Biology subjects

Lin, S.

Publications and source records attributed to Lin, S..

At least 199 records · Page 11Linked to original sources

bioRxiv2021

Distinct and additive effects of calorie restriction and rapamycin in aging skeletal muscle

As global life expectancy continues to climb, maintaining skeletal muscle function is increasingly essential to ensure a good life quality for aging populations. Calorie restriction (CR) is the most potent and reproducible intervention to extend health and lifespan, but is largely unachievable in humans. Therefore, identification of "CR mimetics" has received much attention. CR targets nutrient-sensing pathways centering on mTORC1. The mTORC1 inhibitor, rapamycin, has been proposed as a potential CR mimetic and is proven to counteract age-related muscle loss. Therefore, we tested whether rapamycin acts via similar mechanisms as CR to slow muscle aging. Contrary to our expectation, long-term CR and rapamycin-treated geriatric mice display distinct skeletal muscle gene expression profiles despite both conferring benefits to aging skeletal muscle. Furthermore, CR improved muscle integrity in a mouse with nutrient-insensitive sustained muscle mTORC1 activity and rapamycin provided additive benefits to CR in aging mouse muscles. Therefore, RM and CR exert distinct, compounding effects in aging skeletal muscle, opening the possibility of parallel interventions to counteract muscle aging.

physiology↗

bioRxiv2021

Redundant and specific roles of EGFR ligands in the ERK activation waves during collective cell migration of MDCK cells

Epidermal growth factor receptor (EGFR) plays a pivotal role in collective cell migration by mediating cell-to-cell propagation of extracellular signal-regulated kinase (ERK) activation. Here, we aimed to determine which EGFR ligands mediate the ERK activation waves by gene knockout. Four of the seven known EGFR ligands are expressed in MDCK cells. We found that epidermal growth factor (EGF)-deficient cells exhibited lower basal ERK activity than the cells deficient in heparin-binding EGF (HBEGF), transforming growth factor alpha (TGF) or epiregulin (EREG), but all cell lines deficient in a single EGFR ligand retained the ERK activation waves. Therefore, we knocked out the EGFR ligand genes in decreasing order of expression. ERK activation waves were markedly suppressed, albeit incompletely, only when all four EGFR ligands were knocked-out. Re-expression of the EGFR ligands revealed that all but HBEGF could restore the ERK activation waves. Aiming at complete elimination of the ERK activation waves, we further attempted to knockout Nrg1, a ligand for ErbB3 and ErbB4, and found that Nrg1 deficiency induced growth arrest in the absence of all four EGFR ligand genes expressed in MDCK cells. Collectively, these results showed that EGFR ligands exhibit remarkable redundancy in the propagation of ERK activation waves during collective cell migration.

cell biology↗

bioRxiv2021

Transcriptional landscape of human microglia reveals robust gene expression signatures that implicates age, sex and APOE-related immunometabolic pathway perturbations

Microglia have fundamental roles in health and disease, however effects of age, sex and genetic factors on human microglia have not been fully explored. We applied bulk and single cell approaches to comprehensively characterize human microglia transcriptomes and their associations with age, sex and APOE. We identified a novel microglial signature, characterized its expression in bulk tissue and single cell microglia transcriptomes. We discovered microglial co-expression network modules associated with age, sex and APOE-{varepsilon}4 that are enriched for lipid and carbohydrate metabolism genes. Integrated analyses of modules with single cell transcriptomes revealed significant overlap between age-associated module genes and both pro-inflammatory and disease-associated microglial clusters. These modules and clusters harbor known neurodegenerative disease genes including APOE, PLCG2 and BIN1. Meta-analyses with published bulk and single cell microglial datasets further supported our findings. Thus, these data represent a well-characterized human microglial transcriptome resource; and highlight age, sex and APOE-related microglial immunometabolism perturbations with potential relevance in neurodegeneration.

genetics↗

bioRxiv2021

The Mediator tail module cooperates with proneural factors to determine fate identity in Glioblastoma.

