Biology subjects

Liang, R.

Publications and source records attributed to Liang, R..

50 records · Page 3Linked to original sources

bioRxiv2022

Elevated p21 (CDKN1a) mediates β-thalassemia erythroid apoptosis but its loss does not improve β-thalassemic erythropoiesis

{beta}-thalassemias are common hemoglobinopathies due to mutations in the {beta}-globin gene that lead to hemolytic anemias. Premature death of {beta}-thalassemic erythroid precursors results in ineffective erythroid maturation, increased production of erythropoietin (Epo), expansion of erythroid progenitor compartment, extramedullary erythropoiesis and splenomegaly. However, the molecular mechanism of erythroid apoptosis in {beta}-thalassemia is not well understood. Using a mouse model of {beta}-thalassemia (Hbbth3/+), we show that dysregulated expression of Foxo3 transcription factor and its upstream pro-apoptotic regulator TP53 is implicated in {beta}-thalassemia erythroid apoptosis. In Foxo3-/- /Hbbth3/+ mice, erythroid apoptosis is significantly reduced while erythroid cell maturation, red blood cell and hemoglobin production are substantially improved. However, persistence of elevated reticulocytes and splenomegaly suggests that ineffective erythropoiesis is not resolved in Foxo3-/-/Hbbth3/+. We next focused on cell cycle inhibitor Cdkn1a (p21) and show that p21 that is a target of both Foxo3 and TP53 is markedly upregulated in both mouse and patients-derived {beta}-thalassemic erythroid precursors. To address the contribution of p21 to {beta}-thalassemia pathophysiology, we generated p21-/- /Hbbth3/+ mice. Double mutant p21/Hbbth3/+ mice exhibited embryonic lethality with only a fraction of mice surviving to weaning. Notably, studies in adult mice showed apoptosis and circulating Epo were greatly reduced in erythroid compartments of surviving p21-/- /Hbbth3/+ relative to Hbbth3/+ mice, while ineffective erythroid cell maturation, extramedullary erythropoiesis and splenomegaly were not modified. These combined results indicate that while lack of Foxo3 reduces apoptosis and improves anemia, diminished p21-mediated apoptosis is insufficient to improve red blood cell production in Hbbth3/+ mice. They also suggest that a molecular network constituted by p21, FOXO3 and TP53, control erythroid cell survival and differentiation in {beta}-thalassemia. Overall, these studies provide a new framework for investigating ineffective erythropoiesis in {beta}-thalassemia. Key PointsO_LIElevated p21 mediates {beta}-thalassemia erythroid cell apoptosis C_LIO_LILoss of Foxo3 or p21 reduces {beta}-thalassemia erythroid cell apoptosis but does not improve {beta}-thalassemia ineffective erythropoiesis C_LIO_LIA network of Foxo3, p21 and TP53 controls {beta}-thalassemia erythroid apoptosis C_LIO_LIApoptosis may be uncoupled from ineffective erythropoiesis in {beta}-thalassemia C_LI

developmental biology↗

bioRxiv2022

Identification of cell types in multiplexed in situ images by combining protein expression and spatial information using CELESTA reveals novel spatial biology

Advances in multiplexed in situ imaging are revealing important insights in spatial biology. However, cell type identification remains a major challenge in imaging analysis, with most existing methods involving substantial manual assessment and subjective decisions for thousands of cells. We propose a novel machine learning algorithm, CELESTA, which uses both cells protein expression and spatial information to identify cell type of individual cells. We demonstrate the performance of CELESTA on multiplexed immunofluorescence in situ images of colorectal cancer and head and neck cancer. Using the cell types identified by CELESTA, we identify tissue architecture associated with lymph node metastasis in HNSCC, which we validate in an independent cohort. By coupling our in situ spatial analysis with single-cell RNA-sequencing data on proximal sections of the same tissue specimens, we identify and validate cell-cell crosstalk associated with lymph node metastasis, demonstrating the power of spatial biology to reveal clinically-relevant cellular interactions.

