Biology subjects

Wong, M.

Publications and source records attributed to Wong, M..

At least 73 records · Page 4Linked to original sources

bioRxiv2021

High levels of truncated RHAMM cooperate with dysfunctional p53 to accelerate theprogression of pancreatic cancer

Pancreatic cancer has the lowest survival rate in all types of cancer. Pancreatic cancer patients are often diagnosed at advanced stages. A better therapeutic development for this devastating disease is urgently needed. Receptor for hyaluronan-mediated motility (RHAMM), not expressed in adult pancreas, has been suggested as a prognostic factor and a potential therapeutic target for pancreatic ductal adenocarcinoma (PDAC) and pancreatic neuroendocrine tumor (PNET). In this study, we initially sought to determine whether genetic deletion of RHAMM would slow down pancreatic cancer progression using Rhamm-/- mice. However, we found that Rhamm-/- mice expressed a truncated HMMR{Delta}exon8-16 protein at higher abundance levels than wild-type RHAMM. While HMMR{Delta}exon8-16 did not enable malignant progression of pancreatic intraepithelial neoplasia in p48-Cre; LSL-KRASG12D mice, it accelerated the formation of invasive PDAC and shortened the survival of p48-Cre; LSL-KRASG12D mice with heterozygous p53 knockout. KrasG12D PDAC mice with homozygous p53 knockout mice died around 10 weeks, and the effect of HMMR{Delta}exon8-16 was not apparent in these mice with short-life span. In addition, HMMR{Delta}exon8-16 shortened the survival of PNET-bearing RIP-Tag mice, which had inactivated p53. In our analysis of TCGA dataset, pancreatic cancer patients with mutant TP53 or loss of one copy of TP53 had higher RHAMM expression, which, combined, predicted worse outcomes. Taken together, by collaborating with dysfunctional p53, high levels of HMMR{Delta}exon8- 16 that lacks the centrosome targeting domain and degrons for interaction with the Anaphase-Promoting Complex (APC) accelerated pancreatic cancer progression.

cancer biology↗

bioRxiv2020

Bone marrow adipocytes drive the development of tissue invasive Ly6Chigh monocytes during obesity

During obesity and high fat-diet (HFD) feeding in mice, sustained low-grade inflammation includes not only increased pro-inflammatory macrophages in the expanding adipose tissue, but also bone marrow (BM) production of invasive Ly6Chigh monocytes. As BM adiposity also accrues with HFD, we explored the relationship between the gains in BM white adipocytes and invasive Ly6Chigh monocytes in vivo and through ex vivo paradigms. We find a temporal and causal link between BM adipocyte whitening and the Ly6Chigh monocyte surge, preceding the adipose tissue macrophage rise during HFD. Phenocopying this, ex vivo treatment of BM cells with conditioned media from BM adipocytes or from bona fide white adipocytes favoured Ly6Chigh monocyte preponderance. Notably, the switch towards the Ly6Chigh subset was preceded by monocyte metabolic reprogramming towards glycolysis, reduced oxidative potential and increased mitochondrial fission. In sum, short-term HFD changes BM cellularity, resulting in local adipocyte whitening driving a gradual increase and activation of invasive Ly6Chigh monocytes.

immunology↗

bioRxiv2020

Unscrambling cancer genomes via integrated analysis of structural variation and copy number

Complex somatic genomic rearrangement and copy number alterations (CNA) are hallmarks of nearly all cancers. Whilst whole genome sequencing (WGS) in principle allows comprehensive profiling of these events, biological and clinical interpretation remains challenging. We have developed LINX, a novel algorithm which allows interpretation of short-read paired-end WGS derived structural variant and CNA data by clustering raw structural variant calls into distinct events, predicting their impact on the local structure of the derivative chromosome, and annotating their functional impact on affected genes. Novel visualisations facilitate further investigation of complex genomic rearrangements. We show that LINX provides insights into a diverse range of structural variation events including single and double break-junction events, mobile element insertions, complex shattering and high amplification events. We demonstrate that LINX can reliably detect a wide range of pathogenic rearrangements including gene fusions, immunoglobulin enhancer rearrangements, intragenic deletions and duplications. Uniquely, LINX also predicts chained fusions which we demonstrate account for 13% of clinically relevant oncogenic fusions. LINX also reports a class of inactivation events we term homozygous disruptions which may be a driver mutation in up to 8.8% of tumors including frequently affecting PTEN, TP53 and RB1, and are likely missed by many standard WGS analysis pipelines.