Glioblastoma stem cells (GSCs) exhibit latent neuronal lineage differentiation potential governed by the proneural transcription factor Achaete-scute homolog 1 (ASCL1) and harnessing and promoting terminal neuronal differentiation has been proposed as a novel therapeutic strategy. Here, using a genome-wide CRISPR suppressor screen we identified genes required for ASCL1-mediated neuronal differentiation. This approach revealed a specialized function of the Mediator complex tail module and of its subunits MED24 and MED25 for this process in GSCs, human fetal neural stem cells and pluripotent stem cells. We show that upon induction of neuronal differentiation MED25 is recruited to genomic loci co-occupied by ASCL1 to regulate neurogenic gene expression programs. MED24 and MED25 are sufficient to induce neuronal differentiation in GSC cultures and to mediate neuronal differentiation in multiple contexts. Collectively our data expand our understanding of the mechanisms underlying directed terminal neuronal differentiation in brain tumor stem cells and point to a unique function of the Mediator tail in neuronal reprogramming.

cell biology↗

bioRxiv2021

Fixational Eye Movements Depend on Task and Target

Human fixational eye movements are so small and precise that they require high-speed, accurate tools to fully reveal their properties and functional roles. Where the fixated image lands on the retina and how it moves for different levels of visually demanding tasks is the subject of the current study. An Adaptive Optics Scanning Laser Ophthalmoscope (AOSLO) was used to image, track and present Maltese cross, disk, concentric circles, Vernier and tumbling-E letter fixation targets to healthy subjects. During these different passive (static) or active (discriminating) fixation tasks under natural eye motion, the landing position of the target on the retina was tracked in space and time over the retinal image directly. We computed both the eye motion and the exact trajectory of the fixated targets motion over the retina. We confirmed that compared to passive fixation, active tasks elicited a partial inhibition of microsaccades, leading to longer drifts periods compensated by larger corrective saccades. Consequently the fixation stability during active tasks was larger overall than during passive tasks. The preferred retinal locus of fixation was the same for each task and did not coincide with the location of the peak cone density.

neuroscience↗

bioRxiv2021

The Actin Bundling Protein Fascin Promotes Lung Cancer Growth and Metastasis by Enhancing Glycolysis and PFKFB3 Expression

Fascin is a pro-metastasis actin bundling protein upregulated in essentially all the metastatic carcinoma. It is believed that fascin promotes cancer cell migration and invasion by facilitating membrane protrusions such as filopodia and invadopodia. Aerobic glycolysis is a key feature of cancer metabolism that provides critical intermediate metabolites for tumor growth and cell proliferation. Here we report that fascin increase glycolysis in lung cancer to promote tumor growth and metastasis. Fascin promotes glycolytic flux by increasing the expression and activities of phosphofructose kinase 1 and 2 (PFK1 and 2). The glycolytic function of fascin depends on activation of YAP1 through its canonical actin bundling activity. Fascin promotes the binding of YAP1 to a TEAD1/4 binding motif located 30 bp upstream of the PFKFB3 transcription start site to activate its transcription. Our interrogation of the TCGA database suggest that the fascin-YAP1-PFKFB3 circuit is likely conserved across different types of cancer. We further showed that the glycolytic function of fascin is essential for promotion of lung cancer growth and metastasis. Importantly, pharmacological inhibitors of fascin could be used to suppress the YAP1-PFKFB3 signaling and inhibit glycolysis in cancer cell lines, organoid cultures and xenograft metastasis models. Taken together, our data reveal an important glycolytic role of fascin in lung cancer metabolism, and suggest that pharmacological inhibitors of fascin could be used to reprogram cancer metabolism in lung cancer and potentially other cancer with fascin upregulation.

cancer biology↗

bioRxiv2021

Bayestrat: Population Stratification Correction Using Bayesian Shrinkage Prior for Genetic Association Studies

In genetic association studies with common diseases, population stratification is a major source of confounding. Principle component regression (PCR) and linear mixed model (LMM) are two commonly used approaches to account for population stratification. Previous studies have shown that LMM can be interpreted as including all principle components (PCs) as random-effect covariates. However, including all PCs in LMM may inflate type I error in some scenarios due to redundancy, while including only a few pre-selected PCs in PCR may fail to fully capture the genetic diversity. Here, we propose a statistical method under the Bayesian framework, Bayestrat, that utilizes appropriate shrinkage priors to shrink the effects of non- or minimally confounded PCs and improve the identification of highly confounded ones. Simulation results show that Bayestrat consistently achieves lower type I error rates yet higher power, especially when the number of PCs included in the model is large. We also apply our method to two real datasets, the Dallas Heart Studies (DHS) and the Multi-Ethnic Study of Atherosclerosis (MESA), and demonstrate the superiority of Bayestrat over commonly used methods.

