Interneuron FGF13 regulates seizure susceptibility via a sodium channel-independent mechanism.

Developmental and Epileptic Encephalopathies (DEEs), a class of devastating neurological disorders characterized by recurrent seizures and exacerbated by disruptions to excitatory/inhibitory balance in the brain, are commonly caused by mutations in ion channels. Disruption of, or variants in, FGF13 were implicated as causal for a set of DEEs, but the underlying mechanisms were clouded because FGF13 is expressed in both excitatory and inhibitory neurons, FGF13 undergoes extensive alternative splicing producing multiple isoforms with distinct functions, and the overall roles of FGF13 in neurons are incompletely cataloged. To overcome these challenges, we generated a set of novel cell type-specific conditional knockout mice. Interneuron-targeted deletion of Fgf13 led to perinatal mortality associated with extensive seizures and impaired the hippocampal inhibitory/excitatory balance while excitatory neuron-targeted deletion of Fgf13 caused no detectable seizures and no survival deficits. While best studied as a voltage-gated sodium channel (Nav) regulator, we observed no effect of Fgf13 ablation in interneurons on Navs but rather a marked reduction in K+ channel currents. Re-expressing different Fgf13 splice isoforms could partially rescue deficits in interneuron excitability and restore K+ channel current amplitude. These results enhance our understanding of the molecular mechanisms that drive the pathogenesis of Fgf13-related seizures and expand our understanding of FGF13 functions in different neuron subsets.

An injury-induced tissue niche shaped by mesenchymal plasticity coordinates the regenerative and disease response in the lung.

Severe lung injury causes basal stem cells to migrate and outcompete alveolar stem cells resulting in dysplastic repair and a loss of gas exchange function. This "stem cell collision" is part of a multistep process that is now revealed to generate an injury-induced tissue niche (iTCH) containing Keratin 5+ epithelial cells and plastic Pdgfra+ mesenchymal cells. Temporal and spatial single cell analysis reveals that iTCHs are governed by mesenchymal proliferation and Notch signaling, which suppresses Wnt and Fgf signaling in iTCHs. Conversely, loss of Notch in iTCHs rewires alveolar signaling patterns to promote euplastic regeneration and gas exchange. The signaling patterns of iTCHs can differentially phenotype fibrotic from degenerative human lung diseases, through apposing flows of FGF and WNT signaling. These data reveal the emergence of an injury and disease associated iTCH in the lung and the ability of using iTCH specific signaling patterns to discriminate human lung disease phenotypes.

Dysregulated alveolar epithelial cell progenitor function and identity in Hermansky-Pudlak syndrome pulmonary fibrosis.

Hermansky-Pudlak syndrome (HPS) is a genetic disorder associated with pulmonary fibrosis in specific subtypes, including HPS-1 and HPS-2. Single mutant HPS1 and HPS2 mice display increased fibrotic sensitivity while double mutant HPS1/2 mice exhibit spontaneous fibrosis with aging, which has been attributed to HPS mutations in alveolar epithelial type II (AT2) cells. Unifying mechanisms of AT2 cell dysfunction in genetic and sporadic fibrotic lung diseases remain unknown. Incorporating AT2 cell lineage tracing in HPS mice, we observed a progressive decline in AT2 cell numbers with aging and aberrant differentiation with increased AT2-derived alveolar epithelial type I cells. HPS AT2 cell proliferation was impaired ex vivo and in vivo , suggesting an intrinsic progenitor defect. Transcriptomic analysis of HPS AT2 cells revealed elevated expression of genes associated with aberrant differentiation and cellular senescence. Through lineage tracing and organoid modeling, we demonstrated that HPS AT2 cells were primed to persist in a Krt8 + reprogrammed transitional state, mediated by p53 activity. These findings suggest that pulmonary fibrosis in HPS may be driven by AT2 cell progenitor dysfunction in the setting of p53-mediated senescence, highlighting a novel potential therapeutic target in HPS and suggesting unifying mechanisms underlying HPS and other forms of pulmonary fibrosis.