cancer biology↗

bioRxiv2020

SARS-CoV-2 infects carotid arteries: implications for vascular disease and organ injury in COVID-19

Stroke and central nervous system dysfunction are cardinal symptoms in critically ill corona virus disease 19 (COVID-19) patients. In an autopsy series of 32 COVID-19 patients, we investigated whether carotid arteries were infected with SARS-CoV-2 by employing genomic, virologic, histochemical and transcriptomic analyses. We show that SARS-CoV-2 productively infects and modulates vascular responses in carotid arteries. This finding has far reaching implications for the understanding and clinical treatment of COVID-19.

microbiology↗

bioRxiv2020

DNA damage activates mTORC1 signaling in podocytes leading to glomerulosclerosis

DNA repair is essential for preserving genome integrity and ensuring cellular functionality and survival. Podocytes, post-mitotic glomerular epithelial cells, bear limited regenerative capacity, and their survival is indispensable to maintain the function of the kidneys filtration units. While podocyte depletion is a hallmark of the aging process and of many proteinuric kidney diseases, the underlying factors remain unclear. We investigated DNA repair in podocyte diseases by using a constitutive and an inducible podocyte-specific knockout mouse model for Ercc1, a multifunctional endonuclease cofactor involved in nucleotide excision repair (NER), interstrand crosslink (ICL) repair, and DNA double-strand break (DSB) repair. We assessed the consequences of Ercc1 loss in vivo, complemented by mechanistical in vitro studies of induced DNA damage in cultured podocytes. Furthermore, we characterized DNA damage-related alterations in mouse and human renal tissue of different ages as well as in patient biopsies with minimal change disease and focal segmental glomerulosclerosis. Podocyte-specific Ercc1 knockout resulted in accumulation of DNA damage with ensuing proteinuria, podocyte loss, glomerulosclerosis, renal insufficiency, and reduced lifespan. The response to genomic stress was different to the pattern reported in other cell types, as podocytes activated mTORC1 signaling upon DNA damage in vitro and in vivo. The induced mTORC1 activation was abrogated by inhibiting DNA damage response through DNA-PK and ATM kinases in vitro. Moreover, pharmacological inhibition of mTORC1 modulated the development of glomerulosclerosis in Ercc1-deficient mice. Perturbed DNA repair gene expression and genomic stress was also detected in podocytes of human focal segmental glomerulosclerosis, characterized by podocyte loss. Beyond that, DNA damage accumulation occurred in podocytes of healthy aging mice and humans. These findings reveal that genome maintenance is crucial for podocyte maintenance, linked to the mTORC1 pathway, and involved in the aging process as well as in the development of glomerulosclerosis, potentially serving as a therapeutic target in the future.

cell biology↗

bioRxiv2020

Alpha-ketoglutarate inhibits Regulatory T cell differentiation by coupling lipidome remodelling to mitochondrial metabolism

The differentiation of CD4 T cells to a specific effector fate is metabolically regulated, integrating glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) with transcriptional and epigenetic changes. OXPHOS is tightly coordinated with the tricarboxylic acid (TCA) cycle but the precise role of TCA intermediates in CD4 T cell differentiation remain unclear. Here we demonstrate that -ketoglutarate (KG) inhibited regulatory T cell (Treg) generation while conversely, increasing Th1 polarization. In accord with these data, KG promoted the effector profile of Treg-polarized chimeric antigen receptor-engineered T cells against the ErbB2 tumor antigen. Mechanistically, KG significantly altered transcripts of genes involved in lipid-related processes, inducing a robust lipidome-wide remodelling and decreased membrane fluidity. A massive increase in storage and mitochondria lipids was associated with expression of mitochondrial genes and a significantly augmented OXPHOS. Notably, inhibition of succinate dehydrogenase activity, the bridge between the TCA cycle and the electron transport chain, enforced Treg generation. Thus, our study identifies novel connections between KG, lipidome remodelling and OXPHOS in CD4 T cell fate decisions.

