Biology subjects

Cao, J.

Publications and source records attributed to Cao, J..

At least 127 records · Page 7Linked to original sources

bioRxiv2022

A comprehensive view of cell-type-specific temporal dynamics in human and mouse brains

Progenitor cells play fundamental roles in preserving optimal organismal functions under normal, aging, and disease conditions. However, progenitor cells are incompletely characterized, especially in the brain, partly because conventional methods are restricted by inadequate throughput and resolution for deciphering cell-type-specific proliferation and differentiation dynamics in vivo. Here, we developed TrackerSci, a new technique that combines in vivo labeling of newborn cells with single-cell combinatorial indexing to profile the single-cell chromatin landscape and transcriptome of rare progenitor cells and track cellular differentiation trajectories in vivo. We applied TrackerSci to analyze the epigenetic and gene expression dynamics of newborn cells across entire mouse brains spanning three age stages and in a mouse model of Alzheimers disease. Leveraging the dataset, we identified diverse progenitor cell types less-characterized in conventional single cell analysis, and recovered their unique epigenetic signatures. We further quantified the cell-type-specific proliferation and differentiation potentials of progenitor cells, and identified the molecular programs underlying their aging-associated changes (e.g., reduced neurogenesis/oligodendrogenesis). Finally, we expanded our analysis to study progenitor cells in the aged human brain through profiling [~]800,000 single-cell transcriptomes across five anatomical regions from six aged human brains. We further explored the transcriptome signatures that are shared or divergent between human and mouse oligodendrogenesis, as well as the region-specific down-regulation of oligodendrogenesis in the human cerebellum. Together, the data provide an in-depth view of rare progenitor cells in mammalian brains. We anticipate TrackerSci will be broadly applicable to characterize cell-type-specific temporal dynamics in diverse systems.

genomics↗

bioRxiv2022

Common risk variants in AHI1 are associated with childhood steroid-sensitive nephrotic syndrome

BackgroundSteroid-sensitive nephrotic syndrome (SSNS) is the most common form of kidney disease in children worldwide. Genome-wide association studies (GWAS) have demonstrated association of SSNS with genetic variation at HLA-DQ/DR and have identified several non-HLA loci that aid in further understanding of disease pathophysiology. We sought to identify additional genetic loci associated with SSNS in children of Sri Lankan and European ancestry. MethodsWe conducted a GWAS in a cohort of Sri Lankan individuals comprising 420 pediatric patients with SSNS and 2339 genetic ancestry matched controls obtained from the UK Biobank. We then performed a trans-ethnic meta-analysis with a previously reported European cohort of 422 pediatric patients and 5642 controls. ResultsOur GWAS confirmed the previously reported association of SSNS with HLA-DR/DQ (rs9271602, p=1.12x10-27, odds ratio[OR]=2.75). Trans-ethnic meta-analysis replicated these findings and identified a novel association at AHI1 (rs2746432, p=2.79x10-8, OR=1.37), which was also replicated in an independent South Asian cohort. AHI1 is implicated in ciliary protein transport and immune dysregulation, with rare variation in this gene contributing to Joubert syndrome type 3. ConclusionsCommon variation in AHI1 confers risk of the development of SSNS in both Sri Lankan and European populations. The association with common variation in AHI1 further supports the role of immune dysregulation in the pathogenesis of SSNS and demonstrates that variation across the allele frequency spectrum in a gene can contribute to disparate monogenic and polygenic diseases. AUTHOR SUMMARYSteroid-sensitive nephrotic syndrome (SSNS) is the most common kidney disease in children worldwide, but the cause of disease is not well understood. Genome-wide association studies (GWAS) in SSNS have shown that genes in the classical HLA region (the human immune centre) and several genes outside of this region are associated with the disease, which has allowed us to further understand the cause of disease. We performed a GWAS of Sri Lankan ancestry that included 420 paediatric patients and 2339 ancestry-matched controls and confirmed association at HLA-DQ/DR with SSNS. We then performed a Sri Lankan-European trans-ethnic meta-analysis and identified a new association with SSNS outside of HLA, in AHI1. This finding further supports the role of immune system involvement in the etiology of SSNS and increases our knowledge of the genetic causes of disease. AHI1 is a gene that can also cause ciliary problems and demonstrates that different genetic variants within the same gene can contribute to both single-gene (Joubert syndrome, a rare disease that causes kidney and neurological problems) and multi-gene diseases (SSNS).

