Biology subjects

Wang, B.

Publications and source records attributed to Wang, B..

At least 451 records · Page 25Linked to original sources

bioRxiv2022

Comparing acute IOP-induced lamina cribrosa deformations pre-mortem and post-mortem

PurposeLamina cribrosa (LC) deformations caused by elevated intraocular pressure (IOP) are believed to contribute to glaucomatous neuropathy and have therefore been extensively studied, in many conditions from in-vivo to ex-vivo. We compare acute IOP-induced global and local LC deformations immediately before (pre-mortem) and after (post-mortem) sacrifice by exsanguination. MethodsThe optic nerve heads of three healthy monkeys 12-15 years old were imaged with spectral-domain optical coherence tomography under controlled IOP pre-mortem and post-mortem. Volume scans were acquired at baseline IOP (8-10 mmHg) and at 15, 30, and 40 mmHg IOP. A digital volume correlation technique was used to determine the IOP-induced 3D LC deformations (strains) in regions visible pre-mortem and post-mortem. ResultsBoth conditions exhibited similar nonlinear relationships between IOP increases and LC deformations. Median effective and shear strains were, on average over all eyes and pressures, smaller post-mortem than pre-mortem, by 14% and 11%, respectively (Ps < 0.001). Locally, however, the differences in LC deformation between conditions were variable. Some regions were subjected pre-mortem to triple the strains observed post-mortem, and others suffered smaller deformations pre-mortem than post-mortem. ConclusionsIncreasing IOP acutely caused nonlinear LC deformations with an overall smaller effect post-mortem than pre-mortem. Locally, deformations pre-mortem and post-mortem were sometimes substantially different. We suggest that the differences may be due to weakened mechanical support from the unpressurized central retinal vessels post-mortem. Translational RelevanceAdditional to the important pre-mortem information, comparison with post-mortem provides a unique context essential to understand the translational relevance of all post-mortem biomechanics literature. PrecisThe authors compared in monkeys acute IOP-induced deformations of the lamina cribrosa pre-mortem and post-mortem. Deformation trends were similar pre-mortem and post-mortem, but deformations pre-mortem were generally smaller than those post-mortem, with substantial local variations. The differences are likely due to loss of vessel support post-mortem.

bioengineering↗

bioRxiv2022

GPCR Signaling Promotes a Stress-induced Genetic Program for Phenoptosis in C. elegans

How an organism dies is a fundamental yet poorly understood question in biology. An organism can die of many causes, including stress-induced phenoptosis, also defined as organismic death that is regulated by its genome-encoded programs. The mechanism of stress-induced phenoptosis is still largely unknown. Here we show that transient but severe freezing-thaw stress (FTS) in C. elegans induces rapid and robust phenoptosis that is regulated by G-protein coupled receptor (GPCR) signaling. RNAi screens identify the GPCR-encoding fshr-1 in mediating transcriptional responses to FTS. FSHR-1 increases ligand interaction upon FTS and activates a cyclic AMP-PKA cascade leading to a genetic program to promote organismic death under severe stress. FSHR-1/GPCR signaling up-regulates the bZIP-type transcription factor ZIP-10, linking FTS to expression of genes involved in lipid remodeling, proteostasis and aging. A mathematical model suggests that genes may promote organismic death under severe stress conditions, potentially benefiting growth of the clonal population with individuals less stressed and more reproductively privileged. Our studies reveal roles of FSHR-1/GPCR-mediated signaling in stress-induced gene expression and phenoptosis in C. elegans, providing empirical new insights into mechanisms of stress-induced phenoptosis with evolutionary implications.

