Biology subjects

Qu, J.

Publications and source records attributed to Qu, J..

At least 73 records · Page 4Linked to original sources

bioRxiv2022

Sharing Massive Biomedical Data at Magnitudes Lower Bandwidth Using Implicit Neural Function

Efficient storage and sharing of massive biomedical data would open up their wide accessibility to different institutions and disciplines. However, compressors tailored for natural photos/videos are rapidly limited for biomedical data, while emerging deep learning based methods demand huge training data and are difficult to generalize. Here we propose to conduct Biomedical data compRession with Implicit nEural Function (BRIEF) by representing the original data with compact neural networks, which are data specific and thus have no generalization issues. Benefiting from the strong representation capability of implicit neural function, BRIEF achieves 2 [~] 3 orders of magnitude compression on diverse biomedical data at significantly higher fidelity than existing techniques. Besides, BRIEF is of consistent performance across the whole data volume, supports customized spatially-varying fidelity. BRIEFs multi-fold advantageous features also serve reliable downstream tasks at low bandwidth. Our approach will facilitate low-bandwidth data sharing, and promote collaboration and progress in the biomedical field.

bioinformatics↗

bioRxiv2022

Chromatin profiling identifies transcriptional readthrough as a conserved mechanism for piRNA cluster biogenesis in mosquitoes

The piRNA pathway in mosquitoes differs substantially from other model organisms, with an expanded PIWI gene family and functions in antiviral defense. Here, we defined core piRNA clusters as small RNA source loci that showed ubiquitous expression in both somatic and germline tissues. These core piRNA clusters were enriched for non-retroviral endogenous viral elements (nrEVEs) in antisense orientation and depended on key biogenesis factors, Nxf1, Veneno, Tejas, Yb, and Shutdown. Combined transcriptome and chromatin state analyses identified transcriptional readthrough as a conserved mechanism for piRNA cluster biogenesis in Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, and Anopheles gambiae. Comparative analyses between two Aedes mosquitoes suggested that piRNA clusters function as traps for nrEVEs, allowing adaptation to environmental challenges such as virus infection. Our systematic transcriptome and chromatin state analyses lay the foundation for studies of gene regulation, genome evolution and piRNA functions in these important vector species. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/504762v2_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@388aeborg.highwire.dtl.DTLVardef@28640forg.highwire.dtl.DTLVardef@1c12440org.highwire.dtl.DTLVardef@1331e0b_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LICore piRNA clusters showed ubiquitous expression in both somatic and germline tissues in four vector mosquitoes. C_LIO_LIChromatin profiling identifies transcriptional readthrough as a conserved mechanism for piRNA biogenesis. C_LIO_LIBiogenesis of cluster-derived piRNAs depends on key factors, Nxf1, Veneno, Tejas, Yb, and Shutdown. C_LIO_LIpiRNA clusters function as traps for viral elements downstream of conserved set of genes in Aedes mosquitoes C_LI

genomics↗

bioRxiv2022

Dynamic Phosphorylation Regulates Eukaryotic Translation Initiation Factor 4A Activity During the Cell Cycle

The eukaryotic translation initiation factor 4A (eIF4A) resolves mRNA structures to support protein synthesis, yet little is known about its regulation. Here we analyzed eIF4A phosphorylation during alternate stages of the cell cycle, and found three residues near the DEAD box motif (T73, T146, and S177) underwent substantial phosphorylation changes. Phosphomimetic mutations T73D and T146D led to G2/M phase arrest, and abolished eIF4A interaction with RNA, suggesting eIF4A activity is needed for completion of cell division. In addition to these repressive events, we found that S177, a site immediately adjacent to the DEAD-box, showed diametrically opposed phosphorylation, with only phosphorylated S177 present during G1/S arrest and dephosphorylated S177 peptides during G2/M arrest. Phosphomimetic S177D eIF4A increased polysome levels and enhanced normally reduced eIF4A-eIF4G-interaction during G2/M, while phosphodeficient S177A decreased polysome levels and reduced growth, suggesting phosphorylation of S177 enhances eIF4A-mediated translation during G1/S. Together these results suggest that dynamic phosphorylation of eIF4A S177 serves to stimulate translation during G1/S, while inhibitory phosphorylation of additional sites holds the potential to rapidly transition eIF4A to an inactive state and turn off translation. These results also suggest an important role for eIF4A in coupling translation to cell cycle stages.

