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Chronic HIV infection can dysregulate lipid/cholesterol metabolism in the peripheral system, contributing to the higher incidences of diabetes and atherosclerosis in HIV (+) individuals. Recently, accumulating evidence indicate that HIV proteins can also dysregulate lipid/cholesterol metabolism in the brain and such dysregulation could be linked with the pathogenesis of HIV-associated neurological disorders (HAND)/NeuroHIV. To further characterize the association between lipid/cholesterol metabolism and HAND, we employed HIV-inducible transactivator of transcription (iTAT) and control mice to compare their brain lipid profiles. Our results reveal that HIV-iTAT mice possess dysregulated lipid profiles and have increased numbers of lipid droplets (LDs) accumulation microglia (LDAM) in the brains. HIV protein TAT can upregulate LDs formation through enhancing the lipid/cholesterol synthesis in vitro. Mechanistically, HIV-TAT increases the expression of sterol regulatory element-binding protein 2 (SREBP2) through microRNA-124 downregulation. Cholesterol synthesis inhibition can block HIV-TAT-mediated NLRP3 inflammasome activation and microglial activation in vitro as well as mitigate aging-related behavioral impairment and memory deficiency in HIV-iTAT mice. Taken together, our results indicate an inherent role of lipid metabolism and LDAM in the pathogenesis of NeuroHIV (immunometabolism). These findings suggest that LDAM reversal through modulating lipid/cholesterol metabolism could be a novel therapeutic target for ameliorating NeuroHIV symptoms in chronic HIV (+) individuals.

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Perivascular mural cells including vascular smooth cells (VSMCs) and pericytes are integral components of the vascular system. In the central nervous system (CNS), pericytes are also indispensable for the blood-brain barrier (BBB), blood-spinal cord barrier and blood-retinal barrier, and play key roles in maintaining cerebrovascular and neuronal functions. However, the functional specifications of pericytes between CNS and peripheral organs have not been resolved at the genetic and molecular levels. Hence, the generation of reliable CNS pericyte-specific models and genetic tools remains very challenging. Here, we report a new CNS pericyte marker in mice. This putative cation-transporting ATPase 13A5 (Atp13a5) marker was identified through single cell transcriptomics, based on its specificity to brain pericytes. We further generated a knock-in model with both tdTomato reporter and Cre recombinase. Using this model to trace the distribution of Atp13a5-positive pericytes in mice, we found that the tdTomato reporter reliably labels the CNS pericytes, including the ones in spinal cord and retina but not peripheral organs. Interestingly, brain pericytes are likely shaped by the developing neural environment, as Atp13a5-positive pericytes start to appear around murine embryonic day 15 (E15) and expand along the cerebrovasculature. Thus, Atp13a5 is a specific marker of CNS pericyte lineage, and this Atp13a5-based model is a reliable tool to explore the heterogeneity of pericytes and BBB functions in health and diseases.Significance Statement Pericyte is a key component of the blood-brain barrier (BBB) and highly implicated in neurological and neurodegenerative diseases. However, current genetic tools for brain pericytes often come with limitations, due to the lack of specificity to the pericytes in the brain or central nervous system (CNS), as well as the overlap with other cell types, particularly vascular smooth muscle cells. Here, we identified that Atp13a5 is a CNS-specific pericyte marker based on mouse single-cell transcriptomics, and further validate it using a knock-in model carrying Atp13a5-driven tdTomato reporter and Cre recombinase. The success of the Atp13a5-based model opens new possibility of genetic manipulations targeting only CNS pericytes in vivo and studying their biology and functions in health and diseases more specifically.

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Ribosomopathies arise from the disruptions in ribosome biogenesis within the nucleolus, which is organized via liquid-liquid phase separation (LLPS). The roles of LLPS in ribosomopathies remain poorly understood. Here, we generated human induced pluripotent stem cell (hiPSC) models of ribosomopathy caused by mutations in small nucleolar RNA (snoRNA) gene SNORD118. Mutant hiPSC-derived neural progenitor cells (NPCs) or neural crest cells (NCCs) exhibited ribosomopathy hallmark cellular defects resulting in reduced organoid growth, recapitulating developmental delay in patients. SNORD118 mutations in NPCs disrupted nucleolar morphology and LLPS properties coupled with impaired ribosome biogenesis and a translational downregulation of fibrillarin (FBL), the key LLPS effector acting via the intrinsically disordered region (IDR) motif. IDR-depleted FBL failed to rescue NPC defects, whereas a chimeric FBL with swapped IDR motif from an unrelated protein mitigated ribosomopathy and organoid growth defects. Thus, SNORD118 human iPSC models revealed aberrant phase separation and nucleolar functions as potential pathogenic mechanisms in ribosomopathies.