bioinformatics↗

bioRxiv2022

The SARS-CoV-2 Omicron (B.1.1.529) variant exhibits altered pathogenicity, transmissibility, and fitness in the golden Syrian hamster model

The newly emerging SARS-CoV-2 Omicron (B.1.1.529) variant first identified in South Africa in November 2021 is characterized by an unusual number of amino acid mutations in its spike that renders existing vaccines and therapeutic monoclonal antibodies dramatically less effective. The in vivo pathogenicity, transmissibility, and fitness of this new Variant of Concerns are unknown. We investigated these virological attributes of the Omicron variant in comparison with those of the currently dominant Delta (B.1.617.2) variant in the golden Syrian hamster COVID-19 model. Omicron-infected hamsters developed significantly less body weight losses, clinical scores, respiratory tract viral burdens, cytokine/chemokine dysregulation, and tissue damages than Delta-infected hamsters. The Omicron and Delta variant were both highly transmissible (100% vs 100%) via contact transmission. Importantly, the Omicron variant consistently demonstrated about 10-20% higher transmissibility than the already-highly transmissible Delta variant in repeated non-contact transmission studies (overall: 30/36 vs 24/36, 83.3% vs 66.7%). The Delta variant displayed higher fitness advantage than the Omicron variant without selection pressure in both in vitro and in vivo competition models. However, this scenario drastically changed once immune selection pressure with neutralizing antibodies active against the Delta variant but poorly active against the Omicron variant were introduced, with the Omicron variant significantly outcompeting the Delta variant. Taken together, our findings demonstrated that while the Omicron variant is less pathogenic than the Delta variant, it is highly transmissible and can outcompete the Delta variant under immune selection pressure. Next-generation vaccines and antivirals effective against this new VOC are urgently needed. One Sentence SummaryThe novel SARS-CoV-2 Omicron variant, though less pathogenic, is highly transmissible and outcompetes the Delta variant under immune selection pressure in the golden Syrian hamster COVID-19 model.

microbiology↗

bioRxiv2021

Aldehyde dehydrogenase 3A1 deficiency leads to mitochondrial dysfunction and impacts salivary gland stem cell self-renewal, differentiation and survival

Adult salivary stem/progenitor cells (SSPC) have an intrinsic property to self-renew in order to maintain tissue architecture and homeostasis. Adult salivary glands have been documented to harbor SSPC, which have been shown to play a vital role in the regeneration of the glandular structures post radiation damage. We have previously demonstrated that activation of aldehyde dehydrogenase 3A1 (ALDH3A1) after radiation reduced aldehyde accumulation in SSPC, leading to less apoptosis and improved salivary function. We subsequently found that sustained pharmacological ALDH3A1 activation is critical to enhance regeneration of murine submandibular gland after radiation damage. Further investigation shows that ALDH3A1 function is crucial for SSPC self-renewal and differentiation even in the absence of radiation stress. Salivary glands from Aldh3a1-null mice have fewer acinar structures than wildtype mice. ALDH3A1 deletion or pharmacological inhibition in SSPC leads to a decrease in mitochondrial DNA copy number, lower expression of mitochondrial specific genes and proteins, structural abnormalities, lower membrane potential, and reduced cellular respiration. Loss or inhibition of ALDH3A1 also elevates ROS levels and accumulation of ALDH3A1 substrate 4-hydroxynonenal (4-HNE, a lipid peroxidation product), leading to decreased survival of murine SSPC that can be rescued by treatment with 4-HNE specific carbonyl scavengers. Our data indicate that ALDH3A1 activity protects mitochondrial function and is important for the development and regeneration activity of SSPC. This knowledge will help to guide our translational strategy of applying ALDH3A1 activators in the clinic to prevent radiation-related hyposalivation in head and neck cancer patients.

