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Elevated mitochondrial membrane potential is a therapeutic vulnerability in Dnmt3a-mutant clonal hematopoiesis.
Full text linkThe competitive advantage of mutant hematopoietic stem and progenitor cells (HSPCs) underlies clonal hematopoiesis (CH). Drivers of CH include aging and inflammation; however, how CH-mutant cells gain a selective advantage in these contexts is an unresolved question. Using a murine model of CH (Dnmt3aR878H/+), we discover that mutant HSPCs sustain elevated mitochondrial respiration which is associated with their resistance to aging-related changes in the bone marrow microenvironment. Mutant HSPCs have DNA hypomethylation and increased expression of oxidative phosphorylation gene signatures, increased functional oxidative phosphorylation capacity, high mitochondrial membrane potential (Δψm), and greater dependence on mitochondrial respiration compared to wild-type HSPCs. Exploiting the elevated Δψm of mutant HSPCs, long-chain alkyl-TPP molecules (MitoQ, d-TPP) selectively accumulate in the mitochondria and cause reduced mitochondrial respiration, mitochondrial-driven apoptosis and ablate the competitive advantage of HSPCs ex vivo and in vivo in aged recipient mice. Further, MitoQ targets elevated mitochondrial respiration and the selective advantage of human DNMT3A-knockdown HSPCs, supporting species conservation. These data suggest that mitochondrial activity is a targetable mechanism by which CH-mutant HSPCs gain a selective advantage over wild-type HSPCs
Phenotypic Expansion of Knobloch Syndrome Type 2 in an Individual With a De Novo PAK2 Variant.
Full text linkP21-activated kinase 2 (PAK2) is a serine/threonine kinase essential for a variety of cellular processes including signal transduction, cellular survival, proliferation, and migration. A recent report proposed monoallelic PAK2 variants cause Knobloch syndrome type 2 (KNO2)-a developmental disorder primarily characterized by ocular anomalies. Here, we identified a novel de novo heterozygous missense variant in PAK2, NM_002577.4:c.1273G\u3eA, p.(D425N), by genome sequencing in an individual with features consistent with KNO2. Notable clinical phenotypes observed in this individual were global developmental delay, congenital retinal detachment, mild cerebral ventriculomegaly, hypotonia, failure to thrive, pyloric stenosis, feeding intolerance, patent ductus arteriosus, and mild facial dysmorphism. The p.(D425N) variant lies within the protein kinase domain and is predicted to be functionally damaging by in silico analysis. Previous clinical genetic testing did not report this variant due to unknown relevance of PAK2 variants at the time of testing, highlighting the importance of reanalysis. Our findings substantiate the candidacy of PAK2 variants in KNO2 and expand the KNO2 clinical phenotypic spectrum
TEAL-Seq: targeted expression analysis sequencing.
Full text linkMetagenome sequencing enables the genetic characterization of complex microbial communities. However, determining the activity of isolates within a commun ity presents several challenges, including the wide range of organismal and gene expression abundances, the presence of host RNA, and low microbial biomass at many sites. To address these limitations, we developed “targeted expression analysis sequencing” or TEAL-seq, enabling sensitive species-specific analyses of gene expression using highly multiplexed custom probe pools. For proof of concept, we targeted about 1,700 core and accessory genes of Staphylococcus aureus and S. epidermidis, two key species of the skin microbiome. Two targeting methods were applied to laboratory cultures and human nasal swab specimens. Both methods showed a high degree of specificity, with \u3e90% reads on target, even in the presence of complex microbial or human background DNA/RNA. Targeting using molecular inversion probes demonstra ted excellent correlation in inferred expression levels with bulk RNA-seq. Furthermore, we show that a linear pre-amplification step to increase the number of nucleic acids for analysis yielded consistent and predictable results when applied to complex samples and enabled profiling of expression from as little as 1 ng of total RNA. TEAL-seq is much less expensive than bulk metatranscriptomic profiling, enables detection across a greater dynamic range, and uses a strategy that is readily configurable for determining the transcriptional status of organisms in any microbial community
An intranasal subunit vaccine induces protective systemic and mucosal antibody immunity against respiratory viruses in mouse models.