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Photocatalytic water splitting (PWS) as the holy grail reaction for solar-to-chemical energy conversion is challenged by sluggish oxygen evolution reaction (OER) at water/catalyst interface. Experimental evidence interestingly shows that temperature can significantly accelerate OER, but the atomic-level mechanism remains elusive in both experiment and theory. In contrast to the traditional Arrhenius-type temperature dependence, we quantitatively prove for the first time that the temperature-induced interface microenvironment variation, particularly the formation of bubble-water/TiO2(110) triphase interface, has a drastic influence on optimizing the OER kinetics. We demonstrate that liquid-vapor coexistence state creates a disordered and loose hydrogen-bond network while preserving the proton transfer channel, which greatly facilitates the formation of semi-hydrophobic •OH radical and O-O coupling, thereby accelerating OER. Furthermore, we propose that adding a hydrophobic substance onto TiO2(110) can manipulate the local microenvironment to enhance OER without additional thermal energy input. This result could open new possibilities for PWS catalyst design.

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Animal or human models recapitulating brain ribosomopathies are incomplete, hampering development of urgently needed therapies. Here, we generated genetic mouse and human cerebral organoid models of brain ribosomopathies, caused by mutations in small nucleolar RNA (snoRNA) SNORD118. Both models exhibited protein synthesis loss, proteotoxic stress, and p53 activation and led to decreased proliferation and increased death of neural progenitor cells (NPCs), resulting in brain growth retardation, recapitulating features in human patients. Loss of SNORD118 function resulted in an aberrant upregulation of p-eIF2α, the mediator of integrated stress response (ISR). Using human iPSC cell-based screen, we identified small-molecule 2BAct, an ISR inhibitor, which potently reverses mutant NPC defects. Targeting ISR by 2BAct mitigated ribosomopathy defects in both cerebral organoid and mouse models. Thus, our SNORD118 mutant organoid and mice recapitulate human brain ribosomopathies and cross-validate maladaptive ISR as a key disease-driving mechanism, pointing to a therapeutic intervention strategy.

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Early diagnosis and pharmacological treatment of central nervous system (CNS) diseases has been a long-standing challenge for clinical research due to the presence of the blood-brain barrier. Specific proteins and RNAs in brain-derived extracellular vesicles (EVs) usually reflect the corresponding state of brain disease, and therefore, EVs can be used as diagnostic biomarkers for CNS diseases. In addition, EVs can be engineered and fused to target cells for delivery of cargo, demonstrating the great potential of EVs as a nanocarrier platform. We review the progress of EVs as markers and drug carriers in the diagnosis and treatment of neurological diseases. The main areas include visual imaging, biomarker diagnosis and drug loading therapy for different types of CNS diseases. It is hoped that increased knowledge of EVs will facilitate their clinical translation in CNS diseases.

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Nerves play important roles in organ development and tissue homeostasis. Stem/progenitor cells differentiate into different cell lineages responsible for building the craniofacial organs. The mechanism by which nerves regulate stem/progenitor cell behavior in organ morphogenesis has not yet been comprehensively explored. Here, we use tooth root development in mouse as a model to investigate how sensory nerves regulate organogenesis. We show that sensory nerve fibers are enriched in the dental papilla at the initiation of tooth root development. Through single cell RNA-sequencing analysis of the trigeminal ganglion and developing molar, we reveal several signaling pathways that connect the sensory nerve with the developing molar, of which FGF signaling appears to be one of the important regulators. Fgfr2 is expressed in the progenitor cells during tooth root development. Loss of FGF signaling leads to shortened roots with compromised proliferation and differentiation of progenitor cells. Furthermore, Hh signaling is impaired in Gli1-CreER;Fgfr2fl/fl mice. Modulation of Hh signaling rescues the tooth root defects in these mice. Collectively, our findings elucidate the nerve-progenitor crosstalk and reveal the molecular mechanism of the FGF-SHH signaling cascade during tooth root morphogenesis.