genetics↗

bioRxiv2022

Effects of protoscoleces excretory-secretory products of Echinococcus granulosus on hepatocyte growth, function, and glucose metabolism

Cystic echinococcosis (CE) is one of the most widespread and harmful zoonotic parasitic diseases and it most commonly affects the liver. In this study, we characterized multiple changes in mouse hepatocytes following treatment with excretory-secretory (ES) products of Echinococcus granulosus protoscoleces by a factorial experiment. The cell counting kit-8 assay (CCK-8), the 5-ethynyl-2-deoxyuridine (EdU) assay, and flow cytometry were used to detect the growth of hepatocytes. Inverted microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) were used to observe the morphology and ultrastructure of hepatocytes. An automatic biochemical analyzer and an ELISA detection kit were used to determine six conventional hepatocyte enzymatic indices, the levels of five hepatocyte-synthesized substances, and the contents of glucose and lactate. Western blot analysis was conducted to analyze the protein expression of six rate-limiting enzymes in the glucose metabolism pathway in hepatocytes: glutamic-pyruvic transaminase (ALT), glutamic-oxalacetic transaminase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), gamma-glutamyl transpeptidase (GGT), and leucine arylamidase (LAP). The results of the CCK-8 and EdU assays both showed that ES could inhibit the proliferation of hepatocytes, and flow cytometry indicated that ES could promote apoptosis of hepatocytes. After ES treatment, the ultrastructure of hepatocytes was disrupted to a certain extent. The changes in the cell membrane and microvilli were observed through SEM, and the changes in the nucleus, mitochondria, and rough endoplasmic reticulum were observed through TEM. After ES treatment, the enzymatic activities of the six hepatocyte enzymes were increased in addition to the Fe metabolism and the synthesis of albumin (ALB), uric acid (UA), and urea, whereas the synthesis of transferrin (TRF) was decreased. The expression levels of all six key enzymes in the glucose metabolism pathway in hepatocytes were decreased, and the biological effects were significantly inhibited. We analyzed the causes and possible complications caused by various changes and advocate corresponding measures. We also propose possible mechanisms by which protoscoleces cause hepatocyte necrosis, but the specific mechanism requires further study. Author SummaryEchinococcus granulosus, the most widely distributed and most infected tapeworm, has caused serious economic and social burdens to pastoral areas in China. The metacestodes of Echinococcus granulosus mainly infect the liver of intermediate hosts (humans, cattle, sheep, etc.). Currently, the effects of Echinococcus granulosus on hepatocytes ultrastructure, enzymology, function, and glucose metabolism have not been characterized, and accurate characterization is crucial in the study of related pathogenesis and preventive therapy. Here, we characterize multiple changes in hepatocytes using excretory-secretory (ES) products of Echinococcus granulosus protoscoleces and mouse hepatocyte action by a factorial experiment. We found that ES inhibited hepatocyte proliferation and promoted hepatocyte apoptosis. ES can cause a certain degree of damage to the cell membrane, nucleus, mitochondria, and endoplasmic reticulum of hepatocytes. After ES treatment, six enzymatic indexes of hepatocytes were elevated, they were ALT, AST, LDH, ALP, GGT, and LAP, and Fe, ALB, UA, and urea levels synthesized by hepatocytes were significantly higher and TRF levels were significantly lower. Reduced expression of rate-limiting enzymes in six pathways of glucose metabolism in hepatocytes, including PFK-1, IDH, G-6-PD, GS, GP, and GLUT-2, indicating that ES inhibits glucose metabolism in hepatocytes. Our study not only characterized the effects of ES on hepatocytes in detail but also proposed the possible mechanisms causing these effects, which provided a basis for subsequent studies on related pathogenesis and prevention, and treatment.

cell biology↗

bioRxiv2022

A global view of aging and Alzheimer's pathogenesis-associated cell population dynamics and molecular signatures in the human and mouse brains