developmental biology↗

bioRxiv2022

Haplotype-resolved assemblies and variant benchmark of a Chinese Quartet

As the state-of-the-art sequencing technologies and computational methods enable investigation of challenging regions in the human genome, an update variant benchmark is demanded. Herein, we sequenced a Chinese Quartet, consisting of two monozygotic twin daughters and their biological parents, with multiple advanced sequencing platforms, including Illumina, BGI, PacBio, and Oxford Nanopore Technology. We phased the long reads of the monozygotic twin daughters into paternal and maternal haplotypes using the parent-child genetic map. For each haplotype, we utilized advanced long reads to generate haplotype-resolved assemblies (HRAs) with high accuracy, completeness, and continuity. Based on the ingenious quartet samples, novel computational methods, high-quality sequencing reads, and HRAs, we established a comprehensive variant benchmark, including 3,883,283 SNVs, 859,256 Indels, 9,678 large deletions, 15,324 large insertions, 40 inversions, and 31 complex structural variants shared between the monozygotic twin daughters. In particular, the preciously excluded regions, such as repeat regions and the human leukocyte antigen (HLA) region, were systematically examined. Finally, we illustrated how the sequencing depth correlated with the de novo assembly and variant detection, from which we learned that 30 x HiFi is a balance between performance and cost. In summary, this study provides high-quality haplotype-resolved assemblies and a variant benchmark for two Chinese monozygotic twin samples. The benchmark expanded the regions of the previous report and adapted to the evolving sequencing technologies and computational methods.

genomics↗

bioRxiv2022

Unsupervised Restoration of a Complex Learned Behavior After Large-Scale Neuronal Perturbation

Reliable execution of behaviors requires that brain circuits correct for variations in neuronal dynamics. Genetic perturbation of the majority of excitatory neurons in a brain region involved in song production in adult songbirds with stereotypical songs triggered severe degradation of their songs. The song fully recovered within two weeks, and substantial improvement occurred even when animals were prevented from singing during the recovery period, indicating that offline mechanisms enable recovery in an unsupervised manner. Song restoration was accompanied by increased excitatory synaptic inputs to unmanipulated neurons in the same region. A model inspired by the behavioral and electrophysiological findings suggests that a combination of unsupervised single-cell and population-level homeostatic plasticity rules can support the observed functional restoration after large-scale disruption of networks implementing sequential dynamics. In the model the sequence is restored through a parallel homeostatic process, rather than regrown serially, and predicts that sequences should recover in a saltatory fashion. Correspondingly, we observed such recovery in the songs of manipulated animals, with syllables that rapidly alternate between abnormal and normal durations from rendition to rendition until eventually they permanently settled into their original length. These observations indicate the existence of cellular and systems-level restorative mechanisms that ensure behavioral resilience.

neuroscience↗

bioRxiv2022

Oocyte and cumulus cell cooperativity and metabolic plasticity under the direction of oocyte paracrine factors

Mammalian oocytes develop and mature in a mutually dependent relationship with surrounding cumulus cells. The oocyte actively regulates cumulus cell differentiation and function by secreting soluble paracrine oocyte-secreted factors (OSFs). We characterized the molecular mechanisms by which two model OSFs, cumulin and BMP15, regulate oocyte maturation and cumulus-oocyte cooperativity. Exposure to these OSFs during maturation altered the proteomic and multispectral autofluorescence profiles of both the oocyte and cumulus cells. In oocytes, cumulin significantly upregulated proteins involved in nuclear function. In cumulus cells, both OSFs elicited marked upregulation of a variety of metabolic processes (mostly anabolic), including lipid, nucleotide, and carbohydrate metabolism, while mitochondrial metabolic processes were downregulated. The mitochondrial changes were validated by functional assays confirming altered mitochondrial morphology, respiration, and content, whilst maintaining ATP homeostasis. Collectively, these data demonstrate that OSFs remodel cumulus cell metabolism during oocyte maturation in preparation for ensuing fertilization and embryonic development. HIGHLIGHTSO_LIDuring oocyte maturation, oocyte-secreted factors promote cell cooperativity between the oocyte and cumulus cells by altering the molecular composition of both cell types. C_LIO_LIOocyte-secreted factors downregulate protein catabolic processes, and upregulate DNA binding, translation, and ribosome assembly in oocytes. C_LIO_LIOocyte-secreted factors alter mitochondrial number, morphology, and function in cumulus cells. C_LIO_LIOocyte-secreted factors further enhance metabolic plasticity in cumulus cells by upregulating anabolic pathways for macromolecules and small molecule organics. C_LIO_LIThe oocyte, via oocyte-secreted factors, instructs cumulus cells to increase metabolic workload on its behalf, thereby subduing oocyte metabolism. C_LI

developmental biology↗

bioRxiv2022

Release of lipid nanodiscs from charged nano-droplets in the electrospray ionization process revealed by microsecond atomistic molecular dynamics simulations