molecular biology↗

bioRxiv2022

Development of Equine Polyclonal Antibodies as a Broad-Spectrum Therapy Against SARS-CoV-2 Variants

The Coronavirus disease 19 (COVID-19) pandemic has accumulated over 550 million confirmed cases and more than 6.34 million deaths worldwide. Although vaccinations has largely protected the population through the last two years, the effect of vaccination has been increasingly challenged by the emerging SARS-CoV-2 variants. Although several therapeutics including both monoclonal antibodies and small molecule drugs have been used clinically, high cost, viral escape mutations, and potential side effects have reduced their efficacy. There is an urgent need to develop a low cost treatment with wide-spectrum effect against the novel variants of SARS-CoV-2. Here we report a product of equine polyclonal antibodies that showed potential broad spectrum neutralization effect against the major variants of SARS-CoV-2. The equine polyclonal antibodies were generated by horse immunization with the receptor binding domain (RBD) of SARS-CoV-2 spike protein and purified from equine serum. A high binding affinity between the generated equine antibodies and the RBD was observed. Although designed against the RBD of the early wild type strain sequenced in 2020, the equine antibodies also showed a highly efficient neutralization capacity against the major variants of SARS-CoV-2, including the recent BA.2 Omicron variant (IC50 =1.867g/ml) in viral neutralization assay in Vero E6 cells using live virus cultured. The broad-spectrum neutralization capacity of the equine antibodies was further confirmed using pseudovirus neutralization assay covering the major SARS-CoV-2 variants including wild type, alpha, beta, delta, and omicron, showing effective neutralization against all the tested strains. Ex vivo reconstructed human respiratory organoids representing nasal, bronchial, and lung epitheliums were employed to test the treatment efficacy of the equine antibodies. Antibody treatment protected the human nasal, bronchial, and lung epithelial organoids against infection of the novel SARS-CoV-2 variants challenging public health, the Delta and Omicron BA.2 isolates, by reducing >95% of the viral load. The equine antibodies were further tested for potential side effects in a mouse model by inhalation and no significant pathological feature was observed. Equine antibodies, as a mature medical product, have been widely applied in the treatment of infectious diseases for more than a century, which limits the potential side effects and are capable of large scale production at a low cost. A cost-effective, wide-spectrum equine antibody therapy effective against the major SARS-CoV-2 variants can contribute as an affordable therapy to cover a large portion of the world population, and thus potentially reduce the transmission and mutation of SARS-CoV-2.

immunology↗

bioRxiv2022

Multi-omics analyses identify transcription factor interplay in corneal epithelial fate determination and disease

The transparent corneal epithelium in the eye is maintained through the homeostasis regulated by limbal stem cells, while the non-transparent epidermis relies on epidermal keratinocytes for renewal. Despite their cellular similarities, the precise cell fates of these two types of epithelial stem cells, which give rise to functionally distinct epithelia, remain unknown. We performed a multi-omics analysis of human limbal stem cells from the cornea and keratinocytes from the epidermis, and characterized their molecular signatures, highlighting their similarities and differences. Through gene regulatory network analyses, we identified shared and cell type-specific transcription factors that define specific cell fates, and established their regulatory hierarchy. Single-cell RNA-seq analyses of the cornea and the epidermis confirmed these shared and cell type-specific transcription factors. Notably, the shared and limbal stem cell-specific transcription factors can cooperatively target genes associated with corneal opacity. Importantly, we discovered that FOSL2, a direct PAX6 target gene, is a novel candidate associated with corneal opacity, and it regulates genes implicated in corneal diseases. By characterizing molecular signatures, our study unveils the regulatory circuitry governing the limbal stem cell fate and its association with corneal opacity.

