Biology subjects

Chang, S.

Publications and source records attributed to Chang, S..

At least 73 records · Page 4Linked to original sources

bioRxiv2022

Single-cell chromatin accessibility of developing murine pancreas identifies cell state-specific gene regulatory programs

Numerous studies have characterized the existence of cell subtypes, along with their corresponding transcriptional profiles, within the developing mouse pancreas. The upstream mechanisms that initiate and maintain gene expression programs across cell states, however, remain largely unknown. Here, we generate single-nucleus ATAC-Sequencing data of developing murine pancreas and perform an integrated, multi-omic analysis of both chromatin accessibility and RNA expression to describe the chromatin landscape of both the developing epithelium and mesenchyme at E14.5 at single-cell resolution. We identify candidate transcription factors regulating cell fate and construct gene regulatory networks of active transcription factor binding to regulatory regions of downstream target genes. This work serves as a valuable resource for the field of pancreatic biology in general and contributes to our understanding of lineage plasticity among endocrine cell types. In addition, these data identify which epigenetic states should be represented in the differentiation of stem cells to the pancreatic beta cell fate in order to best recapitulate in vitro the gene regulatory networks that are critical for progression along the beta cell lineage in vivo.

developmental biology↗

bioRxiv2022

Spatial Transcriptomics Using Multiplexed Deterministic Barcoding in Tissue

In this study, we present a multiplexed version of deterministic barcoding in tissue (xDbit) to acquire spatially resolved transcriptomes of nine tissue sections in parallel. New microfluidic chips were developed to spatially encode mRNAs over a total tissue area of 1.17 cm2 with spots of 50 mx50 m. Optimization of the biochemical protocol increased read and gene counts per spot by one order of magnitude compared with previous reports. Furthermore, the introduction of alignment markers allows seamless registration of images and spatial transcriptomic spot coordinates. Together with technological advances, we provide an open-source computational pipeline to transform raw sequencing data from xDbit experiments into the AnnData format. The functionality of xDbit was demonstrated by the acquisition of 18 spatially resolved transcriptomic datasets from five different murine organs, including cerebellum, liver, kidney, spleen, and heart. Factor analysis and deconvolution of xDbit spatial transcriptomes allowed for in-depth characterization of the murine kidney.

genomics↗

bioRxiv2022

Mouse lemur transcriptomic atlas elucidates primate genes, physiology, disease, and evolution

Mouse lemurs (Microcebus spp.) are an emerging primate model organism. However, little is known about their genetics or cellular and molecular biology. In the accompanying paper, we used large-scale single cell RNA-sequencing of 27 organs and tissues to identify over 750 molecular cell types, characterize their full transcriptomic profiles, and study evolution of primate cell types. Here we use the atlas to characterize mouse lemur genes, mutations, physiology, and disease. We uncover thousands of previously unidentified lemur genes and hundreds of thousands of new splice junctions that globally define lemur gene structures and reveal over 85,000 primate splice junctions missing in mice. We systematically explore the lemur immune system, comparing the global expression profiles of key immune genes in health and disease, and molecular mapping of immune cell development, trafficking, and their local and global activation to infection. We characterize primate/lemur-specific physiology and disease including molecular features of the immune program, of lemur adipocytes that exhibit dramatic seasonal rhythms, and of metastatic endometrial cancer that resembles the human malignancy. We identify and describe the expression patterns of over 400 primate genes missing in mice, many with similar expression patterns in human and lemur and some implicated in human disease. Finally, we provide an experimental framework for reverse genetic analysis by identifying naturally-occurring nonsense (null) mutations in three primate genes missing in mice and analyzing their transcriptional phenotypes. This work establishes mouse lemur as a tractable primate model organism for genetic and molecular analysis, and it prioritizes primate genes, splice junctions, physiology, and disease for future study.

genetics↗

bioRxiv2022

Myostatin gene deletion alters gut microbiota stimulating fast-twitch glycolytic muscle growth