TGF-ßR2 signaling coordinates pulmonary vascular repair after viral injury in mice and human tissue.

Disruption of pulmonary vascular homeostasis is a central feature of viral pneumonia, wherein endothelial cell (EC) death and subsequent angiogenic responses are critical determinants of the outcome of severe lung injury. A more granular understanding of the fundamental mechanisms driving reconstitution of lung endothelium is necessary to facilitate therapeutic vascular repair. Here, we demonstrated that TGF-ß signaling through TGF-ßR2 (transforming growth factor-ß receptor 2) is activated in pulmonary ECs upon influenza infection, and mice deficient in endothelial Tgfbr2 exhibited prolonged injury and diminished vascular repair. Loss of endothelial Tgfbr2 prevented autocrine Vegfa (vascular endothelial growth factor α) expression, reduced endothelial proliferation, and impaired renewal of aerocytes thought to be critical for alveolar gas exchange. Angiogenic responses through TGF-ßR2 were attributable to leucine-rich α-2-glycoprotein 1, a proangiogenic factor that counterbalances canonical angiostatic TGF-ß signaling. Further, we developed a lipid nanoparticle that targets the pulmonary endothelium, Lung-LNP (LuLNP). Delivery of Vegfa mRNA, a critical TGF-ßR2 downstream effector, by LuLNPs improved the impaired regeneration phenotype of EC Tgfbr2 deficiency during influenza injury. These studies defined a role for TGF-ßR2 in lung endothelial repair and demonstrated efficacy of an efficient and safe endothelial-targeted LNP capable of delivering therapeutic mRNA cargo for vascular repair in influenza infection.

Persistence of anxiety among Asian Americans: racial and ethnic heterogeneity in the longitudinal trends in mental well-being during the COVID-19 pandemic.

PURPOSE: To examine within-individual time trends in mental well-being and factors influencing heterogeneity of these trends. METHODS: Longitudinal telephone survey of adults over 3 waves from the New York City (NYC) Metropolitan area during the COVID-19 Pandemic. Participants reported depression using the Patient Health Questionnaire (PHQ)-8, anxiety using the Generalized Anxiety Disorder (GAD)-7, and past 30-day increases in tobacco or alcohol use at each wave. Adjusted mixed effects logistic regression models assessed time trends in mental well-being. RESULTS: There were 1227 respondents. Over 3 study waves, there were statistically significant decreasing time trends in the odds of each outcome (adjusted OR (95% CI) 0.47 (0.37, 0.60); p < 0.001 for depression; aOR (95% CI) 0.55 (0.45, 0.66); p < 0.001 for anxiety; aOR (95% CI) 0.50 (0.35, 0.71); p < 0.001 for past 30-day increased tobacco use; aOR (95% CI) 0.31 (0.24, 0.40); p < 0.001 for past 30-day increased alcohol use). Time trends for anxiety varied by race and ethnicity (p value for interaction = 0.05, 4 df); anxiety declined over time among white, Black, Hispanic, and Other race and ethnicity but not among Asian participants. CONCLUSIONS: In a demographically varied population from the NYC Metropolitan area, depression, anxiety and increased substance use were common during the first months of the pandemic, but decreased over the following year. While this was consistently the case across most demographic groups, the odds of anxiety among Asian participants did not decrease over time.

Correlations of receptor desensitization of gain-of-function GABRB3 variants with clinical severity.