genetics↗

bioRxiv2021

Continuous Polony Gels for Tissue Mapping with High Resolution and RNA Capture Efficiency

Current technologies for acquiring spatial transcript information from tissue sections rely on either RNA probes or spatial barcodes. The former methods require a priori knowledge for probeset formulation; the latter have yet to achieve single cell resolution and/or transcript capture efficiencies approaching dissociative, single-cell methods. Here, we describe a novel spatial transcriptome assay called polony (or DNA cluster)-indexed library-sequencing (PIXEL-seq). It improves upon other spatial barcoding methods by employing "continuous" polony oligos arrayed across a customized gel surface. In terms of assay performance, PIXEL-seq attains [≤] 1 {micro}m resolution and captures >1,000 unique molecular identifiers/10x10 {micro}m2. In other words, this global, naive platform achieves subcellular spatial transcriptome mapping while maintaining high transcript capture efficiencies.

genomics↗

bioRxiv2021

A New Protocol for Targeted insertion using CRISPR-Cas9, Oligo Single-Stranded DNA and Protoplast Regeneration

Versatile genome editing can be facilitated by the insertion of DNA sequences into specific locations. Current protocols involving clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins rely on low efficiency homology-directed repair or non-homologous end joining with modified double-stranded DNA oligonucleotides as donors. Our simple protocol eliminates the need for expensive equipment, chemical and enzymatic donor DNA modification or plasmid construction by using polyethylene glycol-calcium to deliver non-modified single-stranded DNA oligonucleotides and CRISPR-Cas9 ribonucleoprotein into protoplasts. Plants regenerated via edited protoplasts achieved targeted insertion frequencies of up to 50.0% in Nicotiana benthamiana and 13.6% in rapid cycling Brassica oleracea without antibiotic selection. Using a 60-nt donor containing 27 nt in each homologous arm, 6 of 22 regenerated N. benthamiana plants showed targeted insertions, and one contained a precise insertion of a 6-bp HindIII site. The inserted sequences were transmitted to the next generation and invite the possibility of future exploration of versatile genome editing by targeted DNA insertion in plants.

plant biology↗

bioRxiv2021

Individual structural MRI abnormality indexes are associated with molecular,histological and cognitive indicators for frontal glioma patients

Although every glioma patient varies in tumor size, location, histological grade and molecular biomarkers, structural abnormalities are commonly conducted in a group-level, leading to the miss of individual structural atrophy. In this study, we introduced an individual-level structural abnormality detection method for glioma patients and proposed several novel abnormality indexes to depict the individual atrophy pattern. Forty-five glioma patients in frontal lobe and fifty-two age-matched healthy controls participated in the study. All patients underwent neurocognitive test and molecular examinations, including 1p/19q co-deletion, isocitrate dehydrogenase (IDH) mutation, telomerase reverse transcriptase (TERT) promoter mutation and O6-methylguanine-DNA methyltransferase (MGMT) promotor methylation. Individual structural abnormality maps for every glioma patient were calculated from the preoperative T1 images, and the individual abnormality index were further computed and explored the associations with clinical indicators. The results manifested that: 1. Every glioma patient show unique atrophy pattern; 2. Glioma patients also share consistent atrophy regions; 3. The atrophy pattern is influenced by some molecular biomarkers. Our study provides an effective way to access the individual structural abnormalities in glioma patients, and displays great potentials in individualized precision medicine for glioma patients.

neuroscience↗

bioRxiv2021

Nodal GABA facilitates axon spike transmission in the spinal cord.