The host genome may influence the composition of the intestinal microbiota, and intestinal microbiota performs an important role in muscle growth and development. Here, we showed that Myostatin (MSTN), a key factor for muscle growth, deletion alters muscularis, plica, and intestinal barrier in pigs. Mice transplanted with MSTN-/- pig intestinal flora showed increase in the cross-sectional area of myofibers and fast-twitch glycolytic muscle mass. The microbes responsible for the production of short chain fatty acids (SCFAs) were enriched in both MSTN-/- pigs and recipient mice, and SCFAs levels were elevated in the colon contents. We demonstrated that valeric acid can stimulate type IIb myofiber growth by activation of the Akt/mTOR pathway via GPR43 and improve muscle atrophy induced by dexamethasone. This is the first study to identify the MSTN gene-gut microbiota-SCFA axis and its regulatory role in fast-twitch glycolytic muscle growth.

microbiology↗

bioRxiv2022

Bioinformatics analysis of immune-related prognostic genes and immunotherapy in renal clear cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is an immunogenic tumor and investigating the immunorelated genes is of great significance. To investigate the immunoprognostic genes of ccRCC, we analyzed the data assimilated from a public database (The Cancer Genome Atlas (TCGA) database and the gene expression omnibus (GEO) database) using bioinformatics. Then, a immunoprognosis model was constructed to identify 4 hub genes with moderate predictive values for the prognosis of ccRCC patients. Interestingly, these 4 genes were associated with the prognosis of ccRCC patients based on Oncomine and Gena Expression Profiling Interactive Analysis (GEPIA) databases. The correlation analysis between the immune infiltrate, immune checkpoints, and immunotherapy and this immunoprognosis model showed that immune infiltrate and could predict the immunotherapy effects. We also conducted a quantitative real-time polymerase chain reaction analysis and found that the expressions of 3 hub genes were associated with tumor progression (P<0.1). In conclusion, 4 genes that may serve as potential biomarkers in ccRCC were identified with respect to prognosis.

cancer biology↗

bioRxiv2022

7-UP: generating in silico CODEX from a small set of immunofluorescence markers

Multiplex immunofluorescence (mIF) assays multiple protein biomarkers on a single tissue section. Recently, high-plex CODEX (co-detection by indexing) systems enable simultaneous imaging of 40+ protein biomarkers, unlocking more detailed molecular phenotyping, leading to richer insights into cellular interactions and disease. However, high-plex imaging can be slower and more costly to collect, limiting its applications, especially in clinical settings. We propose a machine learning framework, 7-UP, that can computationally generate in silico 40-plex CODEX at single-cell resolution from a standard 7-plex mIF panel by leveraging cellular morphology. We demonstrate the usefulness of the imputed biomarkers in accurately classifying cell types and predicting patient survival outcomes. Furthermore, 7-UPs imputations generalize well across samples from different clinical sites and cancer types. 7-UP opens the possibility of in silico CODEX, making insights from high-plex mIF more widely available.

bioinformatics↗

bioRxiv2022

Replication stalling activates SSB for recruitment of DNA damage tolerance factors

Translesion synthesis (TLS) polymerases bypass DNA lesions that block replicative polymerases, allowing cells to tolerate DNA damage encountered during replication. It is well known that most bacterial TLS polymerases must interact with the sliding clamp processivity factor to carry out TLS, but recent work in Escherichia coli has revealed that single-stranded DNA-binding protein (SSB) plays a key role in enriching the TLS polymerase Pol IV at stalled replication forks in the presence of DNA damage. It remains unclear how this interaction with SSB enriches Pol IV in a stalling-dependent manner given that SSB is always present at the replication fork. In this study we use single-molecule imaging in live E. coli cells to investigate this SSB-dependent enrichment of Pol IV. We find that Pol IV is enriched through its interaction with SSB in response to a range of different replication stresses, and that changes in SSB dynamics at stalled forks may explain this conditional Pol IV enrichment. Finally, we show that other SSB-interacting proteins (SIPs) are likewise selectively enriched in response to replication perturbations, suggesting that this mechanism is likely a general one for enrichment of repair factors near stalled replication forks.