Genetic variants associated with developmental and epileptic encephalopathies have been identified in the GABRB3 gene that encodes the ß3 subunit of GABAA receptors. Typically, variants alter receptor sensitivity to GABA resulting in either gain- or loss-of-function, which correlates with patient phenotypes. However, it is unclear how another important receptor property, desensitization, contributes to the greater clinical severity of gain-of-function variants. Desensitization properties of 20 gain-of-function GABRB3 variant receptors were evaluated using two-electrode voltage-clamp electrophysiology. The parameters measured included current decay rates and steady-state currents. Selected variants with increased or reduced desensitization were also evaluated using whole-cell electrophysiology in transfected mammalian cell lines. Of the 20 gain-of-function variants assessed, 13 were found to alter receptor desensitization properties. Seven variants reduced desensitization at equilibrium, which acts to worsen gain-of-function traits. Six variants accelerated current decay kinetics, which limits gain-of-function traits. All affected patients displayed severe clinical phenotypes with intellectual disability and difficult-to-treat epilepsy. Nevertheless, variants that reduced desensitization at equilibrium were associated with more severe clinical outcomes. This included younger age of first seizure onset (median 0.5 months), movement disorders (dystonia and dyskinesia), epilepsy of infancy with migrating focal seizures (EIMFS) and risk of early mortality. Variants that accelerated current decay kinetics were associated with slightly milder phenotypes with later seizure onset (median 4 months), unclassifiable developmental and epileptic encephalopathies or Lennox-Gastaut syndrome and no movement disorders. Our study reveals that gain-of-function GABRB3 variants can increase or decrease receptor desensitization properties and that there is a correlation with the degree of disease severity. Variants that reduced the desensitization at equilibrium were clustered in the transmembrane regions that constitute the channel pore and correlated with greater disease severity, while variants that accelerated current decay were clustered in the coupling loops responsible for receptor activation and correlated with lesser severity.

Gain-of-function MARK4 variant associates with pediatric neurodevelopmental disorder and dysmorphism.

Microtubule affinity-regulating kinase 4 (MARK4) is a serine/threonine kinase that plays a key role in tau phosphorylation and regulation of the mammalian target of rapamycin (mTOR) pathway. Abnormal tau phosphorylation and dysregulation of the mTOR pathway are implicated in neurodegenerative and neurodevelopmental disorders. Here, we report a gain-of-function variant in MARK4 in two siblings with childhood-onset neurodevelopmental disability and dysmorphic features. The siblings carry a germline heterozygous missense MARK4 variant c.604T>C (p.Phe202Leu), located in the catalytic domain of the kinase, which they inherited from their unaffected, somatic mosaic mother. Functional studies show that this amino acid substitution has no impact on protein expression but instead increases the ability of MARK4 to phosphorylate tau isoforms found in the fetal and adult brain. The MARK4 variant also increases phosphorylation of ribosomal protein S6, indicating upregulation of the mTORC1 pathway. In this study, we link a germline monoallelic MARK4 variant to a childhood-onset neurodevelopmental disorder characterized by global developmental delay, intellectual disability, behavioral abnormalities, and dysmorphic features.

Hyperactive mTORC1 in lung mesenchyme induces endothelial cell dysfunction and pulmonary vascular remodeling.

Lymphangioleiomyomatosis (LAM) is a progressive cystic lung disease caused by tuberous sclerosis complex 1/2 (TSC1/2) gene mutations in pulmonary mesenchymal cells, resulting in activation of the mechanistic target of rapamycin complex 1 (mTORC1). A subset of patients with LAM develop pulmonary vascular remodeling and pulmonary hypertension. Little, however, is known regarding how LAM cells communicate with endothelial cells (ECs) to trigger vascular remodeling. In end-stage LAM lung explants, we identified EC dysfunction characterized by increased EC proliferation and migration, defective angiogenesis, and dysmorphic endothelial tube network formation. To model LAM disease, we used an mTORC1 gain-of-function mouse model with a Tsc2 KO (Tsc2KO) specific to lung mesenchyme (Tbx4LME-Cre Tsc2fl/fl), similar to the mesenchyme-specific genetic alterations seen in human disease. As early as 8 weeks of age, ECs from mice exhibited marked transcriptomic changes despite an absence of morphological changes to the distal lung microvasculature. In contrast, 1-year-old Tbx4LME-Cre Tsc2fl/fl mice spontaneously developed pulmonary vascular remodeling with increased medial thickness. Single-cell RNA-Seq of 1-year-old mouse lung cells identified paracrine ligands originating from Tsc2KO mesenchyme, which can signal through receptors in arterial ECs. These ECs had transcriptionally altered genes including those in pathways associated with blood vessel remodeling. The proposed pathophysiologic mesenchymal ligand-EC receptor crosstalk highlights the importance of an altered mesenchymal cell/EC axis in LAM and other hyperactive mTORC1-driven diseases. Since ECs in patients with LAM and in Tbx4LME-Cre Tsc2fl/fl mice did not harbor TSC2 mutations, our study demonstrates that constitutively active mTORC1 lung mesenchymal cells orchestrated dysfunctional EC responses that contributed to pulmonary vascular remodeling.