developmental biology↗

bioRxiv2022

Longitudinal structural change of gray matter before and after hemispherotomy

BackgroundHemispherotomy is an effective surgery developed to treat refractory epilepsy caused by diffuse unihemispheric pathologies. Post-surgery neuroplastic mechanisms supporting behavioral recovery after left and right hemispherotomy as well as their difference remain largely unclear. MethodsIn a large cohort of 57 pediatric patients who underwent hemispherotomy, voxel-wise GMV in unaffected regions (contralesional cerebrum and bilateral cerebellum) and behavioral abilities were assessed before and after surgery. Using linear mixed models, we evaluated changes in GMV and behavioral scores, and how GMV changes correlated with improvements in behavioral scores. In addition, three machine learning regression algorithms were applied to assess whether preoperative GMV can predict postoperative behavior. ResultsIn the two patient groups (29 left hemispherotomy patients, age of surgery mean {+/-} standard deviation = 3.5 {+/-} 2.5; 28 right hemispherotomy patients, age of surgery 4.6 {+/-} 2.5), widespread increases in the postoperative GMV in the contralateral cerebrum and ipsilateral cerebellum but decreases in the postoperative GMV in the contralateral cerebellum were consistently observed. Importantly, the decrease in GMV in the contralateral cerebellum was significantly correlated with improvement in behavioral scores in right but not left hemispherotomy patients. Moreover, the preoperative GMV around the most longitudinally changed locations significantly predicted postoperative behavioral scores in left but not right hemispherotomy patients. ConclusionsOur findings indicate an important role for the contralateral cerebellum in the recovery after right hemispherotomy, and postoperative behavioral scores can be predicted with preoperative GMV features. What is already known on this topicThe contralesional cerebrum plays a key role in the recovery after hemispherotomy. What this study addsDespite suffering from gray matter atrophy, GMV decrease in contralesional cerebellum is associated with improvement in behavioral score in patients after hemispherotomy. How this study might affect research, practice or policyThese results provide novel insight into the prominence of the contralateral cerebellum in the recovery after hemispherotomy and highlight the clinical role of presurgery GMV in the prediction of postsurgery behavior.

neuroscience↗

bioRxiv2022

The DEAD-box RNA helicase Dhx15 controls glycolysis and arbovirus replication in Aedes aegypti mosquito cells

Aedes aegypti mosquitoes are responsible for the transmission of arthropod-borne (arbo)viruses including dengue and chikungunya virus (CHIKV), but in contrast to human hosts, arbovirus infected mosquitoes are able to efficiently control virus replication to sub-pathological levels. Yet, our knowledge about the molecular interactions of arboviruses with their mosquito hosts is largely incomplete. Here, we aimed to identify and characterize novel host genes that control arbovirus replication in Aedes mosquitoes. RNA binding proteins (RBPs) are well known to regulate immune signaling pathways in all kingdoms of life. We therefore performed a knockdown screen targeting 461 genes encoding predicted RBPs in Aedes aegypti Aag2 cells and identified 15 genes with antiviral activity against a Sindbis reporter virus. Amongst these, three DEAD-box RNA helicases, AAEL004419/Dhx15, AAEL008728 and AAEL004859 also acted as antiviral factors in dengue and CHIKV infections. Here, we explore the mechanism of Dhx15 in regulating an antiviral transcriptional response in mosquitoes by silencing Dhx15 in Aag2 cells followed by deep-sequencing of poly-A enriched RNAs. Dhx15 knockdown in uninfected or CHIKV-infected cells resulted in differential expression of 856 and 372 genes, respectively. Interestingly, amongst the consistently downregulated genes, glycolytic process was the most strongly enriched GO term as the expression of all core enzymes of the glycolytic pathway was reduced, suggesting that Dhx15 regulates glycolytic function. A decrease in lactate production supported the observation that Dhx15 silencing functionally impaired glycolysis. Modified rates of glycolytic metabolism have been implicated in controlling the replication of several classes of viruses and strikingly, infection of Aag2 cells with CHIKV by itself also resulted in the decrease of several glycolysis genes. Our data suggests that Dhx15 regulates replication of CHIKV, and possibly other arboviruses, by controlling glycolysis in mosquito cells.