cell biology↗

bioRxiv2021

A chemical strategy toward novel brain-penetrant EZH2 inhibitors

Aberrant gene-silencing through dysregulation of polycomb protein activity has emerged as an important oncogenic mechanism in cancer, implicating polycomb proteins as important therapeutic targets. Recently, an inhibitor targeting EZH2, the methyltransferase component of PRC2, received FDA approval following promising clinical responses in cancer patients. However, the current array of EZH2 inhibitors have poor brain-penetrance limiting their use in patients with CNS malignancies, a number of which have been shown to be sensitive to EZH2 inhibition. To address this need, we have identified a chemical strategy, based on computational modeling of pyridone-containing EZH2 inhibitor scaffolds, to minimize P-glycoprotein activity and here we report the first brain-penetrant EZH2 inhibitor, TDI-6118 (compound 5). Additionally, in the course of our attempts to optimize this compound we discovered TDI-11904 (compound 21); a novel, highly-potent, and peripherally active EZH2 inhibitor based on a 7 member ring structure.

cancer biology↗

bioRxiv2021

HIV proviral burden, genetic diversity and dynamics in viremic controllers who subsequently initiated suppressive antiretroviral therapy

Curing HIV will require eliminating the reservoir of integrated, replication-competent proviruses that persist despite antiretroviral therapy (ART). Understanding the burden, genetic diversity and longevity of persisting proviruses in diverse individuals with HIV is critical to this goal, but these characteristics remain understudied in some groups. Among them are viremic controllers, individuals who naturally suppress HIV to low levels but for whom therapy is nevertheless recommended. We reconstructed within-host HIV evolutionary histories from longitudinal single-genome amplified viral sequences in four viremic controllers who eventually initiated ART, and used this information to characterize the age and diversity of proviruses persisting on therapy. We further leveraged these within-host proviral age distributions to estimate rates of proviral turnover prior to ART. This is an important yet understudied metric, since pre-ART proviral turnover dictates reservoir composition at ART initiation (and thereafter), which is when curative interventions, once developed, would be administered. Despite natural viremic control, all participants displayed significant within-host HIV evolution pre-therapy, where overall on-ART proviral burden and diversity broadly reflected the extent of viral replication and diversity pre-ART. Consistent with recent studies of non-controllers, the proviral pools of two participants were skewed towards sequences that integrated near ART initiation, suggesting dynamic proviral turnover during untreated infection. In contrast, proviruses recovered from the two other participants dated to time-points that were more evenly spread throughout infection, suggesting slow or negligible proviral decay following deposition. HIV cure strategies will need to overcome within-host proviral diversity, even in individuals who naturally controlled HIV replication before therapy. ImportanceHIV therapy is life-long because integrated, replication-competent viral copies persist within long-lived cells. To cure HIV, we need to understand when these viral reservoirs form, how large and genetically diverse they are, and how long they endure. Elite controllers, individuals who naturally suppress HIV to undetectable levels, are being intensely studied as models of HIV remission, but viremic controllers, individuals who naturally suppress HIV to low levels, remain understudied even though they too may hold valuable insights. We combined phylogenetics and mathematical modeling to reconstruct proviral seeding and decay from infection to therapy-mediated suppression in four viremic controllers. We recovered diverse proviruses persisting during therapy that broadly reflected HIVs within-host evolutionary history, where the estimated half-lives of the persistent proviral pool during untreated infection ranged from <1 year to negligible. Cure strategies will need to contend with proviral diversity and between-host heterogeneity, even in individuals who naturally control HIV.

microbiology↗

bioRxiv2021

Selective inhibition reveals the regulatory function of DYRK2 in protein synthesis and calcium entry