Conventional single-cell genomics approaches are limited by throughput and thus may have failed to capture aspects of the molecular signatures and dynamics of rare cell types associated with aging and diseases. Here, we developed EasySci, an extensively improved single-cell combinatorial indexing strategy, for investigating the age-dependent dynamics of transcription and chromatin accessibility across diverse brain cell types. We profiled [~]1.5 million single-cell transcriptomes and [~]400,000 single-cell chromatin accessibility profiles across mouse brains spanning different ages, genotypes, and both sexes. With a novel computational framework designed for characterizing cellular subtypes based on the expression of both genes and exons, we identified > 300 cell subtypes and deciphered their underlying molecular programs and spatial locations especially for rare cell types (e.g., pinealocytes, tanycytes). Leveraging these data, we generated a global readout of age-dependent changes at cell subtype resolution, providing insights into cell types that expand (e.g., rare astrocytes and vascular leptomeningeal cells in the olfactory bulb, reactive microglia, and oligodendrocytes) or are depleted (e.g., neuronal progenitors, neuroblasts, committed oligodendrocyte precursors) as age progresses. Furthermore, we explored cell-type-specific responses to genetic perturbations associated with Alzheimers disease (AD) and identified rare cell types depleted (e.g., mt-Cytb+, mt-Rnr2+ choroid plexus epithelial cells) or enriched (e.g., Col25a1+, Ndrg1+ interbrain and midbrain neurons) in both AD models. Key findings are consistent between males and females, validated across the transcriptome, chromatin accessibility, and spatial analyses. Finally, we profiled a total of 118,240 single-nuclei transcriptomes from twenty-four post-mortem human brain samples derived from control and AD patients, revealing highly cell-type-specific and region-specific gene expression changes associated with AD pathogenesis. Critical AD-associated gene signatures were validated in both human and mice. In summary, these data comprise a rich resource for exploring cell-type-specific dynamics and the underlying molecular mechanisms in normal and pathological mammalian aging.

genomics↗

bioRxiv2022

Synergistic action of the Arabidopsis spliceosome components PRP39a and SmD1 in promoting post-transcriptional transgene silencing

Besides regulating splicing, the conserved spliceosome component SmD1 was shown to promote posttranscriptional silencing of sense transgenes (S-PTGS). Here, we show that the conserved spliceosome component PRP39a also plays a role in S-PTGS. However, PRP39a and SmD1 actions appear distinct in both splicing and S-PTGS. Indeed, RNA-seq analysis of prp39a and smd1 mutants identified different sets of deregulated mRNAs and non-coding RNAs, both at expression level and alternative splicing genome-wide. Moreover, double mutant analyses involving prp39a or smd1 and RNA quality control (RQC) mutants revealed genetic interactions of SmD1 and PRP39a with distinct nuclear RQC machineries, suggesting synergistic rather than redundant roles in the RQC/PTGS interplay. Supporting this hypothesis, a prp39a smd1 double mutant exhibited enhanced suppression of S-PTGS compared with single mutants. Because no major changes in the expression of PTGS or RQC components or in small RNA production were identified in prp39a and smd1 mutants, and because prp39a and smd1 mutations do not alter PTGS triggered by inverted-repeat transgenes directly producing dsRNA (IR-PTGS), PRP39a and SmD1 seem to synergistically promote a step specific to S-PTGS. We propose that, independent of their specific roles in splicing, PRP39a and SmD1 limit 3-to-5 and 5-to-3 degradation of transgene aberrant RNAs, respectively, thus favoring the export of aberrant RNAs to the cytoplasm where their transformation into dsRNA initiates S-PTGS.

plant biology↗

bioRxiv2022

Single cell, whole embryo phenotyping of pleiotropic disorders of mammalian development