immunology↗

bioRxiv2020

Post-transcriptional regulation of human endogenous retroviruses by RNA-Binding Motif Protein 4, RBM4

The human genome encodes for over 1,500 RNA-binding proteins (RBPs), which coordinate regulatory events on RNA transcripts (Gerstberger et al., 2014). Most studies of RBPs concentrate on their action on mRNAs that encode protein, which constitute a minority of the transcriptome. A widely neglected subset of our transcriptome derives from integrated retroviral elements termed endogenous retroviruses (ERVs) that comprise ~8% of the human genome. Some ERVs have been shown to be transcribed under physiological and pathological conditions suggesting that sophisticated regulatory mechanisms to coordinate and prevent their ectopic expression exist. However, it is unknown whether RBPs and ERV transcripts directly interact to provide a post-transcriptional layer of regulation. Here, we implemented a computational pipeline to determine the correlation of expression between individual RBPs and ERVs from single-cell or bulk RNA sequencing data. One of our top candidates for an RBP negatively regulating ERV expression was RNA-Binding Motif Protein 4 (RBM4). We used PAR-CLIP to demonstrate that RBM4 indeed bound ERV transcripts at CGG consensus elements. Loss of RBM4 resulted in elevated transcript level of bound ERVs of the HERV-K and -H families, as well as increased expression of HERV-K envelope protein. We pinpointed RBM4 regulation of HERV-K to a CGG-containing element that is conserved in the long terminal repeats (LTRs) of HERV-K-10 and -K-11, and validated the functionality of this site using reporter assays. In summary, we identified RBPs as potential regulators of ERV function and demonstrate a new role for RBM4 in controlling ERV expression. Significance StatementThe expression of endogenous retroviruses (ERVs) appears to have broad impact on human biology. Nevertheless, only a handful of transcriptional regulators of ERV expression are known and to our knowledge no RNA-binding proteins (RBPs) were yet implicated in positive or negative post-transcriptional regulation of ERVs. We implemented a computational pipeline that allowed us to identify RBPs that modulate ERV expression levels. Experimental validation of one of the prime candidates we identified, RBM4, showed that it indeed bound RNAs made from ERVs and negatively regulated the levels of those RNAs. We hereby identify a new layer of ERV regulation by RBPs.

systems biology↗

bioRxiv2020

An Image-Based Data-Driven Analysis of Cellular Architecture in a Developing Tissue

Quantitative microscopy is becoming increasingly crucial in efforts to disentangle the complexity of organogenesis, yet adoption of the potent and ever-growing toolbox provided by modern data science has been slow, primarily because data-driven tools focus on "big data" whereas microscopy produces "rich data". We tackle this issue using a newly developed algorithm for point cloud-based morphometry to unpack the rich information encoded in high-resolution 3D image data into a straightforward numerical representation. This allowed us to employ machine learning for multi-modal data integration of cell morphology, intracellular organization, gene expression and annotated contextual knowledge that facilitates data exploration. We apply these techniques to construct and explore a quantitative atlas of cellular architecture for the zebrafish posterior lateral line primordium, an experimentally tractable model of complex self-organized organogenesis. In doing so, we are able to retrieve both previously established and novel biologically relevant patterns, demonstrating the potential of our data-driven approach.

developmental biology↗

bioRxiv2019

A Holistic Analysis of the Intestinal Stem Cell Niche Network

Although many studies into the intestinal stem cell (ISC) niche have been carried out, they have focused on the role of a single cell type or molecular signal. However, no holistic comparisons of the predominant cell types and signals present within the intestinal mucosa have been conducted to date. We utilize bulk RNA sequencing to profile 20 different mucosal cell types covering four major cell categories: epithelial, stromal, endothelial and immune. We further examined the stromal signaling environment using scRNAseq to provide a more comprehensive view of the signaling microenvironment within the intestinal mucosa. We identified the primary signals for the major ISC regulatory pathways and their respective cellular sources. Our analysis suggests that a niche network exists, with no single cell type being responsible for ISC self-renewal, proliferation, or differentiation; rather, each cell type within the network carries out specific functions in a highly cooperative and coordinated manner.