Electrospray ionization (ESI) prevents the analyte being split into fragments and is widely used to analyze biomolecules. Nanodiscs provide a native-like environment for membrane proteins, while making them accessible in aqueous solution for analysis. We performed microsecond (s) molecular dynamics simulations at different temperatures, to provide atomistic insight in the release of intact nanodiscs from charged nano-droplets, the late stage of the ESI process. Two distinct main scenarios, at-center and off-center, were observed. The at-center scenario follows the charged residue model, and in the process the nanodisc stays well in the droplet interior, with the membrane scaffold protein (MSP) keeping its annular geometry. As solvent evaporates, lipids turn over to protect the hydrophilic surface. The off-center scenario conforms to a hybrid model of the charged residue model and chain ejection model. The nanodisc migrates to the water/air interface, leading to lipids escaping from the dissociated dimer and the partial ejection of one monomer of MSP. A diversity of morphologies of product gaseous ions was observed, as evidenced by their structural details, collision cross sections and moments of inertia. Our results are in reasonable agreement with results of mass spectrometry in mass-to-charge ratio, charge state and collision cross section. Our work displays the first atomistic view of a heterogeneous system, a lipid nanodisc, releasing from highly charged nano-droplets into the gas phase, which may provide structural supports for the interpretation of mass spectra of complicated systems. What we observe in our simulations could also be generalized to other protein-lipid systems.

biophysics↗

bioRxiv2022

P1 bacteriophage-enabled delivery of CRISPR-Cas9 antimicrobial activity against Shigella flexneri

The discovery of clustered, regularly interspaced, short palindromic repeats (CRISPR) and the Cas9 RNA-guided nuclease provides unprecedented opportunities to selectively kill specific populations or species of bacteria. However, the use of CRISPR-Cas9 to clear bacterial infections in vivo is hampered by the inefficient delivery of cas9 genetic constructs into bacterial cells. Here, we use a broad-host-range P1-derived phagemid to deliver the CRISPR-Cas9 chromosomal-targeting system into Escherichia coli and the dysentery-causing Shigella flexneri to achieve DNA sequence-specific killing of targeted bacterial cells. We show that genetic modification of the helper P1 phage DNA packaging site (pac) significantly enhances the purity of packaged phagemid and improves the Cas9-mediated killing of S. flexneri cells. We further demonstrate that P1 phage particles can deliver chromosomal-targeting cas9 phagemids into S. flexneri in vivo using a zebrafish larvae infection model, where it significantly reduces the bacterial load and promotes host survival. Our study highlights the potential of combining a P1 bacteriophage-based delivery with the CRISPR chromosomal-targeting system to achieve DNA sequence-specific cell lethality and efficient clearance of bacterial infection.

microbiology↗

bioRxiv2022

Optimized Monophasic Pulses with Equivalent Electric Field for Rapid-Rate Transcranial Magnetic Stimulation

ObjectiveTranscranial magnetic stimulation (TMS) with monophasic pulses achieves greater changes in neuronal excitability but requires higher energy and generates more coil heating than TMS with biphasic pulses, and this limits the use of monophasic pulses in rapid-rate protocols. We sought to design a stimulation waveform that retains the characteristics of monophasic TMS but significantly reduces coil heating, thereby enabling higher pulse rates and increased neuromodulation effectiveness. ApproachA two-step optimization method was developed that uses the temporal relationship between the electric field (E-field) and coil current waveforms. The model-free optimization step reduced the ohmic losses of the coil current and constrained the error of the E-field waveform compared to a template monophasic pulse, with pulse duration as a second constraint. The second, amplitude adjustment step scaled the candidate waveforms based on simulated neural activation to account for differences in stimulation thresholds. The optimized waveforms were implemented to validate the changes in coil heating. Main resultsDepending on the pulse duration and E-field matching constraints, the optimized waveforms produced 12% to 75% less heating than the original monophasic pulse. The reduction in coil heating was robust across a range of neural models. The changes in the measured ohmic losses of the optimized pulses compared to the original pulse agreed with numeric predictions. SignificanceThe first step of the optimization approach was independent of any potentially inaccurate or incorrect model and exhibited robust performance by avoiding the highly nonlinear behavior of neural responses, whereas neural simulations were only run once for amplitude scaling in the second step. This significantly reduced computational cost compared to iterative methods using large populations of candidate solutions and more importantly reduced the sensitivity to the choice of neural model. The reduced coil heating and power losses of the optimized pulses can enable rapid-rate monophasic TMS protocols.