The dual-specificity tyrosine phosphorylation-regulated kinase DYRK2 has emerged as a key regulator of cellular processes such as proteasome-mediated protein degradation. To gain further insights into its function, we took a chemical biology approach and developed C17, a potent small-molecule DYRK2 inhibitor, through multiple rounds of structure-based optimization guided by a number of co-crystallized structures. C17 displayed an effect on DYRK2 at a single-digit nanomolar IC50 and showed outstanding selectivity for the human kinome containing 467 other human kinases. Using C17 as a chemical probe, we further performed quantitative phosphoproteomic assays and identified several novel DYRK2 targets, including eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and stromal interaction molecule 1 (STIM1). DYRK2 phosphorylated 4E-BP1 at multiple sites, and the combined treatment of C17 with AKT and MEK inhibitors showed synergistic 4E-BP1 phosphorylation suppression. The phosphorylation of STIM1 by DYRK2 substantially increased the interaction of STIM1 with the ORAI1 channel, and C17 impeded the store-operated calcium entry process. Collectively, these studies further expand our understanding of DYRK2 and provide a valuable tool to further pinpoint its biological function.

biochemistry↗

bioRxiv2021

Fibrillar Aβ causes profound microglial metabolic perturbations in a novel APP knock-in mouse model

Microglial dysfunction is believed to play a pathogenic role in Alzheimers disease (AD). Here, we characterize the amyloid-{beta} related pathology and microglial responses in an engineered APP knock-in mouse model of familial AD. This model recapitulates key pathological features of AD such as a progressive accumulation of parenchymal amyloid plaques and vascular amyloid deposits, altered glial responses and neurodegeneration. Leveraging multi-omics approaches, we found lipid accumulation and an exacerbated disease-associated transcriptomic response in methoxy-X04-positive, phagocytic microglia. Together, these findings highlight the potential of this novel, open-access mouse model to investigate AD pathogenesis and demonstrate that fibrillar A{beta} triggers lipid dysregulation and immuno-metabolic perturbations in phagocytic microglia. HighlightsO_LINovel open-access APP KI mouse model shows salient AD pathological features C_LIO_LIDeep phenotyping of sorted microglia reveals profound lipidomic perturbations in line with Alois Alzheimers original descriptions of glial adipose inclusions C_LIO_LIImmunometabolic perturbations are exacerbated in microglia accumulating fibrillar A{beta} C_LI

neuroscience↗

bioRxiv2020

3D-Visualization of Amyloid-β Oligomer and Fibril Interactions with Lipid Membranes by Cryo-Electron Tomography

Amyloid-{beta} (A{beta}) monomers assemble into mature fibrils via a range of metastable oligomeric and protofibrillar intermediates. These A{beta} assemblies have been shown to bind to lipid bilayers. This can disrupt membrane integrity and cause a loss of cellular homeostasis, that triggers a cascade of events leading to Alzheimers disease. However, molecular mechanisms of A{beta} cytotoxicity and how the different assembly forms interact with the membrane remain enigmatic. Here we use cryo-electron tomography (cryoET) to obtain three-dimensional nano-scale images of various A{beta} assembly types and their interaction with liposomes. A{beta} oligomers bind extensively to the lipid vesicles, inserting and carpeting the upper-leaflet of the bilayer. Furthermore, curvilinear protofibrils also insert into the bilayer, orthogonally to the membrane surface. A{beta} oligomers concentrate at the interface of vesicles and form a network of A{beta}-linked liposomes. While crucially, monomeric and fibrillar A{beta} have relatively little impact on the membrane. Changes to lipid membrane composition highlights a significant role for GM1-ganglioside in promoting A{beta}-membrane interactions. The different effects of A{beta} assembly forms observed align with the highlighted cytotoxicity reported for A{beta} oligomers. The wide-scale incorporation of A{beta} oligomers and curvilinear protofibrils into the lipid bilayer suggests a mechanism by which membrane integrity is lost.

biophysics↗

bioRxiv2020

Alk1 mutant endothelial cells undergo clonal expansion in mouse brain arteriovenous malformations