Full text linkAlthough vaccines are usually given intramuscularly, the intranasal delivery route may lead to better mucosal protection and limit the spread of respiratory virus while easing administration and improving vaccine acceptance. The challenge, however, is to achieve delivery across the selective epithelial cell barrier. Here we report on a subunit vaccine platform, in which the antigen is genetically fused to albumin to facilitate FcRn-mediated transport across the mucosal barrier in the presence of adjuvant. Intranasal delivery in conventional and transgenic mouse models induces both systemic and mucosal antigen-specific antibody responses that protect against challenge with SARS-CoV-2 or influenza A. When benchmarked against an intramuscularly administered mRNA vaccine or an intranasally administered antigen fused to an alternative carrier of similar size, only the albumin-based intranasal vaccine yields robust mucosal IgA antibody responses. Our results thus suggest that this needle-free, albumin-based vaccine platform may be suited for vaccination against respiratory pathogens
The impact of co-housing on murine aging studies.
Full text linkAnalysis of preclinical lifespan studies often assume that outcome data from co-housed animals are independent. In practice, treatments, such as controlled feeding or putative life-extending compounds, are applied to whole housing units, and as a result, the outcomes are potentially correlated within housing units. We consider intra-class (here, intra-cage) correlation in three published and two unpublished lifespan studies of aged mice encompassing more than 20,000 observations. We show that the independence assumption underlying common analytic techniques does not hold in these data, particularly for traits associated with frailty. We describe and demonstrate various analytical tools available to accommodate this study design and highlight a limitation of standard variance components models (i.e., linear mixed models) which are the usual statistical tools for handling correlated errors. Through simulations, we examine the statistical biases resulting from intra-cage correlations with similar magnitudes as observed in these case studies and discuss implications for power and reproducibility
Analysis of lifespan across diversity outbred mouse studies identifies multiple longevity-associated loci.
Full text linkLifespan is an integrative phenotype whose genetic architecture is likely to highlight multiple processes with high impact on health and aging. Here, we conducted a genetic mega-analysis of longevity in Diversity Outbred (DO) mice that included 2,444 animals from 3 independently conducted lifespan studies. We identified 8 loci that contributed significantly to lifespan independently of diet and drug treatment in at least one study. One of these loci also influenced lifespan in a sex-dependent manner, and we detected an additional locus with a diet-specific effect on lifespan. Collectively, these loci explained over half of the estimated heritable variation in lifespan across these studies and provided insight into the genetic architecture of lifespan in DO mice
Complex genetic variation in nearly complete human genomes.
Full text linkDiverse sets of complete human genomes are required to construct a pangenome reference and to understand the extent of complex structural variation. Here we sequence 65 diverse human genomes and build 130 haplotype-resolved assemblies (median continuity of 130 Mb), closing 92% of all previous assembly gaps1,2 and reaching telomere-to-telomere status for 39% of the chromosomes. We highlight complete sequence continuity of complex loci, including the major histocompatibility complex (MHC), SMN1/SMN2, NBPF8 and AMY1/AMY2, and fully resolve 1,852 complex structural variants. In addition, we completely assemble and validate 1,246 human centromeres. We find up to 30-fold variation in α-satellite higher-order repeat array length and characterize the pattern of mobile element insertions into α-satellite higher-order repeat arrays. Although most centromeres predict a single site of kinetochore attachment, epigenetic analysis suggests the presence of two hypomethylated regions for 7% of centromeres. Combining our data with the draft pangenome reference1 significantly enhances genotyping accuracy from short-read data, enabling whole-genome inference3 to a median quality value of 45. Using this approach, 26,115 structural variants per individual are detected, substantially increasing the number of structural variants now amenable to downstream disease association studies
Cysteinyl leukotrienes stimulate gut absorption of food allergens to promote anaphylaxis in mice.
