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Background: Alpha oscillations are dominant rhythms in the human brain, supporting inhibitory control and coordination of neural activity. Altered alpha dynamics are observed across many neuropsychiatric and neurodevelopmental disorders, including Fragile X syndrome (FXS), the most common monogenic cause of autism and intellectual disability. FXS exhibits paradoxical alpha power: elevated absolute but reduced relative power. To resolve this incongruity, we considered that conventional power metrics, relying on averaging, may obscure the underlying critical temporal dynamics of such neural rhythms.

Methods: Here, we investigate alpha oscillations in FXS as a model to decompose nonspecific alpha abnormalities into underlying temporal features. We used cycle-by-cycle ( bycycle ) alpha burst analysis from source-localized resting-state EEG of 70 individuals with FXS (20.5±10.0 years; 32 females, 38 males) and 71 age- and sex-matched typically developing controls (22.2±10.7 years; 30 females, 41 males). Statistical modeling examined group, sex, and regional differences in alpha burst features using generalized linear mixed-effects models.

Results: We reveal that alpha bursts in FXS show reduced count only in males, prolonged periods across sexes, and elevated amplitudes, particularly in males. Spatial mapping identified differential circuit vulnerability: timing-associated dysregulation in cognitive-control regions and amplitude elevations in sensory cortices. Within the FXS group, global alpha burst amplitude correlated with hyperactivity symptoms and inversely with general intelligence scores, and burst count correlated with age.

Limitations: This study is limited by its resting-state design and cross-sectional nature. Future studies should explore task-based modulation of alpha burst features and longitudinal trajectories in FXS. Additionally, fragile X messenger ribonucleoprotein (FMRP) was not quantified for participants, limiting potential stratification by molecular severity.

Conclusions: These findings resolve paradoxical alpha power in FXS into features consistent with interneuron dysfunction, demonstrating the potential for burst-level decomposition in mechanistic hypothesis generation and biomarker development across neurodevelopmental and neuropsychiatric disorders.

Glycolysis is a conserved metabolic pathway that produces ATP and biosynthetic precursors. Multiple allosteric regulators control glycolytic enzymes in vitro . For example, phosphofructokinase (PFK) is allosterically regulated by fructose-2,6-bisphosphate (F26BP), ATP, ADP, AMP, citrate, acyl-CoA, and inorganic phosphate. It is not well understood which properties of homeostasis are enabled by each of these regulators, and whether they perform redundant or distinct functions. Using mathematical modeling and experiments with human cells lacking F26BP, we demonstrate that F26BP alters glycolytic rate independent of cellular ATP demand-a unique function not shared by other regulators. We also identified several downstream glycolytic intermediates as novel regulators of F26BP levels. Our findings clarify the role of F26BP as a unique regulator that controls the glycolytic rate independently of the cellular energy state in response to hormone and biosynthetic precursor levels. The F26BP regulatory circuit enables respiratory fuel selection and biosynthesis from glycolytic intermediates.

PLCβ enzymes cleave PIP2 from the plasma membrane, producing IP3 and DAG, which regulate intracellular Ca ²⁺ levels and protein kinase C activity, respectively. They are regulated by GPCR signaling through the G proteins Gβγ and Gα _q and have been shown to function as coincidence detectors for dual stimulation of Gα _q and Gα _i -coupled receptors via these G proteins. PLCβs are aqueous-soluble enzymes, but partition onto the membrane surface to access their lipid substrate. We previously demonstrated that membrane recruitment and orientation of the catalytic core on the membrane surface underlie Gβγ-dependent regulation of PLCβ enzymes. Using macrophages as a model system, where PLCβ signaling is essential for responses to infection and tissue injury, we investigated the contribution of Gβγ-dependent regulation and membrane recruitment of PLCβ in the context of endogenous signaling. By measuring Ca ²⁺ mobilization, we demonstrate that both Gα _i and Gαq-coupled receptors independently stimulate PLCβ activity, illustrating that Gβγ alone is sufficient to activate PLCβ in certain contexts. Using total internal reflection and stimulated emission depletion microscopy, we demonstrate that most of the PLCβ3 in the cell is localized away from the plasma membrane at rest but is rapidly recruited to the plasma membrane upon stimulation by both Gα _i and Gα _q -coupled receptors, illustrating that both Gβγ and Gα _q recruit PLCβ to the plasma membrane. These results support an updated model for G protein-dependent regulation of PLCβ enzymes, where Gβγ-induced regulation in the absence of Gα _q is context dependent and dictated by the local concentration of receptor, G proteins, and PLCβ.

Significance statement: PLCβ enzymes are critical mediators of signal transduction with roles in neuronal, cardiac, and immunological signaling. Despite this importance, many aspects of their function and regulation remain poorly understood. PLCβs are aqueous soluble but must partition onto the membrane surface to access their lipid substrate, which enables regulation at the partitioning step, the catalytic step, or both. We previously demonstrated that membrane recruitment and orientation of the catalytic core on the membrane surface underlie the PLCβ regulation by one effector, Gβγ. Using macrophages as a model system for physiological signaling, we demonstrate that Gβγ is capable of independently activating PLCβ via membrane recruitment under the conditions of endogenous signaling.

