Biology subjects

Pham, L.

Publications and source records attributed to Pham, L..

10 recordsLinked to original sources

bioRxiv2025

Differential contribution of HCN1 and HCN4 to the synchronisation of sinoatrial pacemaker cells

The hearts ability to beat with high precision, with a regular and steady rhythm relies on the synchronised activity of pacemaker cells in the sinoatrial node (SAN), which communicate with one another through gap junctions. This process ensures that electrical impulses are organised and reach a critical mass to ignite the electrical activity of the surrounding atrial tissue and trigger the regular heartbeat. HypothesisWe hypothesise that hyperpolarisation-activated, cyclic nucleotide-gated (HCN) channels play a pivotal role in maintaining this synchronisation process. This idea aligns with the well-established role of HCN channels in stabilising the membrane potential in the voltage range of the slow diastolic depolarisation, counteracting voltage fluctuations and effectively filtering out variations in the beating rate from neighbouring cells. AimWe focus on two specific HCN channel subtypes--HCN1 and HCN4--and their contributions to the rapid synchronisation of pacemaker cells, a phenomenon known as phasic entrainment. Using two HCN channel-mutant mouse models, we dissect the distinct roles of HCN1 and HCN4 in this process. MethodsWe employed patch-clamp electrophysiology to determine phase response curves (PRCs) to predict the ability of single cells to interact in the SAN. Using computer simulations, the behaviour of the SAN at the network level was determined. ResultsWe found that HCN1, but not HCN4, is essential for the fast synchronisation of pacemaker cells in the SAN and propose a mechanism by which HCN1 channels regulate this process. ConclusionThese findings highlight HCN1 as a critical component for ensuring the precise and rapid coordination needed for synchronisation of the SAN, for a regular and consistent heartbeat. Translational perspectiveOur work provides essential insights into the cellular and molecular mechanisms governing SAN function and lays the groundwork for several clinically relevant applications. Understanding how If blockers may affect heart rate and rhythm stability is crucial for assessing potential side effects of current and future subtype-specific HCN channel inhibitors. Moreover, our findings could support new diagnostic strategies for detecting patients at risk of SAN dysfunction. Ultimately, these findings pave the way for innovative therapeutic approaches, including targeted channel modulation and future cell or gene therapies to restore pacemaker stability.

physiology↗

bioRxiv2025

A New Aneurysm Rupture-Prone Marfan Mouse Model with FBN1Q2467X Nonsense Mutation Reveals Adventitial Inflammation

Marfan syndrome (MFS) is a genetic disorder caused by mutations in fibrillin-1(FBN1), which encodes FBN1, a key structural component of the extracellular matrix. Mutations in FBN1 influence the severity of aortic disease and therapeutic responses, with aortic aneurysm being the leading cause of mortality in patients with MFS. To investigate the mechanisms driving aneurysm progression, we generated a mouse model (Fbn1Q2469X/+) carrying the FBN1Q2467X nonsense mutation identified in MFS patients. This mutation results in FBN1 deficiency. Although Fbn1Q2469X/+ mice appear normal, showing only mild, nonprogressive dilation of the aortic root and ascending aorta with minor reductions in blood pressure, homozygous Fbn1Q2469X/Q2469X mice develop spontaneous thoracic aortic aneurysms (TAA) that progress to rupture between 10 and 25 days of age, with 100% penetrance. Histopathology shows progressive vessel wall degeneration characterized by disorganized vascular smooth muscle cells, collagen loss, and elastic fiber fragmentation from early to late stages. RNA-seq analysis identifies inflammation as the dominant process in late-stage aneurysms. Immunofluorescence assay reveals inflammatory cells prominently localized to the adventitia near rupture sites, linking adventitial inflammation to aneurysm progression. This genetically modified Fbn1Q2469X/Q2469X mouse model consistently develops progressive aortic aneurysms and provides a reliable, cost-effective platform to investigate the molecular mechanisms of aneurysm progression and to evaluate therapeutic strategies in aneurysm diseases, including Marfan syndrome and related disorders.

genetics↗

bioRxiv2024

Targeted H3K9 acetylation at lncSox1 promoter by cell type-specific epigenome editing promotes intermediate progenitor proliferation in developing mouse cortex