molecular biology↗

bioRxiv2022

Arginine methylation of Puf4 drives diverse protein functions

The evolutionarily conserved Pumilio domain-containing RNA binding proteins (RBPs) are involved in many steps of post-transcriptional gene regulation, including RNA stability, polyadenylation, deadenylation, and translation. RBPs are post-translationally modified by methylation of arginine/glycine-rich domains, though the consequences of these modifications are not well known. We determined the arginine methylation and phosphorylation landscape of the Pumilio domain-containing RBP Puf4 from the basidiomycete fungus Cryptococcus neoformans. We found that methyl-deficient Puf4 mutants do not complement critical PUF4 deletion phenotypes, such as resistances to endoplasmic reticulum stress and antifungals, and cell wall remodeling. Methyl-deficient mutants also exhibit unique RNA and protein interactions. Lastly, we identified intra-protein cross talk between post-translationally modified methylated and phosphorylated residues. Overall, we show that post-translational modifications, particularly arginine methylation, of Puf4 regulate the functions of this RBP.

molecular biology↗

bioRxiv2022

MegaBayesianAlphabet: Mega-scale Bayesian Regression methods for genome-wide prediction and association studies with thousands of traits

Large-scale phenotype data are expected to increase the accuracy of genome-wide prediction and the power of genome-wide association analyses. However, genomic analyses of high-dimensional, highly correlated data are challenging. We developed MegaBayesianAlphabet to simultaneously analyze genetic variants underlying thousands of traits using the flexible priors of the Bayesian Alphabet family. As a demonstration, we implemented the BayesC prior in the R package MegaLMM and applied it to both simulated and real data sets. Our analyses show that the resulting model MegaBayesC can effectively use high-dimensional phenotypic data to improve the accuracy of genetic value prediction, the reliability of marker discovery, and the accuracy of marker effect size estimation in genome-wide analyses.

genetics↗

bioRxiv2022

Proteomic network analysis of bronchoalveolar lavage fluid in ex-smokers to discover implicated protein targets and novel drug treatments for chronic obstructive pulmonary disease

RationaleBronchoalveolar lavage of the epithelial lining fluid can sample the profound changes in the airway lumen milieu prevalent in Chronic Obstructive Pulmonary Disease (COPD). Characterizing the proteins in bronchoalveolar lavage fluid in COPD with advanced proteomic methods will identify disease-related changes, provide insight into pathogenetic mechanisms and potential therapeutics that will aid in the discovery of more effective therapeutics for COPD. ObjectivesWe compared epithelial lining fluid proteome of ex-smokers with moderate COPD who are not in exacerbation status COPD, to non-smoking healthy control subjects using advanced proteomics methods and applied proteome-scale translational bioinformatics approaches to identify potential therapeutic protein targets and drugs that modulate these proteins towards the treatment of COPD. MethodsProteomic profiles of bronchalveolar lavage fluid were obtained from 1) never-smoker control subjects with normal lung function (n=10) or 2) individuals with stable moderate (GOLD stage 2, FEV1 50% - 80% predicted) COPD who were ex-smokers for at least one year (n=10). NIHs Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuitys Ingenuity Pathway Analysis (IPA) were the two bioinformatics tools employed for network analysis on the differentially expressed proteins to identify potential crucial hub proteins. The drug-proteome interaction signature comparison and ranking approach implemented in the Computational Analysis of Novel Drug Opportunities (CANDO) platform for multiscale therapeutic discovery was utilized to identify potential repurposable drugs for the treatment of COPD based on the BALF proteome. Subsequently, a literature-based knowledge graph was utilized to rank combinations of drugs that would most likely ameloriate inflammatory processes by inhibition or activation of their functions. ResultsProteomic network analysis demonstrated that 233 of the >1800 proteins identified in the BALF were differentially expressed in COPD versus control, including proteins associated with inflammation, structural elements, and energy metabolism. Functional annotation of the differentially expressed proteins by their implicated biological processes, cellular localization, and transcription factor interactions was accomplished via DAVID. Canonical pathways containing the differential expressed proteins were detailed via the Ingenuity Pathway Analysis application. Topological network analysis demonstrated that four proteins act as central node proteins in the inflammatory pathways in COPD. The CANDO multiscale drug discovery platform was used to analyze the behavioral similarity between the interaction signatures of all FDA-approved drugs and the identified BALF proteins. The drugs with the signatures most similar interaction signatures to approved COPD drugs were extracted with the CANDO platform. The analysis revealed 189 drugs that putatively target the proteins implicated in COPD. The putative COPD drugs that were identified using CANDO were subsequently analyzed using a knowledge based technique to identify an optimal two drug combination that had the most appropriate effect on the central node proteins. ConclusionAnalysis of the BALF proteome revealed novel differentially expressed proteins in the epithelial lining fluid that elucidate COPD pathogenesis. Network analyses identified critical targets that have critical roles in modulating COPD pathogenesis, for which we identified several drugs that could be repurposed to treat COPD using a multiscale shotgun drug discovery approach.