bioengineering↗

bioRxiv2022

Mechanistic Deep Learning Framework on Cell Traits Derived from Single-Cell Mass Cytometry Data

Germline genetic variations can alter cellular differentiation, potentially impacting the response of immune cells to inflammatory challenges. Current variant- and gene-based methods in mouse and human models have established associations with disease phenotypes; however, the underlying mechanisms at the cellular level are less well-understood. Immunophenotyping by multi-parameter flow cytometry, and more recently mass cytometry, has allowed high-resolution identification and characterization of hematopoietic cells. The obtained characterization yields increased dimensionality; however, conventional analysis workflows have been inefficient, incomplete, or unreliable. In this work, we develop a comprehensive machine learning framework - MDL4Cyto - that is tailored to the analysis of mass cytometry data, incorporating statistical, unsupervised learning, and supervised learning models. The statistical modeling can be used to illuminate cell fate decision and cell-type dynamics. The unsupervised learning models along with complementary marker enrichment analyses highlight genetic perturbations that are significantly associated with alterations in cell populations in the hematopoietic system. Furthermore, our supervised learning models, including deep learning and tree-based algorithms, address the bottleneck to data pre-processing that characterizes conventional workflows and generate inferences (e.g., on marker/cell-type interactions) from raw experimental characterization. Notably, we reveal a close relationship among network design, prediction performance, and the underlying biological context. We show that the network architecture extracted from the differentiation cascade of the investigated biological system yields enhanced prediction performance. The presented methodology will enable new insights into hematopoietic differentiation at baseline and following perturbation. HighlightsO_LIAnalysis pipeline on mass cytometry data with high-performance implementation of statistical, unsupervised learning, and supervised learning models C_LIO_LIConcordance of machine learning results with biological contexts C_LIO_LIBiologically-informed neural network designs enhance prediction performance C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/504669v3_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@10e35cborg.highwire.dtl.DTLVardef@1edc8ecorg.highwire.dtl.DTLVardef@220726org.highwire.dtl.DTLVardef@35916_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2022

A megaprotein-based molecular bridge critical for lipid trafficking and cold resilience

Cells adapt to cold by increasing levels of unsaturated phospholipids and membrane fluidity through homeostatic mechanisms conserved in nearly all forms of life. As most eukaryotic enzymes for lipid synthesis and desaturation localize on endoplasmic reticulum (ER) membranes, it remains unknown how ER-resident lipids rapidly distribute to plasma membranes (PM). Here we report an exceptionally large and evolutionarily conserved protein LPD-3 in C. elegans that plays critical roles in lipid trafficking and cold resilience. We identified lpd-3 mutants in a mutagenesis screen for genetic suppressors of the lipid desaturase FAT-7, and found that the 452 kDa megaprotein LPD-3 bridges ER and PM, consisting of a structurally predicted hydrophobic tunnel for lipid trafficking. Loss of LPD-3 caused abnormal cellular distribution of phospholipids, diminished FAT-7 abundance, and organismic vulnerability to cold. These phenotypic defects of lpd-3 mutants were rescued by Lecithin comprising unsaturated phospholipids. Importantly, we found that deficient lpd-3 homologues in Zebrafish and mammalian cells led to defects similar to those observed in C. elegans. As mutations in KIAA1109/BLTP1, the human orthologue of lpd-3, cause Alkuraya-Kucinskas syndrome, we propose that the LPD-3 family proteins may serve as evolutionarily conserved "highway bridges" critical for ER-associated non-vesicular trafficking of lipids and resilience to cold stress in eukaryotic cells.