biochemistry↗

bioRxiv2022

Microglia Detect Externalized Phosphatidylserine on Synapses for Elimination via TREM2 in Alzheimer's Disease Models

Genetic studies implicate phagocytosis pathways in microglia to be a major Alzheimers disease (AD)-associated process. Microglia phagocytose synapses in AD mouse models, suggesting a role for microglia in region-specific synapse loss, a pathological hallmark of AD. However, whether specific synapses are targeted for elimination, and if so, how, remains to be elucidated. Here, we show that synapses externalize phosphatidylserine (PtdSer) upon challenge by {beta}-amyloid oligomers, which are then selectively engulfed by microglia. Mechanistically, we find that Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) is critical for microglia to sense and preferentially engulf AD synapses. In brains of mice and humans, TREM2 dysfunction leads to exacerbation of apoptotic synapses. Our work altogether suggests a fundamental role for microglia as brain-resident macrophages to remove damaged synapses in AD. We provide mechanistic insight into how TREM2 variants associated with increased risk of developing AD may contribute to defective microglia-synapse function. One-Sentence summaryMicroglia selectively engulf synapses in Alzheimer-like mouse brains via PtdSer-TREM2 signaling.

neuroscience↗

bioRxiv2022

Dysregulated H19/Igf2 expression disrupts cardiac-placental axis during development of Silver Russell Syndrome-like mouse models

Dysregulation of the imprinted H19/IGF2 locus can lead to Silver-Russell Syndrome (SRS) in humans. However, the mechanism of how abnormal H19/IGF2 expression contributes to various SRS phenotypes remains unclear, largely due to incomplete understanding of the developmental functions of these two genes. We previously generated a mouse model with humanized H19/IGF2 ICR (hIC1) on the paternal allele that exhibited H19/Igf2 dysregulation together with SRS-like growth restriction and perinatal lethality. Here we dissect the role of H19 and Igf2 in cardiac and placental development utilizing multiple mouse models with varying levels of H19 and Igf2. We report severe cardiac defects such as ventricular septal defects (VSDs) and thinned myocardium, placental anomalies including thrombosis and vascular malformations, together with growth restriction in mouse embryos that correlated with the extent of H19/Igf2 dysregulation. Transcriptomic analysis using cardiac endothelial cells of these mouse models shows that H19/Igf2 dysregulation disrupts pathways related to extracellular matrix (ECM) and proliferation of endothelial cells. Our work links the heart and placenta through regulation by H19 and Igf2, demonstrating that accurate dosage of both H19 and Igf2 is critical for normal embryonic development, especially related to the cardiac-placental axis.

developmental biology↗

bioRxiv2022

Tripartite extended amygdala - basal ganglia CRH circuit drives arousal and avoidance behaviour

An adaptive stress response involves various mediators and circuits orchestrating a complex interplay of physiological, emotional and behavioural adjustments. We identified a population of corticotropin-releasing hormone (CRH) neurons in the lateral part of the interstitial nucleus of the anterior commissure (IPACL) - a subdivision of the extended amygdala, which exclusively innervate the substantia nigra (SN). Specific stimulation of this circuit elicits arousal and avoidance behaviour contingent on CRH receptor 1 (CRHR1) located at axon terminals in the SN, which originate from external globus pallidus (GPe) neurons. The neuronal activity prompting the observed behaviour is shaped by IPACLCRH and GPeCRHR1 neurons coalescing in the SN. These results delineate a novel tripartite CRH circuit functionally connecting extended amygdala and basal ganglia nuclei to drive arousal and avoidance behaviour. One-Sentence SummaryBrain centres involved in emotional and motor control are connected through a stress peptide promoting arousal and avoidance behaviour

neuroscience↗

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↗

bioRxiv2021

Tabula Microcebus: A transcriptomic cell atlas of mouse lemur, an emerging primate model organism