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Disruption of global ribosome biogenesis selectively affects craniofacial tissues with unclear mechanisms. Craniosynostosis is a congenital craniofacial disorder characterized by premature fusion of cranial suture(s) with loss of suture mesenchymal stem cells (MSCs). Here we focused on ribosomopathy disease gene Snord118, which encodes a small nucleolar RNA (snoRNA), to genetically disturb ribosome biogenesis in suture MSCs using mouse and human induced pluripotent stem cell (iPSC) models. Snord118 depletion exhibited p53 activation, increased cell death, reduced proliferation, and premature osteogenic differentiation of MSCs, leading to suture growth and craniosynostosis defects. Mechanistically, Snord118 deficiency causes translational dysregulation of ribosomal proteins and downregulation of complement pathway genes. Further complement pathway disruption by knockout of complement C3a receptor 1 (C3ar1) exacerbated MSC and suture defects in mutant mice, whereas activating the complement pathway rescued MSC cell fate and suture growth defects. Thus, ribosome biogenesis controls MSC fate via the complement pathway to prevent craniosynostosis.

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How early life experience is translated into storable epigenetic information leading to behavioral changes remains poorly understood. Here we found that Zika virus (ZIKV) induced-maternal immune activation (MIA) imparts offspring with anxiety- and depression-like behavior. By integrating bulk and single-nucleus RNA sequencing (snRNA-seq) with genome-wide 5hmC (5-hydroxymethylcytosine) profiling and 5mC (5-methylcytosine) profiling in prefrontal cortex (PFC) of ZIKV-affected male offspring mice, we revealed an overall loss of 5hmC and an increase of 5mC levels in intragenic regions, associated with transcriptional changes in neuropsychiatric disorder-related genes. In contrast to their rapid initiation and inactivation in normal conditions, immediate-early genes (IEGs) remain a sustained upregulation with enriched expression in excitatory neurons, which is coupled with increased 5hmC and decreased 5mC levels of IEGs in ZIKV-affected male offspring. Thus, MIA induces maladaptive methylome remodeling in brain and selectively regulates neuronal activity gene methylation linking to emotional behavioral abnormalities in offspring.

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The dynamic balance between tRNA supply and codon usage demand is a fundamental principle in the cellular translation economy. However, the regulation and functional consequences of this balance remain unclear. Here, we use PARIS2 interactome capture, structure modeling, conservation analysis, RNA-protein interaction analysis, and modification mapping to reveal the targets of hundreds of snoRNAs, many of which were previously considered orphans. We identify a snoRNA-tRNA interaction network that is required for global tRNA modifications, including 2'-O-methylation and others. Loss of Fibrillarin, the snoRNA-guided 2'-O-methyltransferase, induces global upregulation of tRNA fragments, a large group of regulatory RNAs. In particular, the snoRNAs D97/D133 guide the 2'-O-methylation of multiple tRNAs, especially for the amino acid methionine (Met), a protein-intrinsic antioxidant. Loss of D97/D133 snoRNAs in human HEK293 cells reduced target tRNA levels and induced codon adaptation of the transcriptome and translatome. Both single and double knockouts of D97 and D133 in HEK293 cells suppress Met-enriched proliferation-related gene expression programs, including, translation, splicing, and mitochondrial energy metabolism, and promote Met-depleted programs related to development, differentiation, and morphogenesis. In a mouse embryonic stem cell model of development, knockdown and knockout of D97/D133 promote differentiation to mesoderm and endoderm fates, such as cardiomyocytes, without compromising pluripotency, consistent with the enhanced development-related gene expression programs in human cells. This work solves a decades-old mystery about orphan snoRNAs and reveals a function of snoRNAs in controlling the codon-biased dichotomous cellular states of proliferation and development.

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The meninges lie in the interface between the skull and brain, harboring lymphatic vasculature and skull progenitor cells (SPCs). How the skull and brain communicate remains largely unknown. We found that impaired meningeal lymphatics and brain perfusion drive neurocognitive defects in Twist1+/- mice, an animal model of craniosynostosis recapitulating human Saethre-Chotzen syndrome. Loss of SPCs leads to skull deformities and elevated intracranial pressure (ICP), whereas transplanting SPCs back into mutant mice mitigates lymphatic and brain defects through two mechanisms: (1) decreasing elevated ICP by skull correction and (2) promoting the growth and migration of lymphatic endothelial cells (LECs) via SPC-secreted vascular endothelial growth factor-C (VEGF-C). Treating Twist1+/- mice with VEGF-C promotes meningeal lymphatic growth and rescues defects in ICP, brain perfusion, and neurocognitive functions. Thus, the skull functionally integrates with the brain via meningeal lymphatics, which is impaired in craniosynostosis and can be restored by SPC-driven lymphatic activation via VEGF-C.