Mouse models are a critical tool for studying human diseases, particularly developmental disorders, as well as for advancing our general understanding of mammalian biology. However, it has long been suspected that conventional approaches for phenotyping are insufficiently sensitive to detect subtle defects throughout the developing mouse. Here we set out to establish single cell RNA sequencing (sc-RNA-seq) of the whole embryo as a scalable platform for the systematic molecular and cellular phenotyping of mouse genetic models. We applied combinatorial indexing-based sc-RNA-seq to profile 101 embryos of 26 genotypes at embryonic stage E13.5, altogether profiling gene expression in over 1.6M nuclei. The 26 genotypes include 22 mouse mutants representing a range of anticipated severities, from established multisystem disorders to deletions of individual enhancers, as well as the 4 wildtype backgrounds on which these mutants reside. We developed and applied several analytical frameworks for detecting differences in composition and/or gene expression across 52 cell types or trajectories. Some mutants exhibited changes in dozens of trajectories (e.g., the pleiotropic consequences of altering the Sox9 regulatory landscape) whereas others showed phenotypes affecting specific subsets of cells. We also identify differences between widely used wildtype strains, compare phenotyping of gain vs. loss of function mutants, and characterise deletions of topological associating domain (TAD) boundaries. Intriguingly, even among these 22 mutants, some changes are shared by heretofore unrelated models, suggesting that developmental pleiotropy might be "decomposable" through further scaling of this approach. Overall, our findings show how single cell profiling of whole embryos can enable the systematic molecular and cellular phenotypic characterization of mouse mutants with unprecedented breadth and resolution.

genomics↗

bioRxiv2022

Decoding Subject's Own Name in the Primary Auditory Cortex

Current studies have shown that perception of subjects own name (SON) involves multiple multimodal brain regions, while activities in unimodal sensory regions (i.e., primary auditory cortex) and their interaction with multimodal regions during the self-processing remain unclear. To answer this, we combined multivariate pattern analysis and dynamic causal modelling analysis to explore the regional activation pattern and inter-region effective connection during the perception of SON. We found that SON and other names could be decoded from the activation pattern in the primary auditory cortex. In addition, we found an excitatory effect of SON on connections from the anterior insula/inferior frontal gyrus to the primary auditory cortex, and to the temporal parietal junction. Our findings extended the current knowledge of self-processing by showing that primary auditory cortex could discriminate SON from other names. Furthermore, our findings highlighted the importance of influence of the insula on the primary auditory cortex during self-processing.

neuroscience↗

bioRxiv2022

Why is Babesia not killed by artemisinin like Plasmodium?

Babesia spp. are intraerythrocytic apicomplexan organisms digesting hemoglobin similar to intraerythrocytic Plasmodium spp. However, unlike Plasmodium spp., Babesia spp. are not sensitive to artemisinin, The difference between Babesia genomes and Plasmodium genomes revealed that smaller Babesia genomes lack numerous genes, especially haem synthesis-related genes. Single-cell sequencing analysis showed that different groups of B. microti with expressed pentose phosphate pathway (PPP)-related, DNA replication-related, antioxidation-related, glycolysis-related, and glutathione-related genes were not as sensitive to artemether as P. yoelii 17XNL. Especially, PPP-related, DNA replication-related, and glutathione-related genes were inactively expressed compared with P. yoelii 17XNL. Adding iron supply in vivo can promote the reproduction of B. microti. These results suggest that Babesia spp. lack a similar mechanism to that in malaria parasites, by which haem or iron in hemoglobin is utilized, but it likely leads to their insensitivity to artemisinin in turn. Author summaryBabesia and Plasmodium are similar in many ways, from morphology to life history. In particular, both ingest and break down hemoglobin. However, compared with Plasmodium, Babesia cannot form hemozoin with less pathogenicity and insensitivity to artemisinin. Recent studies suggest that artemisinin can kill malaria parasites through free-radical and iron-capture effects, indicating that iron and haem play a key role in the sensitivity of malaria parasites to artemisinin. The Babesia genome is smaller and does not contain haem synthesis-related genes, indicating low requirements and utilization of haem and iron (HI). Moreover, we found that the expression of PPP-related and DNA replication-related genes is not active, distinctly different from malaria parasites. However, adding iron supply in vivo can increase the infection rate of B. microti. Therefore, we hypothesized that Babesia lacks mechanisms for the efficient utilization of HI, resulting in low requirements for HI, and therefore insensitivity to artemisinin.

evolutionary biology↗

bioRxiv2022

Discovery of an AIM2 inflammasome inhibitor for the treatment of DNA-driven inflammatory disease.