Mouse lemurs are the smallest, fastest reproducing, and among the most abundant primates, and an emerging model organism for primate biology, behavior, health and conservation. Although much has been learned about their physiology and their Madagascar ecology and phylogeny, little is known about their cellular and molecular biology. Here we used droplet- and plate-based single cell RNA-sequencing to profile 226,000 cells from 27 mouse lemur organs and tissues opportunistically procured from four donors clinically and histologically characterized. Using computational cell clustering, integration, and expert cell annotation, we defined and biologically organized over 750 mouse lemur molecular cell types and their full gene expression profiles. These include cognates of most classical human cell types, including stem and progenitor cells, and the developmental programs for spermatogenesis, hematopoiesis, and other adult tissues. We also described dozens of previously unidentified or sparsely characterized cell types and subtypes. We globally compared cell type expression profiles to define the molecular relationships of cell types across the body, and explored primate cell and gene expression evolution by comparing mouse lemur cell transcriptomes to those of human, mouse, and macaque. This revealed cell type specific patterns of primate specialization, as well as many cell types and genes for which lemur provides a better human model than mouse. The atlas provides a cellular and molecular foundation for studying this primate model organism, and establishes a general approach for other emerging model organisms.

genetics↗

bioRxiv2021

Fast volumetric imaging with line-scan confocal microscopy by an electro-tunable lens

In microscopic imaging of biological tissues, particularly real-time visualization of neuronal activities, rapid acquisition of volumetric images poses a prominent challenge. Typically, two-dimensional (2D) microscopy can be devised into an imaging system with 3D capability using any varifocal lens. Despite the conceptual simplicity, such an upgrade yet requires additional, complicated device components and suffers a reduced acquisition rate, which is critical to document neuronal dynamics properly. In this study, we implemented an electro-tunable lens (ETL) in the line-scan confocal microscopy, enabling the volumetric acquisition at the rate of 20 frames per second with the maximum volume of interest of 315 x 315 x 80 m3. The axial extent of point-spread-function (PSF) was 17.6 {+/-} 1.6 m and 90.4 {+/-} 2.1 m with the ETL operating in either stationary or resonant mode, respectively, revealing significant depth elongation by the resonant mode ETL microscopy. We further demonstrated the utilities of the ETL system by volume imaging of cleared mouse brain ex vivo samples and in vivo brains. The current study foregrounds the successful application of resonant ETL for constructing a basis for a high-performance 3D line-scan confocal microscopy system, which will enhance our understanding of various dynamic biological processes.

bioengineering↗

bioRxiv2021

Inhibition of PDE4 by Roflumilast ameliorates sleep deprivation-induced cognitive dysfunction in C57BL/6J mice

Sleep deprivation (SD) interferes with long-term memory and cognitive functions by over-activation of phosphodiesterase (PDEs) enzymes. PDE4, a non-redundant regulator of the cyclic nucleotides (cAMP), is densely expressed in the hippocampus and is involved in learning and memory processes. In the present study, we investigated the effects of Roflumilast (ROF), a PDE4B inhibitor, on sleep deprivation induced cognitive dysfunction in a mouse model. Memory assessment was performed using a novel object recognition task and the hippocampal cAMP level was estimated by the ELISA method. The alterations in the expressions of PDE4B, amyloid-beta (A{beta}), CREB, BDNF, and synaptic proteins (Synapsin I, SAP 97, PSD 95) were assessed to gain insights into the possible mechanisms of action of ROF using the Western blot technique. Results show that ROF reversed SD induced cognitive decline in mice. ROF down-regulated PDE4B and A{beta} expressions in the brain. Additionally, ROF improved the cAMP level and the protein expressions of synapsin I, SAP 97, and PSD 95 in the hippocampal region of SD mice. Taken together, these results suggest that ROF can suppress the deleterious effects of SD-induced cognitive dysfunction via the PDE4B-mediated cAMP/CREB/BDNF signaling cascade.

pharmacology and toxicology↗

bioRxiv2021

A multimodal imaging and analysis pipeline for creating a cellular census of the human cerebral cortex