PIWI-interacting RNAs (piRNAs) are important factors in the protection of the genome against nucleic acid invaders such as transposons. piRNAs are encoded in the genome in regions known as piRNA clusters, but major questions remain surrounding their regulation. Two such questions are how the piRNA clusters are recognized as piRNA sources, and how the piRNA response can expand from these regions to facilitate the recognition of novel invading sequences without risking the targeting of essential cellular mRNAs. Here, we investigated the piRNA clusters of the planarian S, mediterranea , and found that the clusters differ regarding the PIWI proteins their piRNAs bind to, as well as their chromatin features. We uncovered a subset of piRNA clusters that had unique chromatin features and atypical transcription. piRNAs from these clusters were depleted of genic content, stabilized by 3' methylation and were not dependent on the ping-pong mechanism. We therefore named these clusters Seed clusters. We identified a second set of piRNA clusters that we called Spread clusters, which had features reminiscent of pseudogenes or aberrant genic transcripts. piRNA generation from these clusters relied on ping-pong, and piRNAs largely remained unmethylated. Further, we found that many more regions in the genome generated single ping-pong events, suggesting that ping-pong is used as a means to diversify the collection of piRNA-generating transcripts beyond the Seed clusters. We propose that this two-tiered organization of the piRNA clusters allows the stable targeting of known genomic threats by the piRNAs generated from the Seed clusters, while the flexible generation of additional diverse piRNAs from Spread clusters as well as other aberrant transcripts increases the sequence space probed by the piRNA system. The absence of methylation on these additional piRNAs decreases their lifespan and limits the chances of a run-away response. This two-tiered organization thus solves a core challenge of the piRNA system and may be a widespread feature of piRNA systems across animals.

Sex differences in brain connectivity are well documented, yet how these differences evolve across the human lifespan remains poorly understood. Rigorously assessing sex-dependent trajectories of brain network organization is challenging due to difficulty in acquiring, processing, and modeling high-dimensional connectomes. Here, we analyzed 15 types of functional and structural connectivity networks from 1286 healthy individuals aged 8-100+ years, using our new AI-based Krakencoder to derive a low-dimensional multimodal "fusion" connectome representation. Sex differences were minimal in early childhood, pronounced in young to mid-adulthood, and diverged across modalities in later life: functional connectivity grew less distinct and structural connectivity grew more distinct from midlife onward. Functional differences were driven predominantly by higher-order association networks (default mode, control), while structural differences concentrated in lower-order cerebellar and subcortical pathways. These findings provide a lifespan-wide, multimodal map of sex differences in brain networks which may help inform sex-specific vulnerability and resilience to brain disorders.

In adult tissues, epithelial stem cells exist within distinct residences, each endowing them with exclusive instructions for regenerative fitness under homeostasis and stress. Key components of these 'niches' are immune cells, which classically protect the host against external and internal threats. Whether and how stem cell:immune cell crosstalk contributes to normal tissue biology remains less clear. Here, we discover functional adaptation of resident lymphocytes within two distinct skin stem cell niches and show that through this communication, each niche adjusts to meet diverse tissue demands. In the upper hair follicle, where microbial load is high, T cells express lymphotoxin-β and stimulate adjacent receptor-positive epithelial stem cells to form an immune-competent niche that controls microbial expansion. By contrast, in the epidermis, these T cells produce amphiregulin to maintain continuous stem cell reconstitution of the skin's barrier. Concomitantly, they express the immune checkpoint protein 'LAG-3', which autorestricts lymphocyte numbers, and hence amphiregulin levels, thereby preventing over-proliferative responses. Finally, when epidermal T cells are absent, dermal lymphocytes restore the imbalance by colonizing and adapting to their new niche. Our findings unveil functional specialization and homeostatic resilience of immune-stem cell niches, each tailored to suit the demands of distinct tissue microenvironments.

Protein-bound uremic toxins, such as indoxyl sulfate and p -cresyl sulfate, are major contributors to chronic kidney disease (CKD) complications and are poorly removed by dialysis due to strong albumin binding. Targeting their gut-derived microbial precursors offers a promising strategy to reduce systemic toxin load. Thauera aminoaromatica S2 is known to anaerobically degrade p -cresol, but its response to indole and its potential as an orally administered microbial therapy remain poorly characterized. Here, we investigated the activity of Thauera aminoaromatica S2 under exposure to both p -cresol and indole in planktonic and hydrogel-encapsulated forms. Low indole levels (0.25 mM) enhanced planktonic growth in the presence of 2 mM p -cresol, whereas co exposure inhibited p-cresol degradation in hydrogel systems, likely due to restricted diffusion and elevated local indole concentrations. Nonetheless, encapsulation enabled tolerance to conditions (2 mM p -cresol + 0.5 mM indole) that abolished planktonic growth, suggesting microenvironmental protection. Incorporation of activated carbon into the hydrogel restored p -cresol removal despite indole exposure, likely through localized indole sequestration. These results highlight the potential of combining encapsulation with adsorptive additives to stabilize microbial function and support the development of microbial therapies aimed at mitigating uremic toxin precursors in CKD.