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Mesenchymal stem cells (MSCs) reside in microenvironments, referred to as niches, which provide structural support and molecular signals. Sensory nerves are niche components in the homeostasis of tissues such as skin, bone marrow and hematopoietic system. However, how the sensory nerve affects the behavior of MSCs remains largely unknown. Here we show that the sensory nerve is vital for mesenchymal tissue homeostasis and maintenance of MSCs in the continuously growing adult mouse incisor. Loss of sensory innervation leads to mesenchymal disorder and a decrease in MSCs. Mechanistically, FGF1 from the sensory nerve directly acts on MSCs by binding to FGFR1 and activates the mTOR/autophagy axis to sustain MSCs. Modulation of mTOR/autophagy restores the MSCs and rescues the mesenchymal tissue disorder of Fgfr1 mutant mice. Collectively, our study provides insights into the role of sensory nerves in the regulation of MSC homeostasis and the mechanism governing it.

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Single entity measurements based on the stochastic collision electrochemistry provide a promising and versatile means to study single molecules, single particles, single droplets, etc. Conceptually, mass transport and electron transfer are the two main processes at the electrochemically confined interface that underpin the most transient electrochemical responses resulting from the stochastic and discrete behaviors of single entities at the microscopic scale. This perspective demonstrates how to achieve controllable stochastic collision electrochemistry by effectively altering the two processes. Future challenges and opportunities for stochastic collision electrochemistry are also highlighted.

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G4C2 repeat expansion in C9orf72 causes the most common familial frontotemporal dementia and amyotrophic lateral sclerosis (C9FTD/ALS). The pathogenesis includes haploinsufficiency of C9orf72, which forms a protein complex with Smcr8, as well as G4C2 repeat-induced gain of function including toxic dipeptide repeats (DPRs). The key in vivo disease-driving mechanisms and how loss- and gain-of-function interplay remain poorly understood. Here, we identified dysregulation of a lysosome-ribosome biogenesis circuit as an early and key disease mechanism using a physiologically relevant mouse model with combined loss- and gain-of-function across the aging process. C9orf72 deficiency exacerbates FTD/ALS-like pathologies and behaviors in C9ORF72 bacterial artificial chromosome (C9-BAC) mice with G4C2 repeats under endogenous regulatory elements from patients. Single nucleus RNA sequencing (snRNA-seq) and bulk RNA-seq revealed that C9orf72 depletion disrupts lysosomes in neurons and leads to transcriptional dysregulation of ribosomal protein genes, which are likely due to the proteotoxic stress response and resemble ribosomopathy defects. Importantly, ectopic expression of C9orf72 or its partner Smcr8 in C9FTD/ALS mutant mice promotes lysosomal functions and restores ribosome biogenesis gene transcription, resulting in the mitigation of DPR accumulation, neurodegeneration as well as FTD/ALS-like motor and cognitive behaviors. Therefore, we conclude that loss- and gain-of-function crosstalk in C9FTD/ALS converges on neuronal dysregulation of a lysosome-ribosome biogenesis circuit leading to proteotoxicity, neurodegeneration and behavioral defects.

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Community-acquired pneumonia (CAP) is a serious clinical concern. A lack of accurate diagnosis could hinder pathogen-directed therapeutic strategies. To solve this problem, we evaluated clinical application of nested multiplex polymerase chain reaction (PCR) in children with severe CAP. We prospectively enrolled 60 children with severe CAP requiring intensive care between December 2019 and November 2021 at a tertiary medical center. Nested multiplex PCR respiratory panel (RP) and pneumonia panel (PP) were performed on upper and lower respiratory tract specimens. We integrated standard-of-care tests and quantitative PCR for validation. The combination of RP, PP, and standard-of-care tests could detect at least one pathogen in 98% of cases and the mixed viral-bacterial detection rate was 65%. The positive percent agreement (PPA), and negative percent agreement (NPA) for RP were 94% and 99%; the PPA and NPA for PP were 89% and 98%. The distribution of pathogens was similar in the upper and lower respiratory tracts, and the DNA or RNA copies of pathogens in the lower respiratory tract were equal to or higher than those in the upper respiratory tract. PP detected bacterial pathogens in 40 (67%) cases, and clinicians tended to increase bacterial diagnosis and escalate antimicrobial therapy for them. RP and PP had satisfactory performance to help pediatricians make pathogenic diagnoses and establish therapy earlier. The pathogens in the upper respiratory tract had predictive diagnostic values for lower respiratory tract infections in children with severe CAP.