Cells are not uniformly distributed in the human cerebral cortex. Rather, they are arranged in a regional and laminar fashion that span a range of scales. Here we demonstrate an innovative imaging and analysis pipeline to construct a reliable cell census across the human cerebral cortex. Magnetic resonance imaging (MRI) is used to establish a macroscopic reference coordinate system of laminar and cytoarchitectural boundaries. Cell counting is obtained with both traditional immunohistochemistry, to provide a stereological gold-standard, and with a custom-made inverted confocal light-sheet fluorescence microscope (LSFM) for 3D imaging at cellular resolution. Finally, mesoscale optical coherence tomography (OCT) enables the registration of the distorted histological cell typing obtained with LSFM to the MRI-based atlas coordinate system.

neuroscience↗

bioRxiv2021

Refractive-index matching enhanced polarization-sensitive optical coherence tomography quantification in human brain tissue

The importance of polarization-sensitive optical coherence tomography (PS-OCT) has been increasingly recognized in human brain imaging. Despite the recent progress of PS-OCT in revealing white matter architecture and orientation, quantification of fine-scale fiber tracts in the human brain cortex has been a challenging problem, due to a low birefringence in the gray matter. In this study, we investigated the effect of refractive index matching by 2,2-thiodiethanol (TDE) immersion on the improvement of PS-OCT measurements in ex vivo human brain tissue. We obtain the cortical fiber orientation maps in the gray matter, which reveals the radial fibers in the gyrus, the U-fibers along the sulcus, as well as distinct layers of fiber axes exhibiting laminar organization. Further analysis shows that index matching reduces the noise in axis orientation measurements by 56% and 39%, in white and gray matter, respectively. Index matching also enables precise measurements of apparent birefringence, which was underestimated in the white matter by 82% but overestimated in the gray matter by 16% prior to TDE immersion. Mathematical simulations show that the improvements are primarily attributed to the reduction in the tissue scattering coefficient, leading to an enhanced signal-to-noise ratio in deeper tissue regions, which could not be achieved by conventional noise reduction methods.

bioengineering↗

bioRxiv2021

Dynamic assembly of the calcium hemostasis modulator 1 channel gates ATP permeation

Calcium hemostasis modulator 1 (CALHM1) is a voltage- and Ca2+-gated ATP channel that plays an important role in neuronal signaling. The currently reported CALHM structures are all in an ATP-conducting state, and the gating mechanism of ATP permeation remains elusive. Here, we report three cryo-EM reconstructions of Danio rerio heptameric CALHM1s with ordered or flexible long C-terminal helices as well as Danio rerio octameric CALHM1 with flexible long C-terminal helices at resolutions of 3.2 [A], 2.9 [A], and 3.5 [A]. Structural analysis revealed that the heptameric CALHM1s are in an ATP nonconducting state in which the pore diameter in the middle is approximately 6.6 [A]. Compared with those inside the octameric CALHM1s, the N- helices inside heptameric CALHM1s are in the "down" position to avoid steric clash with neighboring TM1 helices. Molecular dynamic simulation shows that the pore size is significantly increased for ATP molecule permeation during the movement of the N- helix from the "down" position to the "up" position. Therefore, we proposed a mechanism in which the "piston-like" motion of the N-helix drives the dynamic assembly of the CALHM1 channel for ATP molecule permeation. Our results provide insights into the ATP permeation mechanism of the CALHM1 channel.

biochemistry↗

bioRxiv2021

Cryo-EM structure of the human TACAN channel in a closed state

TACAN is an ion channel involved in sensing mechanical pain. It has recently been shown to represent a novel and evolutionarily conserved class of mechanosensitive channels. Here, we present the cryoelectron microscopic structure of human TACAN (hTACAN). hTACAN forms a dimer in which each protomer consists of a transmembrane globular domain (TMD) that is formed of six helices and an intracellular domain (ICD) that is formed of two helices. Molecular dynamic simulations suggest that a putative ion conduction pathway is located inside each protomer. Single point mutation of the key residue Met207 significantly increased the surface tension activated currents. Moreover, cholesterols were identified at the flank of each subunit. Our data show the molecular assembly of hTACAN and suggest that the wild type hTACAN is in a closed state, providing a basis for further understanding the activation mechanism of the hTACAN channel.

biochemistry↗