bioinformatics↗

bioRxiv2022

Arbovirus-vector protein interactomics identifies Loquacious as a co-factor for dengue virus replication in Aedes mosquitoes

Efficient virus replication in Aedes vector mosquitoes is essential for the transmission of arboviral diseases such as dengue virus (DENV) in human populations. Like in vertebrates, virus-host protein-protein interactions are essential for viral replication and immune evasion in the mosquito vector. Here, 79 mosquito host proteins interacting with DENV non-structural proteins NS1 and NS5 were identified by label-free mass spectrometry, followed by a functional screening. We confirmed interactions with host factors previously observed in mammals, such as the oligosaccharyltransferase complex, and we identified protein-protein interactions that seem to be specific for mosquitoes. Among the interactors, the double-stranded RNA (dsRNA) binding protein Loquacious (Loqs), an RNA interference (RNAi) cofactor, was found to be essential for efficient replication of DENV and Zika virus (ZIKV) in mosquito cells. Loqs did not affect viral RNA stability or translation of a DENV replicon and its proviral activity was independent of its RNAi regulatory activity. Interestingly, Loqs colocalized with DENV dsRNA in viral replication organelles in infected cells and directly interacted with high affinity with DENV RNA in the 3 untranslated region in vitro (KD = 100-200 nM). Our study provides an interactome for DENV NS1 and NS5 and identifies Loqs as a key proviral host factor in mosquitoes. We propose that DENV hijacks a factor of the RNAi mechanism for replication of its own RNA. AUTHOR SUMMARYDengue virus is a mosquito-transmitted virus endemic to the tropics and subtropics, affecting an estimated 390 million people yearly. While the mechanisms of infection, pathogenesis and immune evasion have been extensively studied in humans, replication in Aedes mosquitoes has received much less attention, despite being a critical step in the arbovirus transmission cycle. Here, we used a proteomic approach to identify Aedes mosquito proteins recruited by dengue virus non- structural proteins NS1 and NS5. In addition to previously established host proteins that interact with DENV in mammals, we identified Loquacious, a double-stranded RNA binding protein involved in the antiviral RNAi immune response of mosquitoes. Unexpectedly, our data showed Loquacious functions as a proviral factor that is recruited to replication organelles to facilitate viral RNA replication. We propose that DENV exploits host immune components, such as Loquacious, for its own benefit.

microbiology↗

bioRxiv2022

A reference single-cell regulomic and transcriptomic map of cynomolgus monkeys

Non-human primates (NHP) are attractive laboratory animal models that accurately reflect both developmental and pathological features of humans. Here we present a compendium of cell types from the cynomolgus monkey Macaca fascicularis (denoted as Monkey Atlas) using both single-cell chromatin accessibility (scATAC-seq) and RNA sequencing (scRNA-seq) data at the organism-wide level. The integrated cell map enables in-depth dissection and comparison of molecular dynamics, cell-type composition and cellular heterogeneity across multiple tissues and organs. Using single-cell transcriptomic data, we inferred pseudotime cell trajectories and cell-cell communications to uncover key molecular signatures underlying their cellular processes. Furthermore, we identified various cell-specific cis-regulatory elements and constructed organ-specific gene regulatory networks at the single-cell level. Finally, we performed a comparative analysis of single-cell landscapes among mouse, cynomolgus monkey and human, and we showed that cynomolgus monkey has significantly higher degree of cell-type similarity to human than mouse. Taken together, our study provides a valuable resource for NHP cell biology.

bioinformatics↗

bioRxiv2022

The interactome of Cryptococcus neoformans Rmt5 reveals multiple regulatory points in fungal cell biology and pathogenesis