Inflammation driven by DNA sensors is now understood to be central to disease pathogenesis. Here we describe new inhibitors of pathogenic DNA sensing, primarily of the inflammasome forming sensor AIM2. Molecular modelling and biochemistry has revealed potent inhibitors of AIM2 that work by binding competitively to the DNA binding site. Though less potent, these AIM2 inhibitors, 4-sulfonic calixarenes, also inhibit DNA sensors cGAS and TLR9 demonstrating a broad utility against pathogenic DNA-driven inflammatory responses. The 4-sulfonic calixarenes inhibited AIM2 dependent post-stroke T cell death, highlighting a proof of concept that the 4-sulfonic calixarenes could be effective at combatting post-stroke immunosuppression. By extension, we propose a broad utility against DNA driven inflammation in disease. Finally, we reveal that the ancient drug suramin, by virtue of its structural similarities, is an excellent inhibitor of DNA-dependent inflammation and propose that suramin could be rapidly repurposed to meet an ever increasing clinical need.

immunology↗

bioRxiv2022

Novel leaf-root coordination driven by leaf water storage tissues in mangroves

Interactions among root and leaf traits (in particular, leaf hydraulic and leaf economics traits) are fundamental in generating diverse strategies in land plants, yet it remains a knowledge gap in mangrove plants that experiences saline stress distinct from most other vascular plants. Here, we tested the trait relationships in mangrove plants and compared them with typical land plants (non-mangrove). Consistent with non-mangrove plants, leaf hydraulic and economics traits were decoupled in mangrove plants. However, mangrove leaf economics traits correlated strongly with root hydraulic traits, which are normally decoupled in non-mangrove plants. Moreover, we observed a unique scaling relationship between leaf dry mass per area and root hydraulic traits in mangroves. The novel coordination between leaves and roots arises from the wide-presence of leaf water storage tissues in mangroves, and this potentially represents a new paradigm with which we look into the ecology, physiology and evolution of this important vegetation.

ecology↗

bioRxiv2022

Multi-environmental population phenotyping suggest the higher risks of wheat Rht-B1b and Rht-D1b cultivars in global warming scenarios

After six decades practice, the semi-dwarf alleles Rht-B1b and Rht-D1b (also called Rht-1 and Rht-2) had been applied into around 70% current wheat cultivars, laid the foundation for the worldwide wheat production supply, then the agronomic traits controlled by the two alleles still keep unclear except dwarfing wheat. Here 13 agronomic traits were investigated in 400 wheat accessions with seven environments, uncovered the genetic effects of Rht-B1b and Rht-D1b on wheat structure and yield traits in different genetic backgrounds and environments, and the distinct genetic effects between Rht-B1b and Rht-D1b, suggesting that the introduction of green revolution alleles profoundly shaped the agronomy traits of modern wheat cultivars. The late-sowing assays and regression analysis based on the phenotypic and their meteorological data showed the accessions containing Rht-B1b and Rht-D1b are more sensitive to the temperature increase, and the Rht-D1b may lost additional 8% yield compared the cultivars without the green revolution alleles if the average temperature increases 1 {degrees}C. These results suggest the application of green revolution are facing more challenges to maintain the futural wheat production supply in global warming scenarios.

molecular biology↗

bioRxiv2022

leptin b and its regeneration enhancer illustrate the regenerative features of zebrafish hearts

Zebrafish possess a remarkable regenerative capacity, which is mediated by the induction of various genes upon injury. Injury-dependent transcription is governed by the tissue regeneration enhancer elements (TREEs). Here, we utilized leptin b (lepb), an injury-specific factor, and its TREE to dissect heterogeneity of non-cardiomyocytes (CMs) in regenerating zebrafish hearts. Our single-cell RNA sequencing (scRNA-seq) analysis demonstrated that the endothelium/endocardium(EC) is activated to induce distinct subpopulations upon cardiac injury. We demonstrated that lepb can be utilized as a regeneration-specific marker to subset injury-activated ECs. lepb+ ECs robustly induce pro-regenerative factors, implicating lepb+ ECs as a signaling center to interact with other cardiac cells. Our scRNA-seq analysis identified that lepb is also produced by specific subpopulation of epicardium (Epi) and epicardium-derived cells (EPDCs). To determine lepb labels injury-emerging non-CM cells, we tested the activity of lepb-linked regeneration enhancer (LEN) with chromatin accessibility profiles and transgenic lines. While non-detectable in uninjured hearts, LEN directs EC and Epi/EPDC expression upon injury. The endogenous LEN activity was assessed using LEN deletion lines, demonstrating that LEN deletion abolished injury-dependent expression of lepb, but not other nearby genes. Our integrative analyses identify regeneration-emerging cell types and factors, leading to the discovery of regenerative features of hearts.