The distribution and level of epigenetic (chromatin) marks have implications for differential regulatory effects at specific gene loci. Herein, we applied a protocol which combines in vivo electroporation and a CRISPR-dead (d)Cas9 system to probe and edit a specific chromatin mark in the epigenome of intermediate progenitor cells (IPCs) in developing mouse cortex. We found that the promoter of lncSox1, a long non-coding gene, is a key genomic locus for H3K9 acetylation (H3K9ac) during IPC amplification. CRISPR-dCas9-mediated addition of H3K9ac at lncSox1 promoter resulted in lncSox1 upregulation, with attendant increase in IPC pool and augmented neurogenesis. Thus, we have identified dynamic regulation of lncSox1 as a major downstream target of H3 acetylation, and as part of an epigenetic mechanism involved in IPC proliferation during neocortex expansion. This finding is a proof-of-concept that our epigenome editing-based method can be used for manipulating specific epigenetic effectors to determine their (neuro)biological significance. MOTIVATIONThe abundance of basal progenitors is critical for cortical neurogenesis during brain development. There is growing interest in identifying how specific epigenetic factors regulate the genesis and expansion of basal progenitor cell sub-populations, including intermediate progenitor cells. We established a protocol that allowed us to identify the involvement of a long non-coding RNA (lncSox1) in regulating the proliferation of intermediate progenitor cells under the influence of H3K9 acetylation (H3K9ac). By enhancing H3K9ac at the promoter region of lncSox1 using a CRISPR-dCas9-mediated gene-editing tool, we were able to determine that lncSox1 upregulation is a downstream effect of H3K9ac acetylation and is necessary for intermediate progenitor pool amplification during cortical development. Highlights- Identification of H3 acetylation-dependent expression of ncRNAs in developing cortex. - Establishment of cell Cre/LoxP and CRISPR-dCas9-dependent H3K9ac epigenome editing. - CRISPR-dCas9-mediated addition of H3K9ac at lncSox1 promoter resulted in lncSox1 upregulation. - H3K9 acetylation at lncSox1 promoter enhances proliferation of TBR2-expressing IPCs. - Targeted epigenome editing revealed lncSox1 as a key regulator of cortical development.

neuroscience↗

bioRxiv2024

A cross-species analysis of neuroanatomical covariance sex difference in humans and mice

Structural covariance in brain anatomy is thought to reflect inter-regional sharing of developmental influences - although this hypothesis has proved hard to causally test. Here, we use neuroimaging in humans and mice to study sex-differences in anatomical covariance - asking if regions that have developed shared sex differences in volume also show shared sex differences in volume covariance. This study design illuminates both the biology of sex-differences and theoretical models for anatomical covariance - benefitting from tests of inter-species convergence. We find that volumetric structural covariance is stronger in adult females compared to adult males for both wild-type mice and healthy human subjects: 98% of all comparisons with statistically significant covariance sex differences in mice are female-biased, while 76% of all such comparisons are female-biased in humans (q < 0.05). In both species, a regions covariance and volumetric sex-biases have weak inverse relationships to each other: volumetrically male-biased regions contain more female-biased covariations, while volumetrically female-biased regions have more male-biased covariations (mice: r = -0.185, p = 0.002; humans: r = -0.189, p = 0.001). Our results identify a conserved tendency for females to show stronger neuroanatomical covariance than males, evident across species, which suggests that stronger structural covariance in females could be an evolutionarily conserved feature that is partially related to volumetric alterations through sex. SIGNIFICANCE STATEMENTStructural covariance is a potent readout of coordinated brain development, but hard to probe experimentally. We use sex differences as a naturally occurring test for developmental theories of structural covariance - adopting a cross-species approach for validation and translational benefit. Brain MRI reveals two conserved features of anatomical covariance across humans and mice: (i) tighter inter-regional coordination of brain development in females as evidenced by stronger volume covariance; (ii) a tendency for female-biased covariance to involve regions that are smaller in females - suggesting a previously unknown counterbalancing between these two distinct modes of sex-biased brain organization. These findings advance understanding of coordinated brain development and sex difference in a cross-species framework - facilitating future translational research on both topics.