Protein arginine methylation is a key post-translational modification in eukaryotes that modulates core cellular processes, including translation, morphology, transcription, and RNA fate. However, this has not been explored in Cryptococcus neoformans, a human-pathogenic basidiomycetous encapsulated fungus. We characterized the five protein arginine methyltransferases in C. neoformans and highlight Rmt5 as critical regulator of cryptococcal morphology and virulence. An rmt5{Delta} mutant was defective in thermotolerance, had a remodeled cell wall, and exhibited enhanced growth in an elevated carbon dioxide atmosphere and in chemically induced hypoxia. We revealed that Rmt5 interacts with post-transcriptional gene regulators, such as RNA-binding proteins and translation factors. Further investigation of the rmt5{Delta} mutant showed that Rmt5 is critical for the homeostasis of eIF2 and its phosphorylation state following 3-amino-1,2,4-triazole-induced ribosome stalling. RNA sequencing of one rmt5{Delta} clone revealed stable chromosome 9 aneuploidy that was ameliorated by complementation but did not impact the rmt5{Delta} phenotype. As a result of these diverse interactions and functions, loss of RMT5 enhanced phagocytosis by murine macrophages and attenuated disease progression in mice. Taken together, our findings link arginine methylation to critical cryptococcal cellular processes that impact pathogenesis, including post-transcriptional gene regulation by RNA-binding proteins. SignificanceThe fungal pathogen Cryptococcus neoformans is a huge threat for people living with immune deficits, especially HIV/AIDS. Its virulence potential is dependent on virulence factors, stress adaptation, and thermotolerance. Post-transcriptional gene regulation is important for these pathogenic processes, but the mechanisms that govern post-transcriptional regulator function are unexplored. Protein arginine methylation is a major modification of post-transcriptional regulators that has not been investigated in pathogenic fungi. Here we investigated the role of arginine methylation by arginine methyltransferases on the biology and virulence of C. neoformans. Phenotypic characterization of deletion mutants revealed pleiotropic functions for RMTs in this pathogen. Further investigation of the Rmt5 interactome using proximity-dependent biotinylation revealed interactions with RNA binding proteins and translation factors, thereby impacting virulence-associated processes.

microbiology↗

bioRxiv2021

Inter-species geographic signatures for tracing horizontal gene transfer and long-term persistence of carbapenem resistance

BackgroundCarbapenem-resistant Enterobacterales (CRE) are an urgent global health threat. Inferring the dynamics of local CRE dissemination is currently limited by our inability to confidently trace the spread of resistance determinants to unrelated bacterial hosts. Whole genome sequence comparison is useful for identifying CRE clonal transmission and outbreaks, but high-frequency horizontal gene transfer (HGT) of carbapenem resistance genes and subsequent genome rearrangement complicate tracing the local persistence and mobilization of these genes across organisms. MethodsTo overcome this limitation, we developed a new approach to identify recent HGT of large, near-identical plasmid segments across species boundaries, which also allowed us to overcome technical challenges with genome assembly. We applied this to complete and near-complete genome assemblies to examine the local spread of CRE in a systematic, prospective collection of all CRE, as well as time- and species-matched carbapenem susceptible Enterobacterales, isolated from patients from four U.S. hospitals over nearly five years. ResultsOur CRE collection comprised a diverse range of species, lineages and carbapenem resistance mechanisms, many of which were encoded on a variety of promiscuous plasmid types. We found and quantified rearrangement, persistence, and repeated transfer of plasmid segments, including those harboring carbapenemases, between organisms over multiple years. Some plasmid segments were found to be strongly associated with specific locales, thus representing geographic signatures that make it possible to trace recent and localized HGT events. Functional analysis of these signatures revealed genes commonly found in plasmids of nosocomial pathogens, such as functions required for plasmid retention and spread, as well survival against a variety of antibiotic and antiseptics common to the hospital environment. ConclusionsCollectively, the framework we developed provides a clearer, high resolution picture of the epidemiology of antibiotic resistance importation, spread, and persistence in patients and healthcare networks.

genomics↗

bioRxiv2021

Long-term in vivo imaging of mouse spinal cord through an optically cleared intervertebral window