Screening of Oligomeric (Meth)acrylate Vaccine Adjuvants Synthesized via Catalytic Chain Transfer Polymerization.

This report details the first systematic screening of free-radical-produced methacrylate oligomer reaction mixtures as alternative vaccine adjuvant components to replace the current benchmark compound squalene, which is unsustainably sourced from shark livers. Homo-/co-oligomer mixtures of methyl, butyl, lauryl, and stearyl methacrylate were successfully synthesized using catalytic chain transfer control, where the use of microwave heating was shown to promote propagation over chain transfer. Controlling the mixture material properties allowed the correct viscosity to be achieved, enabling the mixtures to be effectively used in vaccine formulations. Emulsions of selected oligomers stimulated comparable cytokine levels to squalene emulsion when incubated with human whole blood and elicited an antigen-specific cellular immune response when administered with an inactivated influenza vaccine, indicating the potential utility of the compounds as vaccine adjuvant components. Furthermore, the oligomers' molecular sizes were demonstrated to be large enough to enable greater emulsion stability than squalene, especially at high temperatures, but are predicted to be small enough to allow for rapid clearance from the body.

GABAA receptors in epilepsy: Elucidating phenotypic divergence through functional analysis of genetic variants.

Normal brain function requires a tightly regulated balance between excitatory and inhibitory neurotransmissions. γ-Aminobutyric acid type A (GABAA ) receptors represent the major class of inhibitory ion channels in the mammalian brain. Dysregulation of these receptors and/or their associated pathways is strongly implicated in the pathophysiology of epilepsy. To date, hundreds of different GABAA receptor subunit variants have been associated with epilepsy, making them a prominent cause of genetically linked epilepsy. While identifying these genetic variants is crucial for accurate diagnosis and effective genetic counselling, it does not necessarily lead to improved personalised treatment options. This is because the identification of a variant does not reveal how the function of GABAA receptors is affected. Genetic variants in GABAA receptor subunits can cause complex changes to receptor properties resulting in various degrees of gain-of-function, loss-of-function or a combination of both. Understanding how variants affect the function of GABAA receptors therefore represents an important first step in the ongoing development of precision therapies. Furthermore, it is important to ensure that functional data are produced using methodologies that allow genetic variants to be classified using clinical guidelines such as those developed by the American College of Medical Genetics and Genomics. This article will review the current knowledge in the field and provide recommendations for future functional analysis of genetic GABAA receptor variants.

Guided construction of single cell reference for human and mouse lung.

Accurate cell type identification is a key and rate-limiting step in single-cell data analysis. Single-cell references with comprehensive cell types, reproducible and functionally validated cell identities, and common nomenclatures are much needed by the research community for automated cell type annotation, data integration, and data sharing. Here, we develop a computational pipeline utilizing the LungMAP CellCards as a dictionary to consolidate single-cell transcriptomic datasets of 104 human lungs and 17 mouse lung samples to construct LungMAP single-cell reference (CellRef) for both normal human and mouse lungs. CellRefs define 48 human and 40 mouse lung cell types catalogued from diverse anatomic locations and developmental time points. We demonstrate the accuracy and stability of LungMAP CellRefs and their utility for automated cell type annotation of both normal and diseased lungs using multiple independent methods and testing data. We develop user-friendly web interfaces for easy access and maximal utilization of the LungMAP CellRefs.

Dominant negative variants in IKZF2 cause ICHAD syndrome, a new disorder characterised by immunodysregulation, craniofacial anomalies, hearing impairment, athelia and developmental delay.