cell biology↗

bioRxiv2022

Parallel molecular computation on digital data stored in DNA

DNA is an incredibly dense storage medium for digital data, but computing on the stored information is expensive and slow (rounds of sequencing, in silico computation, and DNA synthesis). Augmenting DNA storage with "in-memory" molecular computation, we use strand displacement reactions to algorithmically modify data stored in the topological modification of DNA. A secondary sequence-level encoding allows high-throughput sequencing-based readout. We show multiple rounds of binary counting and cellular automaton Rule 110 computation on 4-bit data registers, as well as selective access and erasure. Avoiding stringent sequence design, we demonstrate large strand displacement cascades (122 distinct steps) on naturally-occurring DNA sequences. Our work merges DNA storage and DNA computing, setting the foundation of entirely molecular algorithms for parallel manipulation of digital information kept in DNA.

bioengineering↗

bioRxiv2022

Alleviating cell lysate-induced inhibition to enable RT-PCR from single cells in picoliter-volume double emulsion droplets

Microfluidic droplet assays enable single-cell PCR and sequencing assays at unprecedented scale, with most methods encapsulating cells within nanoliter sized single emulsion droplets (water-in-oil). Encapsulating cells within picoliter double emulsion (DE) (water-in-oil-in-water) droplets allows sorting on commercially available FACS machines, making it possible to isolate single cells based on phenotypes of interest for downstream sequencing and PCR analyses. However, DE droplets must be within the range of large cells (<20 pl) for sorting on standard cytometers, posing challenges for molecular biology as prior reports suggest that reverse transcription (RT) and PCR amplification cannot proceed efficiently at volumes below 1 nL due to cell lysate-induced inhibition. To overcome this limitation, we used a plate-based RT-PCR assay designed to mimic reactions in picoliter droplets to systematically quantify and ameliorate the inhibition. We find that RT-PCR is blocked by lysate-induced cleavage of nucleic acid probes and primers, but that this cleavage can be efficiently alleviated through heat lysis that simultaneously inactivates inhibitory lysate components. We further show that the magnitude of RT-PCR inhibition depends strongly on cell type, but that RT-PCR can proceed in low-picoscale reaction volumes for most cell lines tested. Finally, we demonstrate one-step RT-PCR from single cells in 20 pL double emulsion droplets with fluorescence detectable via FACS. These results open up exciting new avenues for improving picoscale droplet RT-PCR reactions and expanding microfluidic droplet based single-cell analysis technologies.

bioengineering↗

bioRxiv2022

Effects of Cr stress on bacterial community structure and diversity in rhizosphere soils of Iris pseudacorus

Rhizosphere microorganisms play an important role in improving soil microenvironment, which contributes to plant growth under heavy metal stress. However, the effect of chromium (Cr) on plant rhizosphere bacterial community is still unknown. In this paper, sole-cultivated pattern, two-cultivated pattern and three-cultivated pattern, combined with 16S rRNA high-throughput sequencing technology, the effects of Cr stress on bacterial community structure and diversity in rhizosphere soil of Iris Pseudacorus were analyzed. The results showed that under Cr stress, I. Pseudacorus showed good tolerance and enrichment. However, under Cr stress, the Alpha diversity indices (Shannon, Chao and Sobs) of rhizosphere bacterial community decreased by 9.1%, 30.3% and 28.0% on average, respectively. The change of bacterial community was 22.6% due to Cr stress, and the common species of bacterial community decreased by 4.2%. Proteobacteria, Actinobacteria, Acidobacteria, Firmicutes and Gemmatimonadetes accounted for more than 78.2% of the total sequence. With the increase of plant diversity, Bacteroides and Pseudomonas appeared successively, and the abundance of the dominant species increased obviously. Through the symbiotic network diagram, it was found that the synergistic effect between dominant species in two-cultivated pattern was significantly enhanced, and the soil microenvironment was significantly improved. In conclusion, the results of this study will provide a reference for understanding the response of rhizosphere bacterial community to heavy metal Cr and the interaction between wetland plants and rhizosphere bacteria during wetland phytoremediation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/503686v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1d5949eorg.highwire.dtl.DTLVardef@1103c11org.highwire.dtl.DTLVardef@d7b700org.highwire.dtl.DTLVardef@2f9df4_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2022

Targeting Epsins to Inhibit FGF Signaling while Potentiating TGF-β Signaling Constrains Endothelial-to-Mesenchymal-Transition in Atherosclerosis

