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Background: Pregnancy requires finely tuned immune changes that support implantation, placental development, maternal-fetal tolerance, and preparation for labor, yet the normative trajectories of circulating inflammatory proteins across gestation remain poorly defined. This cross-sectional study investigates how circulating inflammatory proteins vary with gestational age in pregnancy and examines the impacts of fundamental biological characteristics, such as gravidity and fetal sex.

Methods: Data were drawn from 1154 pregnant individuals from six study sites of the National Institutes of Health Environmental influences on Child Health Outcomes (ECHO) Cohort. We used Olink high-throughput proteomic profiling to map cross-sectional associations between protein expression levels and gestational age at blood draw using linear, spline-based, and generalized additive modeling approaches.

Results: Generalized additive models provided the best fit, revealing that immune changes across pregnancy were predominantly nonlinear. Sixty-one proteins showed significant associations with gestational age, with many exhibiting shared inflection points that aligned with major physiological transitions. A small subset of proteins also showed evidence of modification by fetal and maternal characteristics. CD244 displayed different gestational patterns by fetal sex, while CST5 and SIRT2 showed varied gestational associations by maternal gravidity.

Conclusion: The findings highlight pregnancy as a sequence of coordinated immune transitions rather than a simple linear shift and provide one of the most detailed characterizations to date of circulating inflammatory protein dynamics across human gestation. Establishing these normative trajectories offers a crucial reference for detecting early deviations that may signal risk for pregnancy complications and for identifying biomarkers in maternal and fetal health research.

The integration of electroencephalography (EEG) and functional Magnetic Resonance Imaging (fMRI) can be used to characterize temporal and spatial components of neural activity during unfolding mental experience. Here we introduce a multi-session simultaneous EEG-fMRI dataset with measures of continuous behavior and spontaneous mental experience. Data components, organized in Brain Imaging Dataset Structure (BIDS) format, include fMRI, EEG with carbon wire loop sensors for artifact removal, continuous performance task responses, experience sampling ratings, and mental health surveys, from 24 healthy adults. Tasks included the gradual-onset continuous performance task and resting state with intermittent experience sampling of 13 unique thought dimensions (36 repetitions, including 468 total ratings, per participant). The same protocol was completed on two different days, yielding approximately 1.33 hours of simultaneous EEG-fMRI data per individual. The dataset may be used to explore the behavioral and experiential relevance of brain activity during the wakeful resting state. The dataset also provides a means to study the reliability of relationships between fMRI and EEG features across sessions within individuals.

The developmental relationship between nephron progenitors and the renal interstitium remains unresolved, in part due to limitations of existing lineage tracing tools. The widely used transgenic Six2TGC line, which is routinely employed to target the nephron lineage, exhibits mosaic recombination and altered progenitor dynamics. To overcome these shortcomings, we generate a knock-in Six2Cre mouse allele that faithfully recapitulates endogenous Six2 expression, preserves nephron endowment, and achieves near-complete, non-mosaic recombination. Side-by-side lineage tracing with Six2Cre and Six2TGC, combined with RNA velocity analysis of single-cell RNA-sequencing datasets, reveals a brief interval around embryonic day 11 during which Six2-expressing mesenchymal nephron progenitors contribute to the renal interstitium. This contribution is transient and stage-restricted. These findings reveal an early dual potential within nephron progenitors and define a precise developmental window for dissecting mechanisms that coordinate nephron-interstitium integration.

Reconstructing the lineage histories of individual cells can reveal the dynamics of developmental and disease processes. In engineered recording systems, cells stochastically edit synthetic barcode sequences as they proliferate, creating distinct, heritable edit patterns that can be used to reconstruct the lineage trees relating individual cells in a manner analogous to phylogenetic reconstruction. However, recording depth is often limited by the kinetics of the editing process: the rate of editing declines exponentially over time for an array of independently editable targets, leading to most edits occurring in early generations. Here, we introduce the hypercascade, a regenerative molecular recording system that takes advantage of the predictability of A-to-G base editing to progressively create new target sites over time. The hypercascade packs 4 editable target sites in every 20 bp of sequence, enabling high density information storage. More importantly, the hypercascade's regenerative logic leads to an approximately constant rate of mutation accumulation over time. This in turn facilitates reconstruction of deep lineage relationships. We demonstrate this by reconstructing trees spanning 23 days of editing and approximately 17 generations after a single polyclonal engineering step. Finally, simulations show that the hypercascade has the potential to record chromatin state transition dynamics across multiple genomic loci in parallel. The hypercascade thus provides a flexible and broadly useful tool for molecular recording.

Online monitoring and quantification of neural signals has tremendous value both for neurofeedback experiments and for brain-computer interfaces. Unfortunately, established methods of online monitoring primarily involve the use of thresholded neural activity rather than sorted single-neuron spikes. The recent introduction of large-scale, high-density electrophysiology has enabled the recording of activity from hundreds of neurons simultaneously in both model organisms and human participants. This development highlights the need for a robust and easily implementable system for sorting spikes during data collection for live analyses of neuronal signals. Here, we describe a system for live sorting of neuronal activity (LSS) based on the widely used Kilosort platform. The LSS workflow utilizes an initial period of recorded neural data to identify waveform templates using Kilosort. LSS then interfaces with the SpikeGLX API to retrieve small batches (e.g. 50 ms) of data and for processing online. We measured the similarity of single-neuron activity sorted live by LSS to that sorted offline in neurophysiological recordings from macaque visual cortex using Neuropixels probes. We show that LSS closely replicates the post-stimulus time histograms and visual response tuning curves of single-neurons obtained using offline sorting. Furthermore, we show that decoding neural signals online with LSS consistently outperforms online decoding of thresholded activity, and that LSS can achieve the same performance as that obtained with offline sorting.

