14-year-olds. We conclude that as children gained better control over the bike with age, they were better able to match their gap decisions with their crossing movements such that bicycling afforded even safer road-crossing than walking for 14-year-olds.

Spontaneous activity is a hallmark of developing neural systems. In the retina, spontaneous activity comes in the form of retinal waves, comprised of three stages persisting from embryonic day 16 (E16) to eye opening at postnatal day 14 (P14). Though postnatal retinal waves have been well characterized, little is known about the spatiotemporal properties or the mechanisms mediating embryonic retinal waves, designated stage 1 waves. Using a custom-built macroscope to record spontaneous calcium transients from whole embryonic retinas, we show that stage 1 waves are initiated at several locations across the retina and propagate across a broad range of areas. Blocking gap junctions reduced the frequency and size of stage 1 waves, nearly abolishing them. Global blockade of nAChRs similarly nearly abolished stage 1 waves. Thus, stage 1 waves are mediated by a complex circuitry involving subtypes of nAChRs and gap junctions. Stage 1 waves in mice lacking the β2 subunit of the nAChRs (β2-nAChR-KO) persisted with altered propagation properties and were abolished by a gap junction blocker. To assay the impact of stage 1 waves on retinal development, we compared the spatial distribution of a subtype of retinal ganglion cells, intrinsically photosensitive retinal ganglion cells (ipRGCs), which undergo a significant amount of cell death, in WT and β2-nAChR-KO mice. We found that the developmental decrease in ipRGC density is preserved between WT and β2-nAChR-KO mice, indicating that processes regulating ipRGC numbers and distributions are not influenced by spontaneous activity.

Nucleosome DNA unwrapping and its disassembly into hexasomes and tetrasomes is necessary for genomic access and plays an important role in transcription regulation. Previous single-molecule mechanical nucleosome unwrapping revealed a low- and a high-force transitions, and force-FRET pulling experiments showed that DNA unwrapping is asymmetric, occurring always first from one side before the other. However, the assignment of DNA segments involved in these transitions remains controversial. Here, using high-resolution optical tweezers with simultaneous single-molecule FRET detection, we show that the low-force transition corresponds to the undoing of the outer wrap of one side of the nucleosome (∼27 bp), a process that can occur either cooperatively or noncooperatively, whereas the high-force transition corresponds to the simultaneous unwrapping of ∼76 bp from both sides. This process may give rise stochastically to the disassembly of nucleosomes into hexasomes and tetrasomes whose unwrapping/rewrapping trajectories we establish. In contrast, nucleosome rewrapping does not exhibit asymmetry. To rationalize all previous nucleosome unwrapping experiments, it is necessary to invoke that mechanical unwrapping involves two nucleosome reorientations: one that contributes to the change in extension at the low-force transition and another that coincides but does not contribute to the high-force transition.

Aversive responses to bright light (photoaversion) require signaling from the eye to the brain. Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) encode absolute light intensity and are thought to provide the light signals for photoaversion. Consistent with this, neonatal mice exhibit photoaversion before the developmental onset of image vision, and melanopsin deletion abolishes photoaversion in neonates. It is not well understood how the population of ipRGCs, which constitutes multiple physiologically distinct types (denoted M1-M6 in mouse), encodes light stimuli to produce an aversive response. Here, we provide several lines of evidence that M1 ipRGCs that lack the Brn3b transcription factor drive photoaversion in neonatal mice. First, neonatal mice lacking TRPC6 and TRPC7 ion channels failed to turn away from bright light, while two photon Ca2+ imaging of their acutely isolated retinas revealed reduced photosensitivity in M1 ipRGCs, but not other ipRGC types. Second, mice in which all ipRGC types except for Brn3b-negative M1 ipRGCs are ablated exhibited normal photoaversion. Third, pharmacological blockade or genetic knockout of gap junction channels expressed by ipRGCs, which reduces the light sensitivity of M2-M6 ipRGCs in the neonatal retina, had small effects on photoaversion only at the brightest light intensities. Finally, M1s were not strongly depolarized by spontaneous retinal waves, a robust source of activity in the developing retina that depolarizes all other ipRGC types. M1s therefore constitute a separate information channel between the neonatal retina and brain that could ensure behavioral responses to light but not spontaneous retinal waves.SIGNIFICANCE STATEMENT At an early stage of development, before the maturation of photoreceptor input to the retina, neonatal mice exhibit photoaversion. On exposure to bright light, they turn away and emit ultrasonic vocalizations, a cue to their parents to return them to the nest. Neonatal photoaversion is mediated by intrinsically photosensitive retinal ganglion cells (ipRGCs), a small percentage of the retinal ganglion cell population that express the photopigment melanopsin and depolarize directly in response to light. This study shows that photoaversion is mediated by a subset of ipRGCs, called M1-ipRGCs. Moreover, M1-ipRGCs have reduced responses to retinal waves, providing a mechanism by which the mouse distinguishes light stimulation from developmental patterns of spontaneous activity.

The article published in the British Journal of Haematology analyzes the outcomes of patients with secondary central nervous system lymphoma (SCNSL) who received chimeric antigen receptor T‐cell therapy (CAR‐T). The study included 144 adult patients with SCNSL who received CD19‐directed commercial CAR‐T, with the primary endpoint being overall survival. The study found that CAR‐T was feasible and associated with similar toxicities compared to patients with systemic large B cell lymphoma, showing high response rates but not durable responses. The study highlights the need for future research to improve response rates and the durability of remission in SCNSL patients treated with CAR‐T. [Extracted from the article]