BACKGROUNDEpsin endocytic adaptor proteins are implicated in the progression of atherosclerosis; however, the underlying molecular mechanisms have not yet been fully defined. In this study, we determined how epsins enhance endothelial-to-mesenchymal transition (EndoMT) in atherosclerosis and assessed the efficacy of a therapeutic peptide in a preclinical model of this disease. METHODSUsing single cell RNA sequencing (scRNA-seq), combined with molecular, cellular, and biochemical analyses, we investigated the role of epsins in stimulating EndoMT using knock-out mouse models. The therapeutic efficacy of a synthetic peptide targeting atherosclerotic plaques was then assessed in Apoe-/- mice. RESULTSScRNA-seq and lineage tracing revealed that epsins 1 and 2 promote EndoMT, and the loss of endothelial epsins inhibits EndoMT marker expression as well as transforming growth factor-beta signaling in vitro and in atherosclerotic mice, which is associated with smaller lesions in Apoe-/- mouse model. Mechanistically, the loss of endothelial cell epsins results in increased fibroblast growth factor receptor-1 (FGFR1) expression that inhibits TGF-{beta} signaling and EndoMT. Epsins directly bind ubiquitinated FGFR1 through their ubiquitin-interacting motif (UIM), which results in endocytosis and degradation of this receptor complex. Consequently, administration of a synthetic UIM-containing peptide API significantly attenuates EndoMT and progression of atherosclerosis. CONCLUSIONSWe conclude that epsins potentiate EndoMT during atherogenesis by increasing TGF-{beta} signaling through FGFR1 internalization and degradation. Inhibition of EndoMT by reducing epsin-FGFR1 interaction with a therapeutic peptide may represent a novel treatment strategy for atherosclerosis.

cell biology↗

bioRxiv2022

Targeting Epsins by nanotherapy regulates lipid metabolism and promotes 1 ABCG1-mediated cholesterol efflux to fortify atheroma regression

BACKGROUNDExcess cholesterol accumulation in lesional macrophages elicits complex responses in atherosclerosis. Epsins, a family of endocytic adaptors, fuel the progression of atherosclerosis; however, the underlying mechanism and therapeutic potential of targeting Epsins remains unknown. In this study, we determined the role of Epsins in macrophage-mediated metabolic regulation. We then developed an innovative method to therapeutically-target macrophage Epsins with specially-designed S2P-conjugated lipid nanoparticles (NPs), which encapsulate small interfering RNAs to suppress Epsins. METHODSWe used single cell RNA sequencing (scRNA-seq) with our newly developed algorithm MEBOCOST to study cell-cell communications mediated by metabolites from sender cells and sensor proteins on receiver cells. Biomedical, cellular and molecular approaches were utilized to investigate the role of macrophage Epsins in regulating lipid metabolism and transport. We performed this study using myeloid-specific Epsin double knockout (LysM-DKO) mice and mice with a genetic reduction of ABCG1 (LysM-DKO-ABCG1fl/+). The NPs targeting lesional macrophages were developed to encapsulate interfering RNAs to treat atherosclerosis. RESULTSWe revealed that Epsins regulate lipid metabolism and transport in atherosclerotic macrophages. Inhibiting Epsins by nanotherapy halts inflammation and accelerates atheroma resolution. Harnessing lesional macrophage-specific NP delivery of Epsin siRNAs, we showed that silencing of macrophage Epsins markedly diminished atherosclerotic plaque size and promoted plaque regression. Mechanistically, we demonstrated that Epsins bound to CD36 to facilitate lipid uptake by enhancing CD36 endocytosis and recycling. Conversely, Epsins promoted ABCG1 degradation via lysosomes and hampered ABCG1-mediated cholesterol efflux and reverse cholesterol transport. In a LysM-DKO-ABCG1fl/+ mouse model, enhanced cholesterol efflux and reverse transport due to Epsin deficiency was suppressed by the reduction of ABCG1. CONCLUSIONSOur findings suggest that targeting Epsins in lesional macrophages may offer therapeutic benefits for advanced atherosclerosis by reducing CD36-mediated lipid uptake and increasing ABCG1-mediated cholesterol efflux. Novelty and SignificanceO_ST_ABSWHAT IS KNOWN?C_ST_ABSO_LIEpsin endocytic adaptor proteins are upregulated in human and mouse atherosclerotic lesions C_LIO_LILesional macrophages internalize lipids primarily through scavenger receptor-mediated endocytosis such as CD36 and SR-A C_LIO_LIMacrophage-mediated cholesterol efflux and reverse cholesterol transport is crucial to atheroma resolution C_LI WHAT NEW INFORMATION DOES THIS ARTICLE CONTRIBUTE?O_LIScRNA-seq combined with the newly-developed algorithm MEBOCOST reveals that Epsins are involved in macrophage-mediated lipid metabolic regulation C_LIO_LIMacrophage epsins promote lipid uptake by targeting CD36 endocytosis and membrane recycling via the Epsin ENTH domain C_LIO_LIEpsins bind ubiquitinated ABCG1--resulting in the endocytosis and lysosomal degradation of this cholesterol transporter, which reduces cholesterol efflux. C_LIO_LIMacrophage-specific, nanoparticle-mediated RNAi delivery exhibits a therapeutic benefit for the treatment of atherosclerosis C_LIO_LIAtherosclerotic plaque regression using this nanoparticle delivery platform represents a clinically-relevant approach for the treatment of advance atherosclerosis C_LI BRIEF SUMMARYDespite the development of new cholesterol-lowering therapies, including the recently approved PCSK9 small interfering RNA (siRNA) antagonists, patients still face a tremendous risk of developing major acute cardiovascular events resulting from chronic inflammation in the plaque. We employed a novel nanomedicine platform containing a stabilin-2 targeting peptide (S2P) to deliver Epsin-specific siRNAs to lesional macrophages. We discovered that inhibition of these adaptor proteins in lesional macrophages significantly diminished plaque size and necrotic core area, increased fibrous cap thickness, and promoted plaque regression.