No full textINTRODUCTION Allergic reactions to food are mediated by cross-linking of preformed food-specific immunoglobulin E (IgE) antibodies bound to tissue mast cells upon allergen exposure. However, some people who have food-specific IgE do not have any allergic reaction to that food and are considered “sensitized tolerant.” How this population remains unresponsive to allergens is unclear, but understanding the underlying mechanisms could identify approaches for treating food allergy. RATIONALE To identify the genetic causes of a sensitized tolerant state, we studied an unexplained aspect of food allergy in mouse models. With rare exceptions, the commonly used C57BL/6 mouse strain demonstrates anaphylaxis to food allergens when challenged systemically, but not orally, despite robust IgE production, thus potentially modeling a sensitized tolerant state. The intestinal epithelium is composed of tight junctions that allow ions and small molecules to pass through paracellular transport. Proteins are primarily absorbed as amino acids or small polypeptides that are unable to cross-link IgE, which recognize whole epitopes. Therefore, anaphylaxis can only occur when submucosal mast cells encounter minimally digested food allergens; delivery of intact allergens has been shown to occur by transcellular transport through intestinal secretory cells. We thus hypothesized that transcellular allergen transport is genetically regulated and thereby susceptibility to anaphylaxis. RESULTS We found that oral anaphylaxis–resistant C57BL/6 mice have gut barriers that are impermeable to intact food allergens relative to susceptible strains such as C3H/HeJ even before allergic sensitization. Resistance correlated with reduced transport of allergens through secretory cells of the small intestine. Using a forward genetic screen of oral anaphylaxis, we identified a resistance gene, dipeptidase 1 (Dpep1), which encodes an enzyme expressed in the intestinal epithelium that catabolizes a cysteinyl leukotriene (CysLT) lipid inflammatory molecule. Although CysLTs are important mediators of allergic responses, a mechanistic connection between CysLTs and food allergen transport is unknown. We found that oral anaphylaxis–susceptible mice had elevated CysLTs in the gut, suggesting impaired DPEP1 enzymatic activity. Indeed, blockade of DPEP1 with cilastatin enhanced allergen absorption in anaphylaxis-resistant mice. Conversely, inhibition of leukotriene synthesis with zileuton reduced allergen absorption and prevented anaphylaxis after oral challenge in susceptible mice. CONCLUSION We discovered a mechanism by which CysLTs promote allergen absorption in the gut, thus increasing susceptibility to anaphylaxis after allergen ingestion. This function was distinct from their classical role in mediating anaphylaxis symptoms. Although multiple genetic and environmental factors likely modulate anaphylaxis, our data implicate DPEP1 as one modulator of intestinal allergen absorption in mice. Our work indicates that the intestinal barrier can protect sensitized individuals from experiencing allergic symptoms upon food ingestion, which could be achieved by reducing gut CysLTs with zileuton. This suggests that blocking leukotriene synthesis could be a treatment for food allergies. Moreover, our investigations have advanced the understanding of what regulates oral anaphylaxis in mouse models, paving the way for physiological allergen exposures to be modeled in C57BL/6 genetically modified mice
Male caregiving experience alters hippocampal neuroplasticity and transcription independent of reproduction in a biparental species.
Full text linkIn mammals, measurable changes in brain and behavior accompany the transition to parenthood. In the biparental California mouse, Peromyscus californicus, fathers experience enhanced neuroplasticity, including increased hippocampal dendritic spine density, reduced anxiety, and improved memory. Here, we first investigate whether siring offspring or pup interaction drives structural neuroplasticity in fathers and find that hippocampal spine density is positively associated with caregiving experience, even in the absence of reproduction. Next, we evaluate the transcriptional response to caregiving in the hippocampus of these males and identify 158 differentially expressed genes between fathers and non-fathers, many of which are associated with neurogenesis, neuronal signaling, and dendritic plasticity. Importantly, 11 of the differentially expressed genes in fathers are similarly regulated in non-fathers that interacted with unrelated pups, suggesting that even limited interaction with pups, in the absence of siring a litter, can induce transcriptional changes in the male brain. Moreover, of the differentially expressed genes that encode for transcription factors and cofactors, we repeatedly find that their expression correlates with spine density in males with varied caregiving experience, thus linking these transcriptional changes to neuroplasticity. Together, this work demonstrates that caregiving-regardless of whether the pups are the male\u27s own biological offspring-drives hippocampal neuroplasticity and transcriptional changes in males of a biparental species
Recruitment, rewiring and deep conservation in flowering plant gene regulation.
No full textTranscription factors (TFs) are proteins that bind DNA to control where and when genes are expressed. In plants, dozens of TF families interact with distinct sets of binding sites (TFBSs) that reflect each TF\u27s role in organismal function and species-specific adaptations. However, defining these roles and understanding broader patterns of regulatory evolution remain challenging, as predicted TFBSs may lack a clear impact on transcription, and experimentally derived TF binding maps to date are modest in scale or restricted to model organisms. Here we present a scalable TFBS assay that we leveraged to create an atlas of nearly 3,000 genome-wide binding site maps for 360 TFs in ten species spanning 150 million years of flowering plant evolution. We found that TF orthologues from distant species retain nearly identical binding preferences, while on the same timescales the gain and loss of TFBSs are widespread. Within lineages, however, conserved TFBSs are over-represented and found in regions harbouring signatures of functional regulatory elements. Moreover, genes with conserved TFBSs showed striking enrichment for cell-type-specific expression in 14 single-nucleus RNA atlases, providing a robust marker of each TF\u27s activity and developmental role. Finally, we compare distant lineages, illustrating how ancient regulatory modules were recruited and rewired to enable adaptations underlying the evolutionary success of grasses