Biology subjects

Yan, W.

Publications and source records attributed to Yan, W..

At least 91 records · Page 5Linked to original sources

bioRxiv2021

miRNA-dependent poly(A) length control in uncoupling transcription and translation of haploid male germ cells

As one of the post-transcriptional regulatory mechanisms, transcription and translations uncoupling plays an essential role in development and adulthood physiology. However, it remains elusive how thousands of mRNAs get translationally silenced while stability is maintained for up to hours or even days before translation. In addition to oocytes and neurons, developing spermatids have significant uncoupling of transcription and translation for delayed translation. Therefore, spermiogenesis represents an excellent in vivo model for investigating the mechanism underlying uncoupled transcription and translation. Through full-length poly(A) deep sequencing, we discovered dynamic changes in poly(A) length through deadenylation and re-polyadenylation. Deadenylation appeared to be mediated by microRNAs (miRNAs), and transcripts with shorter poly(A) tails tend to be sequestered into ribonucleoproteins (RNPs) for translational repression and stabilization. In contrast, re-polyadenylation allows for translocation of the translationally repressed transcripts from RNPs to polysomes for translation. Overall, our data suggest that miRNA-dependent poly(A) length control represents a novel mechanism underlying uncoupled translation and transcription in haploid male germ cells.

molecular biology↗

bioRxiv2021

Immunoengineered Nanobead System for the Isolation and Detection of Circulating Tumor Cells

Highly efficient capture and detection of circulating tumor cells (CTCs) remain elusive mainly because of their extremely low concentration in the peripheral blood of patients. Herein, we present an approach for the simultaneous capturing, isolation, and detection of CTCs using an immuno-fluorescent magnetic nanobead system (iFMNS) coated with a monoclonal anti-EpCAM antibody. The developed antibody nanobead system allows magnetic isolation and fluorescent-based quantification of CTCs. The expression of EpCAM on the surface of captured CTCs could be directly visualized without additional immune-fluorescent labeling. Our approach is shown to result in a 70 - 95% capture efficiency of CTCs, and 95% of the captured cells remain viable. Using our approach, the isolated cells could be directly used for culture, reverse transcription-polymerase chain reaction (RT-PCR), and immunocytochemistry (ICC) identification. We applied iFMNS for testing CTCs in peripheral blood samples from a lung cancer patient, which suggested that this approach would be a promising tool for CTCs enrichment and detection in one step.

bioengineering↗

bioRxiv2021

A sexual dimorphic role of the miR-465 cluster in placental development

A sexually dimorphic role of miRNAs in placental development has never been reported. Here, we show that ablation of the miR-465 cluster caused selective degeneration of female conceptuses as early as embryonic day (E)8.5, leading to a male-biased sex ratio (60% males) among miR-465 KO mice. Given that the miR-465 cluster miRNAs were predominantly expressed in the extraembryonic tissue and ablation of these miRNAs led to dysregulation of numerous critical placental genes, our data strongly suggest the miR-465 cluster is required for full developmental potential of the female, but not the male, extraembryonic tissue/placenta.

developmental biology↗

bioRxiv2021

Identification of Nocardia species using Autof MS 1000 and Bruker MALDI-TOF MS systems: A comparison

Nocardia is an important cause of clinically invasive disease, but for most clinical laboratories, identification of these isolates to the species level is challenging. Recently, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely used for identification of most bacterial and fungal isolates. In this multicenter study, we evaluated the identification of Nocardia isolates using Autof MS1000 and Bruker Biotyper. A total of 86 non-duplicate Nocardia isolates from 7 hospital laboratories were evaluated. Further, we carried out sequence analysis of 16S rRNA, gyrB, secA1, hsp65, and rpoB genes as a reference method for Nocardia species identification. The 86 isolates were directly spotted on the target plate and plate protein extraction was performed. Data were analyzed by SPSS 19.0. In total, 72 (83.7%) strains (score [≥] 9.0) and 70 (81.4%) strains (score [≥] 2.0) were correctly identified by the Autof MS1000 and Bruker Biotyper systems, respectively, at the species level. There was no significant difference (P > 0.05) between the two systems using the same protein extraction method. In conclusion, the Autof MS 1000 and Bruker MALDI-TOF systems showed no difference in identification of Nocardia spp. to the species level and could meet the most important clinical requirement for species identification.