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Craniosynostosis is a major congenital craniofacial disorder characterized by the premature fusion of cranial suture(s). Patients with severe craniosynostosis often have impairments in hearing, vision, intracranial pressure and/or neurocognitive functions. Craniosynostosis can result from mutations, chromosomal abnormalities or adverse environmental effects, and can occur in isolation or in association with numerous syndromes. To date, surgical correction remains the primary treatment for craniosynostosis, but it is associated with complications and with the potential for re-synostosis. There is, therefore, a strong unmet need for new therapies. Here, we provide a comprehensive review of our current understanding of craniosynostosis, including typical craniosynostosis types, their clinical manifestations, cranial suture development, and genetic and environmental causes. Based on studies from animal models, we present a framework for understanding the pathogenesis of craniosynostosis, with an emphasis on the loss of postnatal suture mesenchymal stem cells as an emerging disease-driving mechanism. We evaluate emerging treatment options and highlight the potential of mesenchymal stem cell-based suture regeneration as a therapeutic approach for craniosynostosis.

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The recent development and application of methods based on the general principle of "crosslinking and proximity ligation" (crosslink-ligation) are revolutionizing RNA structure studies in living cells. However, extracting structure information from such data presents unique challenges. Here, we introduce a set of computational tools for the systematic analysis of data from a wide variety of crosslink-ligation methods, specifically focusing on read mapping, alignment classification, and clustering. We design a new strategy to map short reads with irregular gaps at high sensitivity and specificity. Analysis of previously published data reveals distinct properties and bias caused by the crosslinking reactions. We perform rigorous and exhaustive classification of alignments and discover eight types of arrangements that provide distinct information on RNA structures and interactions. To deconvolve the dense and intertwined gapped alignments, we develop a network/graph-based tool Crosslinked RNA Secondary Structure Analysis using Network Techniques (CRSSANT), which enables clustering of gapped alignments and discovery of new alternative and dynamic conformations. We discover that multiple crosslinking and ligation events can occur on the same RNA, generating multisegment alignments to report complex high-level RNA structures and multi-RNA interactions. We find that alignments with overlapped segments are produced from potential homodimers and develop a new method for their de novo identification. Analysis of overlapping alignments revealed potential new homodimers in cellular noncoding RNAs and RNA virus genomes in the Picornaviridae family. Together, this suite of computational tools enables rapid and efficient analysis of RNA structure and interaction data in living cells.

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The potential distribution at the electrode interface is a core factor in electrochemistry, and it is usually treated by the classic Gouy-Chapman-Stern (G-C-S) model. Yet the G-C-S model is not applicable to nanosized particles collision electrochemistry as it describes steady-state electrode potential distribution. Additionally, the effect of single nanoparticles (NPs) on potential should not be neglected because the size of a NP is comparable to that of an electrode. Herein, a theoretical model termed as Metal-Solution-Metal Nanoparticle (M-S-MNP) is proposed to reveal the dynamic electrode potential distribution at the single-nanoparticle level. An explicit equation is provided to describe the size/distance-dependent potential distribution in single NPs stochastic collision electrochemistry, showing the potential distribution is influenced by the NPs. Agreement between experiments and simulations indicates the potential roles of the M-S-MNP model in understanding the charge transfer process at the nanoscale.

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Zika virus (ZIKV) infection during pregnancy is linked to various developmental brain disorders. Infants who are asymptomatic at birth might have postnatal neurocognitive complications. However, animal models recapitulating these neurocognitive phenotypes are lacking, and the circuit mechanism underlying behavioral abnormalities is unknown. Here, we show that ZIKV infection during mouse pregnancy induces maternal immune activation (MIA) and leads to autistic-like behaviors including repetitive self-grooming and impaired social memory in offspring. In the medial prefrontal cortex (mPFC), ZIKV-affected offspring mice exhibit excitation and inhibition imbalance and increased cortical activity. This could be explained by dysregulation of inhibitory neurons and synapses, and elevated neural activity input from mPFC-projecting ventral hippocampus (vHIP) neurons. We find structure alterations in the synaptic connections and pattern of vHIP innervation of mPFC neurons, leading to hyperconnectivity of the vHIP-mPFC pathway. Decreasing the activity of mPFC-projecting vHIP neurons with a chemogenetic strategy rescues social memory deficits in ZIKV offspring mice. Our studies reveal a hyperconnectivity of vHIP to mPFC projection driving social memory deficits in mice exposed to maternal inflammation by ZIKV.

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