Gene therapy using adeno-associated virus (AAV) vectors shows promise for cancer treatment through molecular intervention, yet achieving sufficient and targeted delivery to brain tumors via systemic administration remains limited by the biological barriers. Here, we investigate whether microbubble-enhanced focused ultrasound (MB-FUS) enhances targeted delivery of systemically administered AAV to orthotopic gliomas, using quantitative PET imaging of 64Cu-radiolabeled AAV9 vectors and fluorescent reporter expression to assess biodistribution and functional efficacy. PET imaging at 21 hours post-injection revealed 3.2-fold higher 64Cu-AAV accumulation in MB-FUS-treated tumors compared to non-sonicated tumors (n=3, p=0.004). Quantitative PCR analysis of tumor tissue at the same timepoint confirmed enhanced vector delivery, demonstrating 6.4-fold increased genome copies in MB-FUS-treated tumors (p=0.0003). Optical reporter gene imaging at 17 days post-treatment revealed that enhanced vector delivery translated to significantly increased transgene expression, with 5.3-fold higher transduction in MB-FUS-treated tumors (p=0.0002). These results establish that MB-FUS enables spatially-targeted AAV delivery with quantifiable enhancement of both acute vector biodistribution and downstream transgene expression. The integration of radiolabeled AAV with PET imaging provides a non-invasive methodology for real-time assessment of vector delivery and optimization of treatment protocol for brain cancer gene therapy.

Activated CD4+ T cells have long been considered the principal source of HIV production before treatment and reactivated latently infected cells the source of virus rebound on treatment interruption, based largely on studies of peripheral blood T cells. Here we show before ART in the lymphoid tissue reservoir that HIV-producing resting CD4+ T cells are essentially the sole source of virus production in germinal centers. Virus is produced in infection complexes of virus bearing follicular dendritic cells (FDCs) that induce high multiplicity multi-HIV-DNA copy infections in overlying resting T cells. HIV production is associated with destruction by PANoptosis (pyroptosis, necroptosis and apoptosis) of interacting antibody-producing populations of FDCs, T cells and B cells. PANoptosis is mediated by the PANoptosome, visualized and defined here as a cellular structure incorporating cytoplasmic and nuclear contents and programmed cell death pathway components in a nodal latticework that executes cell emptying and disruption. During ART, HIV-producing resting T cells persist in lymphoid tissues when virus is undetectable in peripheral blood. These HIV-producing resting T cells that persist during ART represent an immediate source of virus to reignite infection on treatment interruption and are thus identified as an important new target for functional cure strategies.

The action potential (AP) is thought to be generated at the axon initial segment and to faithfully propagate along the axon. However, data from both invertebrate and mammalian systems show that the axon is an underappreciated locus of activity modulation and neuronal computation. We assessed axonal AP propagation in neocortical parvalbumin-expressing interneurons (PV-INs) during prolonged, high-frequency activity through paired whole-cell somatic and axon-attached patch clamp recordings in acute brain slices from mouse and human. We found that PV-IN axonal AP propagation remains robust during prolonged activity at moderate frequencies, such as during the entrainment to PV-IN firing patterns recorded in awake, behaving mice in vivo. However, prolonged, high-frequency activity during evoked trains of APs and during seizure-like events resulted in changes in the waveform of the axonal (but not somatic) AP, at least in part due to intrinsic use-dependent mechanisms. This use-dependent decrement in the axonal AP waveform is associated with decreases in calcium influx at PV-IN boutons and subsequent PV-IN-mediated synaptic transmission, indicating this phenomenon may lead to a dissociation between somatic and axonal excitability that could shape PV-IN contributions to circuit dynamics during periods of high activity.

Segregation distortion, the disproportionate inheritance of selfish genetic elements, is an important evolutionary force. While many species carry distorters, it is not clear if humans do. Major limitations for detecting human distortion are the small size of human families and the lack of genetic markers in most subjects. Here, we present evidence of strong distortion in a large human pedigree. We analyzed pedigrees from the Utah Population Database and identified lineages with a high chance of carrying a distorter. In particular, we identified a family that preferentially produced male offspring at a 2:1 ratio. This pattern is consistent with a distorting Y-chromosome, a rarity in species with degenerate Y-chromosomes. The detection of such non-Mendelian inheritance patterns suggests that human genomes may harbor segregation distorters.

Cholesterol is essential for organization of neurotransmitter release machinery, yet how it regulates the balance among different forms of synaptic exo- and endocytosis remains poorly understood. Moreover, which pre-synaptic processes rely on neuronal vs astrocyte-derived cholesterol is unknown. Using nanoscale-precision imaging of single-vesicle release in hippocampal synapses we demonstrate that astrocytic cholesterol is a critical determinant of both temporal and spatial aspects of presynaptic dynamics by differentially modulating the two main forms of synchronous release, uni-vesicular (UVR) and multi-vesicular (MVR), effectively fine-tuning their balance. Disruption of astrocytic cholesterol trafficking to neurons combined with its re-supplementation demonstrated that astrocyte-derived cholesterol is necessary and sufficient to determine the UVR/MVR balance. Moreover, astrocytic cholesterol determines the spatial distribution of vesicle release by modulating utilization of different release sites across the active zone. Astrocytic cholesterol also regulates the balance of the two main forms of single-vesicle endocytosis, fast and ultra-fast. These findings suggest that astrocytic cholesterol supply is a critical modulator of synaptic strength that fine-tunes the balance of different forms of synaptic vesicle exo- and endocytosis.