neuroscience↗

bioRxiv2024

Single-Molecule Barcoding Technology for Single-Cell Genomics

Recent advances in barcoding technologies have significantly enhanced the scalability of single-cell genomic experiments. However, large-scale experiments are still rare due to high costs, complex logistics, and laborintensive procedures. To facilitate the routine application of the largest scalability, it is critical to simplify the production and use of barcoding reagents. Here, we introduce AmpliDrop, a technology that initiates the barcoding process using a pool of inexpensive single-copy barcodes and integrates barcode multiplicity generation with tagging of cellular content into a single reaction driven by DNA polymerase during library preparation. The barcoding reactions are compartmentalized using an electronic pipette or a robotic or standalone liquid handling system. These innovations eliminate the need for barcoded beads and complex combinatorial indexing workflows and provide flexibility for a wide range of scales and tube formats, as well as compatibility with automation. We show that AmpliDrop is capable of capturing transcriptomes and chromatin accessibility, and it can also be adapted for user-customized applications, including antibody-based protein detection, bacterial or viral DNA detection, and CRISPR perturbations without dual guide RNA-expression vectors. We validated AmpliDrop by investigating the influence of short-term static culturing on cell composition in human forebrain organoids, revealing metabolic reprogramming in lineage progenitors.

molecular biology↗

bioRxiv2023

Comparative neuroimaging of sex differences in human and mouse brain anatomy

In vivo neuroimaging studies have established several reproducible volumetric sex differences in the human brain, but the causes of such differences are hard to parse. While mouse models are useful for understanding the cellular and mechanistic bases of sex-biased brain development in mammals, there have been no attempts to formally compare mouse and human sex differences across the whole brain to ascertain how well they translate. Addressing this question would shed critical light on use of the mouse as a translational model for sex differences in the human brain and provide insights into the degree to which sex differences in brain volume are conserved across mammals. Here, we use cross-species structural magnetic resonance imaging to carry out the first comparative neuroimaging study of sex-biased neuroanatomical organization of the human and mouse brain. In line with previous findings, we observe that in humans, males have significantly larger and more variable total brain volume; these sex differences are not mirrored in mice. After controlling for total brain volume, we observe modest cross-species congruence in the volumetric effect size of sex across 60 homologous brain regions (r=0.30; e.g.: M>F amygdala, hippocampus, bed nucleus of the stria terminalis, and hypothalamus and F>M anterior cingulate, somatosensory, and primary auditory cortices). This cross-species congruence is greater in the cortex (r=0.33) than non-cortex (r=0.16). By incorporating regional measures of gene expression in both species, we reveal that cortical regions with greater cross-species congruence in volumetric sex differences also show greater cross-species congruence in the expression profile of 2835 homologous genes. This phenomenon differentiates primary sensory regions with high congruence of sex effects and gene expression from limbic cortices where congruence in both these features was weaker between species. These findings help identify aspects of sex-biased brain anatomy present in mice that are retained, lost, or inverted in humans. More broadly, our work provides an empirical basis for targeting mechanistic studies of sex-biased brain development in mice to brain regions that best echo sex-biased brain development in humans.

neuroscience↗

bioRxiv2023

Targeted Phasing of 2-200 Kilobase DNA Fragments with a Short-Read Sequencer and a Single-Tube Linked-Read Library Method

In the human genome, heterozygous sites are genomic positions with different alleles inherited from each parent. On average, there is a heterozygous site every 1-2 kilobases (kb). Resolving whether two alleles in neighboring heterozygous positions are physically linked--that is, phased--is possible with a short-read sequencer if the sequencing library captures long-range information. TELL-Seq is a library preparation method based on millions of barcoded micro-sized beads that enables instrument-free phasing of a whole human genome in a single PCR tube. TELL-Seq incorporates a unique molecular identifier (barcode) to the short reads generated from the same high-molecular-weight (HMW) DNA fragment (known as linked-reads). However, genome-scale TELL-Seq is not cost-effective for applications focusing on a single locus or a few loci. Here, we present an optimized TELL-Seq protocol that enables the cost-effective phasing of enriched loci (targets) of varying sizes, purity levels, and heterozygosity. Targeted TELL-Seq maximizes linked-read efficiency and library yield while minimizing input requirements, fragment collisions on microbeads, and sequencing burden. To validate the targeted protocol, we phased seven 180-200 kb loci enriched by CRISPR/Cas9-mediated excision coupled with pulse-field electrophoresis, four 20 kb loci enriched by CRISPR/Cas9-mediated protection from exonuclease digestion, and six 2-13 kb loci amplified by PCR. The selected targets have clinical and research relevance (BRCA1, BRCA2, MLH1, MSH2, MSH6, APC, PMS2, SCN5A-SCN10A, and PKI3CA). These analyses reveal that targeted TELL-Seq provides a reliable way of phasing allelic variants within targets (2-200 kb in length) with the low cost and high accuracy of short-read sequencing.