Movement and posture depend on sensory feedback that is regulated by specialized GABAergic neurons (GAD2+) that form axo-axonic contacts onto myelinated proprioceptive sensory axons and are thought to be inhibitory. However, we report here that activating GAD2+ neurons, directly with optogenetics or indirectly by cutaneous stimulation, facilitates sensory feedback to motoneurons in awake rodents and humans. GABAA receptors and GAD2+ innervation at or near nodes of Ranvier of sensory axons cause this facilitation, preventing spike propagation failure at the many axon branch points, which is otherwise common without GABA. In contrast, GABAA receptors are generally lacking from axon terminals (unlike GABAB) and do not presynaptically inhibit transmitter release onto motoneurons. GABAergic innervation near nodes and branch points allows individual branches to function autonomously, with GAD2+ neurons regulating which branches conduct, adding a computational layer to the neuronal networks generating movement and likely generalizing to other CNS axons.

neuroscience↗

bioRxiv2021

A new method for improving extraction efficiency and purity of urine and plasma cell-free DNA

This study assessed three commercially available cell-free DNA (cfDNA) extraction kits and the impact of a PEG-based DNA cleanup procedure (DNApure) on cfDNA quality and yield. Six normal donor urine and plasma samples, and specimens from four pregnant (PG) women carrying male fetuses underwent extractions with the JBS cfDNA extraction kit (kit J), MagMAX Cell-Free DNA Extraction kit (kit M), and QIAamp Circulating Nucleic Acid Kit (kit Q). Recovery of a PCR product spike-in, endogenous TP53, and Y-chromosome DNA was used to assess kit performance. Nucleosomal-sized DNA profiles varied among the kits, with prominent multi-nucleosomal-sized peaks present in urine and plasma DNA isolated by kits J and M only. Kit J recovered significantly more spike-in DNA compared with kit M or Q (p<0.001) from urine, and similar amounts from plasma (p=0.12). Applying DNApure to kit M- and Q-isolated DNA significantly improved the amplification efficiency of spike-in DNA from urine (p<0.001) and plasma (p[≤]0.013). Furthermore, kit J isolated significantly more Y-chromosome DNA from PG urine compared to kit Q (p=0.05). We conclude that DNApure provides an efficient means of improving the yield and purity of cfDNA and minimizing effects of pre-analytical biospecimen variability on liquid biopsy assay performance.

molecular biology↗

bioRxiv2020

An in vivo CRISPR screening platform for prioritizing therapeutic targets in AML

CRISPR-Cas9-based genetic screens have successfully identified cell type-dependent liabilities in cancers, including acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival. Because most of these screens have been performed in vitro, evaluating the physiological relevance of these targets is critical. We have established a CRISPR screening approach using orthotopic xenograft models to prioritize AML-enriched dependencies in vivo, complemented by the validation in CRISPR-competent AML patient-derived xenograft (PDX) models tractable for genome editing. Our integrated pipeline has revealed several targets with translational value, including SLC5A3 as a metabolic vulnerability for AML addicted to exogenous myo-inositol and MARCH5 as a critical guardian to prevent apoptosis in AML. MARCH5 repression enhanced the efficacy of BCL2 inhibitors such as venetoclax, highlighting the clinical potential of targeting MARCH5 in AML. Our study provides a valuable strategy for discovery and prioritization of new candidate AML therapeutic targets. Statement of significanceThere is an unmet need to improve the clinical outcome of AML. We developed an integrated in vivo screening approach to prioritize and validate AML dependencies with high translational potential. We identified SLC5A3 as a metabolic vulnerability and MARCH5 as a critical apoptosis regulator in AML, representing novel therapeutic opportunities.

cancer biology↗

bioRxiv2020

Real-time monitoring epidemic trends and key mutations in SARS-CoV-2 evolution by an automated tool

With the global epidemic of SARS-CoV-2, it is important to monitor the variation, haplotype subgroup epidemic trends and key mutations of SARS-CoV-2 over time effectively, which is of great significance to the development of new vaccines, the update of therapeutic drugs, and the improvement of detection reagents. The AutoVEM tool developed in the present study could complete all mutations detections, haplotypes classification, haplotype subgroup epidemic trends and key mutations analysis for 131,576 SARS-CoV-2 genome sequences in 18 hours on a 1 core CPU and 2G internal storage computer. Through haplotype subgroup epidemic trends analysis of 131,576 genome sequences, the great significance of the previous 4 specific sites (C241T, C3037T, C14408T and A23403G) was further revealed, and 6 new mutation sites of highly linked (T445C, C6286T, C22227T, G25563T, C26801G and G29645T) were discovered for the first time that might be related to the infectivity, pathogenicity or host adaptability of SARS-CoV-2. In brief, we proposed an integrative method and developed an efficient automated tool to monitor haplotype subgroup epidemic trends and screen out the key mutations in the evolution of SARS-CoV-2 over time for the first time, and all data could be updated quickly to track the prevalence of previous key mutations and new key mutations because of high efficiency of the tool. In addition, the idea of combinatorial analysis in the present study can also provide a reference for the mutation monitoring of other viruses.