developmental biology↗

bioRxiv2022

Droplet-based microfluidics as a media optimization tool for cyanobacteria

The urgent need to increase sustainability in biotechnology has led to an increased interest in photosynthetic production platforms. Cyanobacteria are particularly attractive for their fast photoautotrophic growth and genetic accessibility. However, the lack of systematic strain optimization is holding back progress towards industrialization. To overcome this bottleneck, here we introduce a droplet-based microfluidics platform capable of one- (1D) and two-dimension (2D) screening of key parameters in cyanobacterial cultivation. We successfully grew three different unicellular, biotechnologically relevant cyanobacteria: Synechocystis sp. PCC 6803, Synechococcus elongatus UTEX 2973 and Synechococcus sp. UTEX 3154. Highly-resolved 1D screening of nitrate, phosphate, carbonate, and salt content show that nitrate and/or phosphate can be limiting in standard cultivation media. Finally, we show that 2D screening results from our microfluidic setup translate well to a laboratory scale high-density cultivation setup. This study demonstrates that droplet-based microfluidics by micro segmented-flow are a powerful tool for high-throughput optimization of cyanobacterial cultivation.

microbiology↗

bioRxiv2022

Structural insights into the assembly of gp130 family cytokine signaling complexes

The gp130 family cytokine signaling complexes have limited structural information despite their crucial roles in various cellular processes. We determined cryo-EM structures of several complexes of this family, containing full ectodomains of both signaling receptors bound to their respective ligands CNTF, CLCF1, LIF, IL-27, and IL-6. Our structures reveal that gp130 serves as a central receptor by engaging Site 2 of CNTF, CLCF1, LIF, and IL-6, and Site 3 of IL-27 and IL-6. The acute bends at both signaling receptors in all complexes bring the membrane-proximal domains to a ~30 [A] range but with distinct distances and orientations, which might determine biological specificities of these cytokines. We also reveal how CLCF1 engages its secretion chaperone CRLF1. Our data provide valuable insights for therapeutically targeting gp130-mediated signaling.

biochemistry↗

bioRxiv2022

Evidence for the digital programming of cellular differentiation: characterization of binary switches in lineage-determining transcription factors

Previous studies of the murine Ly49 and human KIR gene clusters have revealed a role for bidirectional promoters in the control of variegated gene expression. Whether or not competing promoters control other instances of cell fate determination remains an outstanding question. Although divergent transcripts within 300 bp are found in ~6% of human genes, an analysis of human transcription factor (TF) genes (1640) revealed that 33% possess stable divergent transcripts with a transcription start site (TSS) less than 300 bp upstream, with many separated by less than 30 bp, indicating an enrichment for potential binary switches in TFs. We have performed a detailed examination of putative bidirectional promoter switches in three lineage-determining TF genes: AHR, GATA3, and ROR{gamma}T. These genes also contain additional pairs of opposing promoters that would prevent simultaneous transcription of sense and antisense, and thus may represent simple on/off switches rather than probabilistic switches. In situ RNA hybridization of human tissues revealed mutually exclusive expression of sense versus antisense transcription, indicating switching between stable sense or antisense transcriptional states. Single-cell RNAseq confirmed the separate sense/antisense states, and revealed the identity of cells with active switch elements. Differential gene expression analysis of cells with antisense switch transcripts revealed an enrichment for genes found in immature/stem cells and a lack of genes associated with terminally differentiated cells. Taken together, these data indicate that there is a digital component to the differentiation program mediated by binary promoter switches in lineage-determining transcription factors.

developmental biology↗

bioRxiv2022

Nr4a1 regulates inhibitory circuit structure and function in the mouse brain.