genomics↗

bioRxiv2021

H3 acetylation selectively promotes basal progenitor proliferation and neocortex expansion by activating TRNP1 expression

Increase in the size of human neocortex, acquired in evolution, accounts for the unique cognitive capacity of humans. This expansion appears to reflect the evolutionarily-enhanced proliferative ability of basal progenitors (BPs) in mammalian cortex, which may have been acquired through epigenetic alterations in BPs. However, whether or how the epigenome in BPs differs across species is not known. Here, we report that histone H3 acetylation is a key epigenetic regulation in BP amplification and cortical expansion. Through epigenetic profiling of sorted BPs, we show that H3K9 acetylation is low in murine BPs and high in human BPs. Elevated H3K9ac preferentially increases BP proliferation, increasing the size and folding of the normally smooth mouse neocortex. Mechanistically, H3K9ac drives BP amplification by increasing expression of the evolutionarily regulated gene, TRNP1, in the developing cortex. Our findings demonstrate a previously unknown mechanism that controls cortical architecture. One Sentence SummaryH3K9ac promotes basal progenitor amplification, neocortex expansion and gyrification by activating TRNP1 expression in evolution.

developmental biology↗

bioRxiv2019

Ultra-low input single tube linked-read library method enables short-read NGS systems to generate highly accurate and economical long-range sequencing information for de novo genome assembly and haplotype phasing

Long-range sequencing information is required for haplotype phasing, de novo assembly and structural variation detection. Current long-read sequencing technologies can provide valuable long-range information but at a high cost with low accuracy and high DNA input requirement. We have developed a single-tube Transposase Enzyme Linked Long-read Sequencing (TELL-Seq) technology, which enables a low-cost, high-accuracy and high-throughput short-read next generation sequencer to routinely generate over 100 Kb long-range sequencing information with as little as 0.1 ng input material. In a PCR tube, millions of clonally barcoded beads are used to uniquely barcode long DNA molecules in an open bulk reaction without dilution and compartmentation. The barcode linked reads are used to successfully assemble genomes ranging from microbes to human. These linked-reads also generate mega-base-long phased blocks and provide a cost-effective tool for detecting structural variants in a genome, which are important to identify compound heterozygosity in recessive Mendelian diseases and discover genetic drivers and diagnostic biomarkers in cancers.

genomics↗

bioRxiv2019

Simultaneous Targeting of XPO1 and BCL2 as an Effective Treatment Strategy for Double-Hit Lymphoma

Double-hit lymphoma (DHL) is among the most aggressive and chemoresistant lymphoma subtypes. DHLs carry genomic abnormalities in MYC, BCL2 and/or BCL6 oncogenes. Due to simultaneous overexpression of these driver oncogenes, DHLs are highly resistant to the frontline therapies. Most DHLs overexpress both MYC and BCL2 driver oncogenes concurrently. We reasoned that simultaneous suppression of the two driver oncogenes would be more effective in eradicating DHLs than inactivation of single oncogene. XPO1 is a receptor for nuclear cytoplasmic transport of protein and RNA species. Recently, XPO1 inhibition was shown to downregulate MYC expression in several cancer cell lines. We therefore examined the role of XPO1 as a therapeutic target in suppressing MYC function and the potential synergistic effects of simultaneous suppression of XPO1 and BCL2 in the treatment of DHL. Here, we demonstrate that XPO1 inhibition abrogates MYC protein expression and induces massive tumor cell apoptosis. Combined use of XPO1 and BCL2 inhibitors is highly effective in eradicating DHL cells in cell culture. Notably, in a mouse model of DHL bearing primary tumor cells derived from lymphoma patients, combined treatment with XPO1 and BCL2 inhibitors blocks tumor progression, prevents brain metastasis, and extends host survival. Thus, our study confirms the simultaneous targeting of MYC and BCL2 driver oncogenes through the combined use of XPO1 and BCL2 inhibitors as a unique approach for the treatment of DHLs.

cancer biology↗