RationaleMutation in human arteriovenous malformation (AVM) causative genes in a fraction of endothelial cells (ECs) causes AVMs in mice. It is unclear how a small number of mutant ECs can lead to AVM formation. ObjectiveTo understand how a fraction of mutant ECs causes AVM, we tested the following hypotheses: (1) activin receptor-like kinase 1 (Alk1 or Acvlr1) mutant brain ECs undergo clonal expansion upon angiogenic stimulation, (2) Alk1 mutant ECs display growth advantage, (3) the burden of Alk1 mutant ECs correlates with AVM severity, and (4) Alk1 mutant bone marrow (BM) derived ECs alone is sufficient to cause AVM. Methods and ResultsWe used PdgfbiCreER;Alk1f/f;confetti+/- mice which express an EC-specific tamoxifen (TM)-inducible Cre recombinase, a Cre-regulated confetti transgene, and Alk1 floxed alleles. Brain AVMs were induced by direct brain injection of an adeno-associated viral vector expressing vascular endothelial growth factor (AAV-VEGF) followed with intra-peritoneal injection of TM two weeks later. Color-predominance of confetti reporter in AVMs compared to control brain ECs suggested that clonal expansion was associated with AVM development. We treated PdgfbiCreER;Alk1f/f with different doses of TM to create a mosaic of wild-type (WT) and mutant ECs and found that equal numbers of Alk1+ and Alk1- ECs were proliferating. Increase of TM dose increased the number of Alk1- ECs, the abnormal vessels in brain AVMs, the number of arteriovenous shunts in the intestines, and mouse mortality. To test if mutation of Alk1 in BM-derived ECs can cause brain AVM, we transplanted WT mice with BM of PdgfbiCreER;Alk1f/f mice. After AAV-VEGF and TM treatment, these mice developed AVMs in their brains and arteriovenous shunts in their intestines. ConclusionClonal expansion of Alk1 mutant ECs could partly explain why a fraction of mutant ECs causes AVM. Mutation of AVM causal genes in BM-derived ECs is sufficient to cause AVM formation.

cell biology↗

bioRxiv2020

Limited Environmental Serine Confers Sensitivity to PHGDH Inhibition in Brain Metastasis

A hallmark of metastasis is the adaptation of tumor cells to new environments. Although it is well established that the metabolic milieu of the brain is severely deprived of nutrients, particularly the amino acids serine and its catabolite glycine, how brain metastases rewire their metabolism to survive in the nutrient-limited environment of the brain is poorly understood. Here we demonstrate that cell-intrinsic de novo serine synthesis is a major determinant of brain metastasis. Whole proteome comparison of triple-negative breast cancer (TNBC) cells that differ in their capacity to colonize the brain reveals that 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the rate-limiting step of glucose-derived serine synthesis, is the most significantly upregulated protein in cells that efficiently metastasize to the brain. Genetic silencing or pharmacological inhibition of PHGDH attenuated brain metastasis and improved overall survival in mice, whereas expression of catalytically active PHGDH in a non-brain trophic cell line promoted brain metastasis. Collectively, these findings indicate that nutrient availability determines serine synthesis pathway dependence in brain metastasis, and suggest that PHGDH inhibitors may be useful in the treatment of patients with cancers that have spread to the brain. Statement of SignificanceOur study highlights how limited serine and glycine availability within the brain microenvironment potentiates tumor cell sensitivity to serine synthesis inhibition. This finding underscores the importance of studying cancer metabolism in physiologically-relevant contexts, and provides a rationale for using PHGDH inhibitors to treat brain metastasis.