cell biology↗

bioRxiv2019

Gradual decrease in the inter-population diversity of HIV-1 Env among group M lineages worldwide

HIV-1 group M was transmitted to humans nearly one century ago. The virus has since diversified to form distinct clades, which spread to multiple regions worldwide. Of the different proteins encoded by HIV-1, the envelope glycoproteins (Envs) have diversified most rapidly in all infected populations. We compared the range of variants that emerged during the AIDS pandemic in diverse HIV-1 clades and distinct geographic regions. Our analyses focused on two components of Env that contain multiple epitopes of broadly-neutralizing antibodies: the glycan shield and apex domain. Interestingly, at each Env position, the amino acid in the inferred clade ancestor was replaced by a unique combination of emerging variants. Key antigenic sites and genetic signatures of vaccine protection have gradually evolved toward conserved frequency distributions (FDs) of all amino acids. FDs are specific for position and clade and are highly conserved in populations from different regions. Remarkably, founder effects of Env mutations in distinct clades and recently-infected regions were significantly reduced during the epidemic by evolution of each site toward the position-specific FD. These findings suggest that the selective pressures that guide evolution of Env are conserved in different populations. They are sufficiently strong to reduce founder effects at the clade and regional levels and have significantly altered the distribution of Env forms that circulate worldwide. Consequently, the intra-population diversity of the Env protein continues to increase whereas the inter-population diversity is gradually decreasing.\n\nImportanceThe Env protein of HIV-1 is the primary target in AIDS vaccine design. Due to frequent mutations, new Env variants continuously emerge in the population. The increasing number of Env forms and apparent randomness of the changes limit our ability to design broadly-effective vaccines. We examined the populations-level changes that occurred in Env during the AIDS epidemic. Each position of the molecule has evolved toward a specific combination of amino acids. Similar changes occurred in different HIV-1 subtypes and geographic regions toward the same sets of forms, often from distinct ancestral sequences. Such conserved patterns of evolution define a new framework for designing vaccines that are tailored to the unique combination of Env variants expected to circulate in each population.

evolutionary biology↗

bioRxiv2019

PGRMC1 phosphorylation status and cell plasticity 2: genomic integrity and CpG methylation

Progesterone receptor membrane component 1 (PGRMC1) is often elevated in cancers, and exists in alternative states of phosphorylation. A motif centered on PGRMC1 Y180 was evolutionarily acquired concurrently with the embryological gastrulation organizer that orchestrates vertebrate tissue differentiation. Here, we show that mutagenic manipulation of PGRMC1 phosphorylation alters cell metabolism, genomic stability, and CpG methylation. Each of several mutants elicited distinct patterns of genomic CpG methylation. Mutation of S57A/Y180/S181A led to increased net hypermethylation, reminiscent of embryonic stem cells. Pathways enrichment analysis suggested modulation of processes related to animal cell differentiation status and tissue identity, as well as cell cycle control and ATM/ATR DNA damage repair regulation. We detected different genomic mutation rates in culture. A companion manuscript shows that these cell states dramatically affect protein abundances, cell and mitochondrial morphology, and glycolytic metabolism. We propose that PGRMC1 phosphorylation status modulates cellular plasticity mechanisms relevant to early embryological tissue differentiation.