genomics↗

bioRxiv2020

Pseudomonas aeruginosa secreted protein PA3611 promotes bronchial epithelial cells epithelial-mesenchymal transition through TGF-β1 inducing p38/miRNA/NF-κB pathway

Pseudomonas aeruginosa (PA) is one of the important pathogens, which has been proven to colonize and cause infection in the respiratory tract of patients with structural lung diseases, and further lead to bronchial fibrosis. Epithelial-Mesenchymal Transition (EMT) of bronchial epithelial cells plays a vital role in the process of bronchial fibrosis. Up to the present, the research on bronchial epithelial cells EMT caused by secreted virulence factors of PA has not been reported. In our present study, we found that PA3611 protein stimulation induced the bronchial epithelial cells EMT with up-regulation of mesenchymal cell markers and down-regulation of epithelial cell markers. Meantime, TGF-{beta}1 secretion was markedly increased, I{kappa}B expression was significantly decreased, and NF-{kappa}B p65 subunit phosphorylation was markedly enhanced, in addition, the levels of miR-3065-3p and miR-6802-3p expression and p38 MAPK phosphorylation were obviously increased in bronchial epithelial cells after PA3611 stimulation, further research revealed that PA3611 promoted EMT occur through TGF-{beta}1 induced p38/miRNA/NF-{kappa}b pathway. The function of PA3611 was also verified in PA-infected rats and results showed that {Delta}PA3611 could reduce lung inflammation and EMT. Overall, our results revealed that PA3611 promotes EMT via simulating the production of TGF-{beta}1 induced p38/miRNA/NF-{kappa}B pathway-dependent manner, suggesting that PA3611 acts as a crucial virulence factor in bronchial epithelial cells EMT process and has potential use as a target for clinical treatment of bronchial EMT and fibrosis caused by chronic PA infection. Author summaryStructural lung disease can increase the chance of chronic infection, including infected by Pseudomonas aeruginosa, which can cause lung structure damages and affect lung functions in further, and forming a vicious circle of intertwining, ultimately, it leads to pulmonary fibrosis. EMT of bronchial epithelial cells plays a vital role in the process of bronchial fibrosis. However, the relationship and mechanism of PA infection leads to the destruction of lung structure and bronchial epithelial cells EMT are still not very clear. We found pseudomonas aeruginosa secreted protein PA3611 can stimulate bronchial epithelial cells EMT through up-regulation of mesenchymal cell markers -SMA and Vimentin expression and down-regulation of epithelial cell markers E-cadherin and Zonula Occludens-1. Meantime, TGF-{beta}1 secretion was markedly increased, I{kappa}B expression was significantly decreased, and NF-{kappa}B p65 subunit phosphorylation was markedly enhanced, in addition, the levels of miR-3065-3p and miR-6802-3p expression and p38 MAPK phosphorylation were obviously increased in bronchial epithelial cells after PA3611 stimulation, further studies suggested that PA3611 was shown to promote EMT occur through TGF-{beta}1 induced p38/miRNA/NF-Kb pathway. Our results revealed that PA3611 promotes EMT via simulating the production of TGF-{beta}1 induced p38/miRNA/NF-{kappa}B pathway-dependent manner, suggesting that PA3611 acts as a crucial virulence factor in bronchial epithelial cells EMT process and as a potential target for the treatment of chronic structural lung diseases.

microbiology↗

bioRxiv2020

The functional and phenotypic diversity of single T-cell infiltrates in human colorectal cancer as correlated with clinical outcome