BACKGROUND: Helios (encoded by IKZF2), a member of the Ikaros family of transcription factors, is a zinc finger protein involved in embryogenesis and immune function. Although predominantly recognised for its role in the development and function of T lymphocytes, particularly the CD4+ regulatory T cells (Tregs), the expression and function of Helios extends beyond the immune system. During embryogenesis, Helios is expressed in a wide range of tissues, making genetic variants that disrupt the function of Helios strong candidates for causing widespread immune-related and developmental abnormalities in humans. METHODS: We performed detailed phenotypic, genomic and functional investigations on two unrelated individuals with a phenotype of immune dysregulation combined with syndromic features including craniofacial differences, sensorineural hearing loss and congenital abnormalities. RESULTS: Genome sequencing revealed de novo heterozygous variants that alter the critical DNA-binding zinc fingers (ZFs) of Helios. Proband 1 had a tandem duplication of ZFs 2 and 3 in the DNA-binding domain of Helios (p.Gly136_Ser191dup) and Proband 2 had a missense variant impacting one of the key residues for specific base recognition and DNA interaction in ZF2 of Helios (p.Gly153Arg). Functional studies confirmed that both these variant proteins are expressed and that they interfere with the ability of the wild-type Helios protein to perform its canonical function-repressing IL2 transcription activity-in a dominant negative manner. CONCLUSION: This study is the first to describe dominant negative IKZF2 variants. These variants cause a novel genetic syndrome characterised by immunodysregulation, craniofacial anomalies, hearing impairment, athelia and developmental delay.

Religiosity, Mental Health and Substance Use among Black and Hispanic Adults during the First Six Months of the COVID-19 Pandemic in New York City.

The purpose of this study was to investigate the association between personal religiosity, mental health, and substance use outcomes among Black and Hispanic adults during the first six months of the COVID-19 outbreak in New York City (NYC). Phone interviews were conducted with 441 adults to obtain information on all variables. Participants self-reported race/ethnicity as Black/African American (n = 108) or Hispanic (n = 333). Logistic regression were used to examine associations between religiosity, mental health, and substance use. There was a significant inverse association of religiosity and substance use. Religious people had a lower prevalence of drinking alcohol (49.0%) compared to non-religious people (67.1%). Religious people also had substantially lower prevalence of cannabis or other drug use (9.1%) in comparison to non-religious people (31%). After adjusting for age, sex, race/ethnicity, and household income, the association of religiosity with alcohol use and with cannabis/other drug use remained statistically significant. Despite restricted access to in-person religious activities and congregational supports, the findings suggest that religiosity itself may be helpful from a public health perspective, independent of serving as a conduit for other social services.

Generation of tandem alternative splice acceptor sites and CLTC haploinsufficiency: A cause of CLTC-related disorder.

Tandem splice acceptors (NAGNn AG) are a common mechanism of alternative splicing, but variants that are likely to generate or to disrupt tandem splice sites have rarely been reported as disease causing. We identify a pathogenic intron 23 CLTC variant (NM_004859.4:c.[3766-13_3766-5del];[=]) in a propositus with intellectual disability and behavioral problems. By RNAseq analysis of peripheral blood mRNA, this variant generates transcripts using cryptic proximal splice acceptors (NM_004859.4: r.3765_3766insTTCACAGAAAGGAACTAG, and NM_004859.4:r.3765_3766insAAAGGAACTAG). Given that the propositus expresses 38% the level of CLTC transcripts as unaffected controls, these variant transcripts, which encode premature termination codons, likely undergo nonsense mediated mRNA decay (NMD). This is the first functional evidence for CLTC haploinsufficiency as a cause of CLTC-related disorder and the first evidence that the generation of tandem alternative splice sites causes CLTC-related disorder. We suggest that variants creating tandem alternative splice sites are an underreported disease mechanism and that transcriptome-level analysis should be routinely pursued to define the pathogenicity of such variants.

Comparative reductions of norovirus, echovirus, adenovirus, Campylobacter jejuni and process indicator organisms during water filtration in alluvial sand.