Spinal cord, as part of the central nervous system, accounts for the main communication pathway between the brain and the peripheral nervous system. Spinal cord injury is a devastating and largely irreversible neurological trauma, and can result in lifelong disability and paralysis with no available cure. In vivo spinal cord imaging in mouse models without introducing immunological artifacts is critical to understand spinal cord pathology and discover effective treatments. We developed a minimal-invasive intervertebral window by retaining ligamentum flavum to protect the underlying spinal cord. By introducing an optical clearing method, we achieved repeated two-photon fluorescence and stimulated Raman scattering imaging at subcellular resolution with up to 16 imaging sessions over 167 days and observed no inflammatory response. Using this optically cleared intervertebral window, we studied the neuron-glia dynamics following laser axotomy and observed strengthened contact of microglia with the nodes of Ranvier during axonal degeneration. By enabling long-term, repetitive, stable, high-resolution and inflammation-free imaging of mouse spinal cord, our method provides a reliable platform in the research aiming at understanding and treatment of spinal cord pathology.

neuroscience↗

bioRxiv2021

Regional Cell Atlas of Human Intestine Shapes Distinct Immune Surveillance

Regional intestinal immune surveillance remains obscure. In this study, we integrated single-cell RNA sequencing and spatial transcriptomics to create a regional atlas of fetal and adult intestines, consisting of 59 cell subsets, of which eight new subsets and ILCs transition states were identified. Results revealed that microenvironment determines in-situ cell differentiation and shapes the regional molecular characteristics, allowing different intestinal segments with diverse functions. We characterized the regional expression of mucins, immunoglobulins, and antimicrobial peptides (AMPs) and their shift during development and in inflammatory bowel disease. Notably, -defensins expressed most abundantly in small intestinal LGR5+ stem cells, rather than in Paneth cells, and down-regulated as cell maturing. Common upstream transcription factors controlled the AMPs expression, illuminating the concurrent change of AMPs during epithelial differentiation, and the spatial co-expression patterns. We demonstrated the correspondence of cell focus of risk genes to diseases location susceptibility and identified distinct cell-cell crosstalk and spatial heterogeneity of immune cell homing in different gut segments. Overall, a cross-spatiotemporal approach to transcriptomes at single-cell resolution revealed that the regional milieu of the human intestine determined cellular and molecular cues of immune surveillance, dictating gut homeostasis and disease.

cell biology↗

bioRxiv2021

Direct focus sensing and shaping for high-resolution multi-photon imaging in deep tissue

High-resolution optical imaging of deep tissue in-situ such as the living brain is fundamentally challenging because of the aberration and scattering of light. In this work, we develop an innovative adaptive optics three-photon microscope based on direct focus sensing and shaping that can accurately measure and effectively compensate for both low- and high-order specimen-induced aberrations and recover near-diffraction-limited performance at depth. A conjugate adaptive optics configuration with remote focusing enables in vivo imaging of fine neuronal structures in the mouse cortex through the intact skull up to a depth of 750 {micro}m below pia, making high-resolution microscopy in cortex near non-invasive. Functional calcium imaging with high sensitivity and accuracy, and high-precision laser-mediated microsurgery through the intact skull were demonstrated. Moreover, we also achieved in vivo high-resolution imaging of the deep cortex and subcortical hippocampus up to 1.1 mm below pia within the intact brain.

bioengineering↗

bioRxiv2021

The endoplasmic reticulum-resident protein TMEM-120/TMEM120A promotes fat storage in C. elegans and mammalian cells

The synthesis of triacylglycerol (TAG) is essential for the storage of excess fatty acids, which can subsequently be used for energy or cell growth. A series of enzymes act in the endoplasmic reticulum (ER) to synthesize TAG, prior to its transfer to lipid droplets (LDs), which are conserved organelles for fat storage. Here, we report that the deficiency of TMEM-120/TMEM120A, a protein with 6-transmembrane helices, retards TAG synthesis and LD expansion in C. elegans. A missense mutation near the predicted coenzyme A binding site of TMEM-120 confers strong loss of function phenotypes. GFP fusion proteins of TMEM-120, expressed at the endogenous level in live worms, were observed throughout the ER network. Using Stimulated Raman Scattering, we discovered a specific requirement of TMEM-120 in the storage of exogenous fatty acids in LDs. Knockdown of TMEM120A impedes adipogenesis of pre-adipocytes in vitro, while its over-expression is sufficient to promote LD expansion. Pharmacological studies indicate that TMEM120A most likely acts upstream of diacylglycerol O-acyltransferase 1 (DGAT1). Our results suggest that TMEM-120/TMEM120A plays a conserved role in increasing the efficiency of TAG synthesis.

cell biology↗