Although degree of T-cell infiltration in CRC was shown to correlate with a positive prognosis, the contribution of phenotypically and functionally distinct T cell subtypes within tumors remains unclear. We analyzed 37,931 single T cells with respect to transcriptome, TCR sequence and 23 cell surface proteins, from tumors and adjacent normal colon of 16 patients. Our comprehensive analysis revealed two phenotypically distinct cytotoxic T cell populations within tumors, including positively prognostic effector memory cells and non-prognostic resident memory cells. These cytotoxic T cell infiltrates transitioned from effector memory to resident memory in a stage-dependent manner. We further defined several Treg subpopulations within tumors. While Tregs overall were associated with positive clinical outcomes, CD38+ peripherally-derived Tregs, phenotypically related to Th17 cells, correlated with poor outcomes independent of cancer stage. Thus, our data highlight the diversity of T cells in CRC and demonstrate the prognostic significance of distinct T cell subtypes, which could inform therapeutic strategies.

immunology↗

bioRxiv2020

Cell-type, single-cell, and spatial signatures of brain-region specific splicing in postnatal development

Alternative RNA splicing varies across brain regions, but the single-cell resolution of such regional variation is unknown. Here we present the first single-cell investigation of differential isoform expression (DIE) between brain regions, by performing single cell long-read transcriptome sequencing in the mouse hippocampus and prefrontal cortex in 45 cell types at postnatal day 7 (www.isoformAtlas.com). Using isoform tests for brain-region specific DIE, which outperform exon-based tests, we detect hundreds of brain-region specific DIE events traceable to specific cell-types. Many DIE events correspond to functionally distinct protein isoforms, some with just a 6-nucleotide exon variant. In most instances, one cell type is responsible for brain-region specific DIE. Cell types indigenous to only one anatomic structure display distinctive DIE, where for example, the choroid plexus epithelium manifest unique transcription start sites. However, for some genes, multiple cell-types are responsible for DIE in bulk data, indicating that regional identity can, although less frequently, override cell-type specificity. We validated our findings with spatial transcriptomics and long-read sequencing, yielding the first spatially resolved splicing map in the postnatal mouse brain (www.isoformAtlas.com). Our methods are highly generalizable. They provide a robust means of quantifying isoform expression with cell-type and spatial resolution, and reveal how the brain integrates molecular and cellular complexity to serve function.

neuroscience↗

bioRxiv2020

LoVIS-Seq reveals clonal dynamics of human-HSPC in humanized mice and vector integration bias

BackgroundCurrent understanding of hematopoiesis is largely derived from mouse models that are physiologically distant from humans. Humanized mice provide the most physiologically relevant small animal model to study human diseases, most notably preclinical gene-therapy studies. However, the clonal repopulation dynamics of human hematopoietic stem and progenitor cells (HSPC) in these animal models is only partially understood. Using a new clonal tracking methodology designed for small sample volumes, we aim to reveal the underlying clonal dynamics of human cell repopulation in a mouse environment. MethodsHumanized BLT (bone marrow-liver-thymus) mice were generated by transplanting lentiviral vector transduced human fetal liver HSPC (FL-HSPC) in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice implanted with a piece of human fetal thymus. We developed a methodology to track vector integration sites (VIS) in a mere 25{micro}l of mouse blood for longitudinal and quantitative clonal analysis of human HSPC repopulation in mouse environment. We explored transcriptional and epigenetic features of human HSPC for possible VIS bias. Results897 HSPC clones were longitudinally tracked in BLT mice--providing a first-ever demonstration of clonal dynamics and competitive expansion of therapeutic and control vector-modified human cell populations simultaneously repopulating in the same humanized mice. The polyclonal repopulation stabilized at 19 weeks post-transplant and the contribution of the largest clone doubled within 4 weeks. Moreover, 550 ([~]60%) clones persisted over 6 weeks and were highly shared between different organs. The normal clonal profiles confirmed the safety of our gene therapy vectors. Multi-omics analysis of human FL-HSPC revealed that 54% of vector integrations in repopulating clones occurred within {+/-}1kb of H3K36me3-enriched regions. ConclusionsHuman repopulation in mice is polyclonal and stabilizes more rapidly than that previously observed in humans. VIS preference for H3K36me3 has no apparent negative effects on HSPC repopulation. Our study provides a methodology to longitudinally track clonal repopulation in small animal models extensively used for stem cell and gene-therapy research and with lentiviral vectors designed for clinical applications. Results of this study provide a framework for understanding the clonal behavior of human HPSC repopulating in a mouse environment, critical for translating results from humanized mice models to the human settings.

molecular biology↗