Central neurons express unique repertoires of secreted and transmembrane proteins that define their synaptic connectivity. However, how these molecular programs are regulated remains poorly understood. Our study shows that, in inhibitory GABAergic interneurons in the mouse forebrain, transcription of synaptic organizer molecules is controlled by Nr4a1, a nuclear receptor whose expression is transiently induced by sensory experience and required for normal learning. Nr4a1 exserts opposite effects on local axonal wiring of Parvalbumin- and Somatostatin-positive interneurons that innervate different subcellular domains of their postsynaptic partners. Loss of Nr4a1 activity in these interneurons leads to cell-type-specific transcriptional switches in multiple gene families, including those involved in surface adhesion and repulsion. Our findings reveal a mechanism by which inducible transcription factors dynamically alter the combinatorial synaptic organizing codes for structural plasticity.

neuroscience↗

bioRxiv2022

Tethering distinct molecular profiles of single cells by their lineage histories to investigate sources of cell state heterogeneity

Gene expression heterogeneity is ubiquitous within single cell datasets, even among cells of the same type. Heritable expression differences, defined here as those which persist over multiple cell divisions, are of particular interest, as they can underlie processes including cell differentiation during development as well as the clonal selection of drug-resistant cancer cells. However, heritable sources of variation are difficult to disentangle from non-heritable ones, such as cell cycle stage, asynchronous transcription, and measurement noise. Since heritable states should be shared by lineally related cells, we sought to leverage CRISPR-based lineage tracing, together with single cell molecular profiling, to discriminate between heritable and non-heritable variation in gene expression. We show that high efficiency capture of lineage profiles alongside single cell gene expression enables accurate lineage tree reconstruction and reveals an abundance of progressive, heritable gene expression changes. We find that a subset of these are likely mediated by structural genetic variation (copy number alterations, translocations), but that the stable attributes of others cannot be understood with expression data alone. Towards addressing this, we develop a method to capture cell lineage histories alongside single cell chromatin accessibility profiles, such that expression and chromatin accessibility of closely related cells can be linked via their lineage histories. We call this indirect "coassay" approach "THE LORAX" and leverage it to explore the genetic and epigenetic mechanisms underlying heritable gene expression changes. Using this approach, we show that we can discern between heritable gene expression differences mediated by large and small copy number changes, trans effects, and possible epigenetic variation.

genomics↗

bioRxiv2022

A Single Multipurpose FSH-Blocking Therapeutic for Osteoporosis and Obesity

Pharmacological and genetic studies over the past decade have established FSH as an actionable target for diseases affecting millions, notably osteoporosis, obesity and Alzheimers disease (AD). Blocking FSH action prevents bone loss, fat gain and AD-like features in mice. We recently developed a first-in-class, humanized, epitope-specific FSH blocking antibody, MS-Hu6, with a KD of 7.52 nM. Using a GLP-compliant platform, we now report the efficacy of MS-Hu6 in preventing obesity and osteoporosis in mice, and parameters of acute safety in monkeys. Biodistribution studies using 89Zr-labelled, biotinylated or unconjugated MS-Hu6 in mice and monkeys showed localization to bone, bone marrow and fat depots. MS-Hu6 displayed a {beta} phase t[1/2] of 13 days (316 hours) in humanized Tg32 mice, and bound endogenous FSH. We tested 215 variations of excipients using the protein thermal shift assay to generate a final formulation that rendered MS-Hu6 stable in solution upon freeze-thaw and at different temperatures, with minimal aggregation, and without self-, cross-, or hydrophobic interactions or appreciable binding to relevant human antigens. MS-Hu6 showed the same level of "humanness" as human IgG1 in silico, and was non-immunogenic in ELISPOT assays for IL-2 and IFN{gamma} in human peripheral blood mononuclear cell cultures. We conclude that MS-Hu6 is efficacious, durable and manufacturable, and is therefore poised for future human testing as a multipurpose therapeutic.

pharmacology and toxicology↗