cell biology↗

bioRxiv2019

Isolation of muscle stem cells from rat skeletal muscles

BackgroundMuscle stem cells (MuSCs) are involved in homeostatic maintenance of skeletal muscles and play a central role in muscle regeneration in response to injury. Thus, understanding MuSC autonomous properties is of fundamental importance for studies of muscle degenerative diseases and muscle plasticity. Rat, as an animal model, has been widely used in the skeletal muscle field, however an efficient approach for MuSC isolation through fluorescence-activated cell sorting from rat muscles has never been described. This work aims to develop and validate an effective protocol for MuSC isolation from rat skeletal muscles.\n\nMethodsTibialis anterior, gastrocnemius, diaphragm, and the individual components of the pelvic floor muscle complex (coccygeus, iliocaudalis, and pubocaudalis) were harvested from female rats and digested for isolation of MuSCs. Three protocols, employing different cell surface markers (CD106, CD56, and CD29), were compared for their ability to isolate a pure MuSC population.\n\nResultsCells obtained using the protocol that relies only on VCAM-1 (CD106) as a positive marker showed high expression of Pax7 upon isolation, ability to progress through myogenic lineage while in culture, and complete differentiation in serum deprived conditions. The protocol was further validated in other skeletal muscles proving to be reproducible.\n\nConclusionsCD106 is an efficient marker for reliable isolation of MuSCs from a variety of rat skeletal muscles.

cell biology↗

bioRxiv2019

Polycomb contraction differentially regulates terminal human hematopoietic differentiation programs

Lifelong production of the many types of mature blood cells from less differentiated progenitors is a hierarchically ordered process that spans multiple cell divisions. The nature and timing of the molecular events required to integrate the environmental signals, transcription factor activity, epigenetic modifications, and changes in gene expression involved are thus complex and still poorly understood. We now show that the more primitive types of human cells in this system display a unique repressive H3K27me3 signature that is retained by mature lymphoid cells but is lost in terminally differentiated monocytes and erythroblasts. Additional intervention data implicate that control of this chromatin state change is a requisite part of the process, whereby normal human hematopoietic progenitor cells make lymphoid and myeloid fate decisions.

cell biology↗

bioRxiv2019

Lifetime changes in CD4 count, viral load suppression and adherence among adolescents living with HIV in urban Peru

IntroductionViral load suppression and adherence to combined antiretroviral therapy (cART) have been shown to be lower in adolescents than in other age groups; however, this relationship has not been documented longitudinally from childhood to adolescence and has rarely been examined outside of high-resource settings and sub-Saharan Africa. To address this knowledge gap, we quantified longitudinal changes in CD4 cell count, viral load suppression, and cART adherence in adolescents living with HIV in urban, Peru.\n\nMethodsWe conducted a retrospective chart review among adolescents ages 10-18 years on cART and receiving care at a large, public sector pediatric hospital as of December 2015. We abstracted clinical notes indicating nonadherence and viral load and CD4 counts from childhood to adolescence. We modeled the association between age and each outcome with restricted cubic splines accounting for multiple observations per person, and graphed study outcomes by age.\n\nResultsA median of 7.7 years (25th percentile=4.9, 75th percentile=10.2) of follow up were observed for 128 adolescents. Nearly 70% of patients had at least one nonadherence note and the proportion with nonadherence increased log-linearly with age (p<0.0001). The peak proportion with viral load suppression was 84% (95% CI: 79, 88) at age 13, which dropped to 67% (95% CI: 47, 83) by age 18. Mean CD4 count decreased at age 13, dropping from 723 cells/mm3 (95% CI: 666, 784) to 429 cells/mm3 (95% CI: 356, 517) by age 18.\n\nConclusionThis is the first report from Latin America to examine longitudinal changes in HIV outcomes from childhood into adolescence. Consistent with the limited evidence from other settings, decreases in viral load suppression and mean CD4 count occurred in early adolescence in tandem with increases in nonadherence. Adolescent-friendly cART adherence support interventions to target this critical period are urgently needed.