cancer biology↗

bioRxiv2018

Life Inside A Dinosaur Bone: A Thriving Microbiome

Fossils were long thought to lack original organic material, but the discovery of organic molecules in fossils and sub-fossils, thousands to millions of years old, has demonstrated the potential of fossil organics to provide radical new insights into the fossil record. How long different organics can persist remains unclear, however. Non-avian dinosaur bone has been hypothesised to preserve endogenous organics including collagen, osteocytes, and blood vessels, but proteins and labile lipids are unstable during diagenesis or over long periods of time. Furthermore, bone is porous and an open system, allowing microbial and organic flux. Some of these organics within fossil bone have therefore been identified as either contamination or microbial biofilm, rather than original organics. Here, we use biological and chemical analyses of Late Cretaceous dinosaur bones and sediment matrix to show that dinosaur bone hosts a diverse microbiome. Fossils and matrix were freshly-excavated, aseptically-acquired, and then analysed using microscopy, spectroscopy, chromatography, spectrometry, DNA extraction, and 16S rRNA amplicon sequencing. The fossil organics differ from modern bone collagen chemically and structurally. A key finding is that 16S rRNA amplicon sequencing reveals that the subterranean fossil bones host a unique, living microbiome distinct from that of the surrounding sediment. Even in the subsurface, dinosaur bone is biologically active and behaves as an open system, attracting microbes that might alter original organics or complicate the identification of original organics. These results suggest caution regarding claims of dinosaur bone soft tissue preservation and illustrate a potential role for microbial communities in post-burial taphonomy.

paleontology↗

bioRxiv2018

Molecular Mechanisms Governing Shade Responses in Maize

Light is one of the most important environmental factors affecting plant growth and development. Plants use shade avoidance and shade tolerance strategies to adjust their growth and development thus increase their success in the competition for incoming light. To investigate the mechanism of shade responses in maize (Zea mays), we examined the anatomical and transcriptional dynamics of the early shade response in seedlings of the B73 inbred line. Transcriptome analysis identified 912 differentially expressed genes, including genes involved in light signaling, auxin responses, and cell elongation pathways. Grouping transcription factor family genes and performing enrichment analysis identified multiple types of transcription factors that are differentially regulated by shade and predicted putative core genes responsible for regulating shade avoidance syndrome. For functional tests, we ectopically over-expressed ZmHB53, a type II HD-ZIP transcription factor gene significantly induced by shade, in Arabidopsis thaliana. Transgenic Arabidopsis plants overexpressing ZmHB53 exhibited narrower leaves, earlier flowering, and enhanced expression of shade-responsive genes, suggesting that ZmHB53 participates in the regulation of shade responses in maize. This study increases our understanding of the regulatory network of the shade response in maize and provides a useful resource for maize genetics and breeding.\n\nHighlightOur findings not only increase the understanding of the regulatory network of the shade avoidance in maize, and also provide a useful resource for maize genetics and breeding.

plant biology↗

bioRxiv2018

Chronic estrus disrupts uterine gland development and homeostasis

Female mice homozygous for an engineered Gnrhr E90K mutation have reduced gonadotropin-releasing hormone signaling, leading to infertility. Their ovaries have numerous antral follicles but no corpora lutea, indicating a block to ovulation. These mutants have high levels of circulating estradiol and low progesterone, indicating a state of persistent estrus. This mouse model provided a unique opportunity to examine the lack of cyclic levels of ovarian hormones on uterine gland biology. Although uterine gland development appeared similar to controls during prepubertal development, it was compromised during adolescence in the mutants. By 20 weeks of age, uterine gland development was comparable to controls, but pathologies, including squamous neoplasia, tubal neoplasia, and cribriform glandular structures, were observed. Induction of ovulations by periodic human chorionic gonadotropin treatment did not rescue post-pubertal uterine gland development. Interestingly, progesterone receptor knockout mice, which lack progesterone signaling, also have defects in post-pubertal uterine gland development. However, progesterone treatment did not rescue post-pubertal uterine gland development. These studies indicate that chronically elevated levels of estradiol with low progesterone and therefore an absence of cyclic ovarian hormone secretion disrupts post-pubertal uterine gland development and homeostasis.

developmental biology↗