cell biology↗

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

HiCAT: A tool for automatic annotation of centromere structure

Significant improvements in long-read sequencing technologies have unlocked complex genomic areas, such as centromeres, in the genome and introduced the centromere annotation problem. Currently, centromeres are annotated in a semi-manual way. Here, we propose HiCAT, a generalizable automatic centromere annotation tool, based on hierarchical tandem repeat mining and maximization of tandem repeat coverage to facilitate decoding of centromere architecture. We applied HiCAT to human CHM13-T2T and gapless Arabidopsis thaliana genomes. Our results not only were generally consistent with previous inferences but also greatly improved annotation continuity and revealed additional fine structures, demonstrating HiCATs performance and general applicability.

bioinformatics↗

bioRxiv2022

Yeast and Mammalian Epsins Use Different Determinants for Localization and Function: Role of Clathrin/AP2/Ubiquitin Binding Motifs and Poly-Glutamine Stretches

Epsins are endocytic adaptor proteins involved in the internalization of important membrane proteins such as EGFR and Notch ligands. Therefore, this protein family impacts critical signaling pathways and processes such as cell migration and cytokinesis and is ultimately required for embryo development in mammals and cell viability in yeast. Intriguingly, although Epsins are conserved and display similar binding determinants, the process of endocytosis in yeast and mammals exhibit some dramatic mechanistic differences. Therefore, we wondered if the function of Epsins in these organisms are similar and are similarly regulated or they also differ. Since proper and timely localization is needed for function, we determined what elements target Epsins to endocytic sites in yeast vs mammals. Specifically, using a systematic/combinatorial mutagenesis approach we produced a collection of yeast and human Epsin mutated variants that was tested for localization at endocytic sites and for function. Our results showed that the intrinsically disordered carboxy-terminus holds the major determinants (involved in binding of ubiquitin, AP2, clathrin and EH domain-containing proteins) for proper intracellular localization of different Epsin paralogs and homologs in yeast and mammals, while also having a major impact on function. Importantly, we established hierarchies of carboxy-terminal binding determinants for sustaining Epsin localization which turned to be different for human vs. yeast cells; favoring clathrin and AP2 binding in the former and recognition of cargo and EH domain-containing proteins for the latter. Further, we found evidence in both systems that yeast Epsins also use for localization regions of the protein that were until now of unknown functional relevance, i.e., glutamine-rich sequences. Interestingly, some molecular determinants within the Epsin molecule seem to have functional importance beyond its contribution to localization to endocytic sites. Based on these findings, we propose working models for Epsin function and recruitment to membranes/endocytic sites at different maturation stages.

cell biology↗