epidemiology↗

bioRxiv2019

WNT/β-CATENIN modulates the axial identity of ES derived human neural crest

The WNT/{beta}-CATENIN pathway is critical for neural crest (NC) formation. However, the effects of the magnitude of the signal remains poorly defined. Here we evaluate the consequences of WNT magnitude variation in a robust model of human NC formation. This model is based on human embryonic stem cells induced by WNT signaling through the small molecule CHIR9902. In addition to its known effect on NC formation, we find that the WNT signal modulates the anterior-posterior axial identity of NCCs in a dose dependent manner, with low WNT leading to anterior OTX+, HOX-NC, and high WNT leading to posterior OTX-, HOX+ NC. Differentiation tests of posterior NC confirm expected derivatives including posterior specific adrenal derivatives, and display partial capacity to generate anterior ectomesenchymal derivatives. Furthermore, unlike anterior NC, posterior NC transit through a TBXT+/SOX2+ neuromesodermal precursor-like intermediate. Finally, we analyze the contributions of other signaling pathways in posterior NC formation, and suggest a critical role for FGF in survival/proliferation, and a requirement of BMP for NC maturation. As expected RA and FGF are able to modulate HOX expression in the posterior NC, but surprisingly, RA supplementation prohibits anterior, but only reduces, posterior NC formation. This work reveals for the first time that the amplitude of WNT signaling can modulate the axial identity of NC cells in humans.

developmental biology↗

bioRxiv2018

Mechanobiological Conditioning of Mesenchymal Stem Cells Enhances Therapeutic Angiogenesis by Inducing a Hybrid Pericyte-Endothelial Phenotype

Stem cell therapies have great promise for revolutionizing treatments for cardiovascular disease and other disorders but have not yet achieved their potential due to poor efficacy and heterogeneity in patient response. Here, we used a novel high throughput screening system to optimize the conditioning of mesenchymal stem cells using a combinatorial set of biochemical factors, pharmacological inhibitors and biomechanical forces. Our studies revealed that a combination of specific kinase inhibitors and a complex mechanical strain waveform dramatically increased the population of mesenchymal stem cells that express markers for both pericytes and endothelial cells. These mechanically and pharmacologically conditioned mesenchymal stem cells had superior properties in enhancing endothelial tube formation, production of angiogenic growth factors and induction of angiogenesis following implantation. Overall, our work supports that combinatorial optimization of mechanical conditioning and pharmacological treatments can significantly enhance the regenerative properties of mesenchymal stem cells.

bioengineering↗

bioRxiv2018

High Throughput Mechanobiological Screens Enable Mechanical Priming of Pluripotency in Mouse Fibroblasts

Transgenic methods for direct reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) are effective in cell culture systems but ultimately limit the utility of iPSCs due to concerns of mutagenesis and tumor formation. Recent studies have suggested that some transgenes can be eliminated by using small molecules as an alternative to transgenic methods of iPSC generation. We developed a high throughput platform for applying complex dynamic mechanical forces to cultured cells. Using this system, we screened for optimized conditions to stimulate the activation of Oct-4 and other transcription factors to prime the development of pluripotency in mouse fibroblasts. Using high throughput mechanobiological screening assays, we identified small molecules that can synergistically enhance the priming of pluripotency of mouse fibroblasts in combination with mechanical loading. Taken together, our findings demonstrate the ability of mechanical forces to induce reprograming factors and support that biophysical conditioning can act cooperatively with small molecules to priming the induction pluripotency in somatic cells.

bioengineering↗

bioRxiv2018

The double tubular contractile structure of the type VI secretion system displays striking flexibility and elasticity

The double tubular structure of the type VI secretion system (T6SS) is considered as one of the longest straight and rigid intracellular structures in bacterial cells. Contraction of the T6SS outer sheath occurs almost instantly and releases sufficient power to inject the inner needle-like Hcp tube and its associated effectors into target bacterial cells through piercing the stiff cell envelope. The molecular mechanism triggering T6SS contraction remains elusive. Here we report that the double tubular T6SS structure is strikingly flexible and elastic, forming U-, circular-, or S-shapes while maintaining functional for contraction and substrate delivery. We show that physical contact with cytoplasmic membrane induced a range of T6SS structure deformation, but the resultant mechanical pressing force on the T6SS baseplate did not trigger contraction. Our results also reveal a stalling intermediate stage of sheath-tube extension following which the structure contracts or resumes to extend. These observations suggest that the recruitment equilibrium of sheath-tube precursors to the extending structure is key to stability/contraction and lead us to propose a model of T6SS contraction, termed ESCAPE (extension-stall-contraction and precursor equilibrium). Our data highlight the remarkable flexibility of the double tubular T6SS structure and its length control mechanism distinct from the other evolutionarily related contractile cell-puncturing systems.

microbiology↗