Sand filtration is a cost-effective means of reducing microbial pathogens in drinking-water treatment. Our understanding of pathogen removal by sand filtration relies largely on studies of process microbial indicators, and comparative data from pathogens are sparse. In this study, we examined the reductions of norovirus, echovirus, adenovirus, bacteriophage MS2 and PRD1, Campylobacter jejuni, and Escherichia coli during water filtration through alluvial sand. Duplicate experiments were conducted using 2 sand columns (50 cm long, 10 cm diameter) and municipal tap water sourced from chlorine-free untreated groundwater (pH 8.0, 1.47 mM) at filtration rates of 1.1-1.3 m/day. The results were analysed using colloid filtration theory and the HYDRUS-1D 2-site attachment-detachment model. The average log10 reduction values (LRVs) of the normalised dimensionless peak concentrations (Cmax/C0) over 0.5 m were: MS2: 0.28; E. coli: 0.76; C. jejuni: 0.78; PRD1: 2.00; echovirus: 2.20; norovirus: 2.35; and adenovirus: 2.79. The relative reductions largely corresponded to the organisms' isoelectric points rather than their particle sizes or hydrophobicities. MS2 underestimated virus reductions by 1.7-2.5 log, and the LRVs, mass recoveries relative to bromide, collision efficiencies, and attachment and detachment rates differed mostly by ∼1 order of magnitude. Conversely, PRD1 reductions were comparable with those of all 3 viruses tested, and its parameter values were mostly within the same orders of magnitude. E. coli seemed an adequate process indicator for C. jejuni with similar reductions. Comparative data describing pathogen and indicator reductions in alluvial sand have important implications for sand filter design, risk assessments of drinking-water supplies from riverbank filtration and the determination of safe setback distances for drinking-water supply wells.

Biophysical forces mediated by respiration maintain lung alveolar epithelial cell fate.

Lungs undergo mechanical strain during breathing, but how these biophysical forces affect cell fate and tissue homeostasis are unclear. We show that biophysical forces through normal respiratory motion actively maintain alveolar type 1 (AT1) cell identity and restrict these cells from reprogramming into AT2 cells in the adult lung. AT1 cell fate is maintained at homeostasis by Cdc42- and Ptk2-mediated actin remodeling and cytoskeletal strain, and inactivation of these pathways causes a rapid reprogramming into the AT2 cell fate. This plasticity induces chromatin reorganization and changes in nuclear lamina-chromatin interactions, which can discriminate AT1 and AT2 cell identity. Unloading the biophysical forces of breathing movements leads to AT1-AT2 cell reprogramming, revealing that normal respiration is essential to maintain alveolar epithelial cell fate. These data demonstrate the integral function of mechanotransduction in maintaining lung cell fate and identifies the AT1 cell as an important mechanosensor in the alveolar niche.

Human germline heterozygous gain-of-function STAT6 variants cause severe allergic disease.

STAT6 (signal transducer and activator of transcription 6) is a transcription factor that plays a central role in the pathophysiology of allergic inflammation. We have identified 16 patients from 10 families spanning three continents with a profound phenotype of early-life onset allergic immune dysregulation, widespread treatment-resistant atopic dermatitis, hypereosinophilia with esosinophilic gastrointestinal disease, asthma, elevated serum IgE, IgE-mediated food allergies, and anaphylaxis. The cases were either sporadic (seven kindreds) or followed an autosomal dominant inheritance pattern (three kindreds). All patients carried monoallelic rare variants in STAT6 and functional studies established their gain-of-function (GOF) phenotype with sustained STAT6 phosphorylation, increased STAT6 target gene expression, and TH2 skewing. Precision treatment with the anti-IL-4Rα antibody, dupilumab, was highly effective improving both clinical manifestations and immunological biomarkers. This study identifies heterozygous GOF variants in STAT6 as a novel autosomal dominant allergic disorder. We anticipate that our discovery of multiple kindreds with germline STAT6 GOF variants will facilitate the recognition of more affected individuals and the full definition of this new primary atopic disorder.