Functional screening through systematic deletion, editing or addition of libraries of genes is a powerful approach for discovering gene functions and developing improved molecular tools. However, due to the need for high throughput, such campaigns are typically conducted in vitro, leading to many discoveries, especially tools and therapeutics, which fail to translate in vivo. Tissue context, cellular physiology, and systemic regulation shape both tool performance and gene function in ways that simplified culture systems cannot predict. Pooled in vivo screening methods have the potential to enable screening within living animals while preserving the physiological context, but current approaches using viral vectors face three critical limitations: multi-transgene insertions per cell confound genotype-phenotype association, viral packaging constrains transgene size, and cell-type tropism restricts and biases targeting. Here, we introduce a zebrafish library transgenesis method that overcomes these limitations through delayed site-specific mosaic integration. We exploit a temporal delay between library microinjection with PhiC31 mRNA, and library integration, to allows the library to spread episomally throughout the developing embryo before integration begins. This produces mosaic animals where each cell independently integrates one randomly-selected library member, enforced by a single genomic AttP landing site. We demonstrate delivery of multi-kilobase transgenes with high library coverage of 1,378-1,989 unique integrants per animal, and single-transgene-per-cell in ∼99% of brain cells. This method provides a platform for direct in vivo screening of large transgene libraries with single-transgene precision, with potential applications in both biological discovery and tool development.

Glycosyltransferases (GTs) catalyze the formation of new glycosidic bonds and thus are vital for synthesizing nature's vast repertoire of glycans and glycoconjugates and for engineering glycan-related medicines and materials. However, obtaining detailed structural and functional insights for the >750,000 known GTs is limited by difficulties associated with their efficient recombinant expression. Members of the GT-C fold, in particular, pose the most significant expression challenges due to the integration and folding requirements of their multiple membrane-spanning regions. Here, we address this challenge by engineering water-soluble variants of an archetypal GT-C fold enzyme, namely the oligosaccharyltransferase PglB from Campylobacter jejuni ( Cj PglB), which possesses 13 hydrophobic transmembrane helices. To render Cj PglB water-soluble, we leveraged two advanced protein engineering methods: one that is universal called SIMPLEx ( s olubilization of IMP s with high levels of ex pression) and the other that is custom tailored called WRAPs ( w ater-soluble R Fdiffused a mphipathic p roteins). Each approach was able to transform Cj PglB into a water-soluble enzyme that could be readily expressed in the cytoplasm of Escherichia coli cells at yields in the 3-6 mg/L range. Importantly, solubilization was achieved without the need for detergents and with retention of catalytic function. Collectively, our findings demonstrate that both SIMPLEx and WRAPs are promising platforms for advancing the molecular characterization of even the most structurally complex GTs, while also enabling broader GT-mediated glycosylation capabilities within synthetic glycobiology applications.

High-grade serous carcinoma (HGSC) is the most common and aggressive form of ovarian cancer. Advanced HGSCs exhibit pronounced cellular heterogeneity, including a subset of cancer-propagating cells (CPCs, also known as cancer stem cells) that are highly tumorigenic and display stem cell-associated properties such as self-renewal and chemoresistance. In contrast, a substantial fraction of HGSC cells is non-tumorigenic. The role of these non-cancer-propagating cells (non-CPCs) and their relationship to CPCs remain poorly understood. Here, we demonstrate that neoplastic cells expressing the intermediate filament protein keratin 5 (KRT5) represent bona fide CPCs. KRT5⁺ cells form cancer organoids over successive passages, are tumorigenic in serial dilution xenograft assays, and are resistant to the antineoplastic agents, doxorubicin and cisplatin. Single-cell lineage-tracing experiments show that KRT5⁺ CPCs give rise to KRT5⁻ cells. KRT5⁺ and KRT5⁻ populations exhibit distinct gene expression profiles, with KRT5⁻ cells characterized by expression of SPP1 , which encodes the secreted factor osteopontin (OPN). Treatment with OPN enhances HGSC organoid growth and chemoresistance, whereas SPP1 knockdown reverses these effects. Together, these findings support a model in which HGSC contains two hierarchically related cell populations: KRT5⁺, OPN-responsive CPCs and KRT5⁻, non-tumorigenic cells that form a niche producing OPN. Targeting pathways that sustain both stem-like tumor cells and their supportive niche may enable reduced dosing of highly toxic chemotherapeutic agents while enhancing therapeutic efficacy in HGSC.