microbiology↗

bioRxiv2020

Single-nuclei RNA-sequencing of plants

Single-cell genomics provides unprecedented potential for research on plant development and environmental responses. Here, we introduce a generic procedure for plant nuclei isolation combined with nanowell-based library preparation. Our method enables the transcriptome analysis of thousands of individual plant nuclei. It serves as alternative to the use of protoplast isolation, which is currently a standard methodology for plant single-cell genomics, although it can be challenging for some plant tissues. We show the applicability of our nuclei isolation method by using different plant materials from several species. The potential of our snRNA-seq method is shown through the characterization of transcriptomes of seedlings and developing flowers from Arabidopsis thaliana. We evaluated the transcriptome dynamics during the early stages of anther development, identify stage-specific transcription factors regulating this process and the prediction of their target genes. Our nuclei isolation procedure can be applied in different plant species and tissues, thus expanding the toolkit for plant single-cell genomics experiments. SIGNIFICANCE STATEMENTWe introduce an optimized plant nuclei isolation procedure followed by single nuclei RNA-seq that can be applied to different plant tissues without the need for protoplast isolation.

genomics↗

bioRxiv2020

Maternal transmission of a paramutant phenotype requires intact DNMT2 functions in the male germline

INTRODUCTION Abstract INTRODUCTION Results DISCUSSION METHODS AND MATERIALS SUPPLEMENTARY MATERIALS Funding Author contributions Competing interests Data and materials availability Supplemental Figures REFERENCES DNA methylation, as a major epigenetic modification in the genome of higher eukaryotes, is catalyzed by DNA methyltransferase enzymes (DNMTs) by addition of a methyl group to the 5th carbon position of cytosine, resulting 5-methylcytosine (5mC) (1, 2). DNMT1 is the most abundant DNA methyltransferase in mammalian cells, and mainly methylate hemi-methylated CpG dinucleotides as a maintenance methyltransferase, although DNMT1 has also been ...

genetics↗

bioRxiv2020

Overcoming resistance to immune checkpoint therapy in PTEN-null prostate cancer by sequential intermittent anti-PI3Ka/b/δ and anti-PD-1 treatment

Prostate cancers generally lack T cell infiltration and display resistance to immune checkpoint therapies (ICT). We found that intermittent but not daily dosing of PI3K/{beta}/{delta} inhibitor BAY1082439 on a Pten-null spontaneous prostate cancer model could overcome ICT resistance and unleash CD8+ T cell-dependent anti-tumor immunity in vivo. Mechanistically, BAY1082439 converts Pten-null cancer cell-intrinsic immune-suppression to immune-stimulation by promoting IFN/{gamma} pathway activation, {beta}2-microglubin expression and CXCL10/CCL5 secretion. Together with its preferential Treg inhibition activity, BAY1082439 promotes clonal expansion of tumor-associated CD8+ T cells. Once primed, tumors remain as T cell-inflamed and become responsive to anti-PD-1 therapy. Our data suggest that intermittent PI3K inhibition can alleviate Pten-null cancer cell-intrinsic immunosuppressive activity and turn "cold" tumors into T cell-inflamed ones, paving the way for successful ICT. SignificanceThe combination of ICT and targeted therapies holds great promises for broad and long-lasting therapeutic effects for cancers. However, combining ICT with anti-PI3K inhibitors have been difficult because the multifaceted effects of PI3K on both cancer cells and immune cells within the tumor microenvironment. Here we show a carefully designed anti-PI3K treatment, both in its specificity and dosing schedule, to inhibit cancer cell growth while promoting anti-tumor immunity, is critically important for successful ICT. Since the PI3K pathway is one of the most frequently altered signaling pathways in human cancers, our work may shed light on treating those cancers with PI3K activation and overcome resistance to ICT. HighlightsO_LIIntermittent PI3K/{beta}/{delta} inhibitor BAY1082439 treatment overcomes ICT resistance C_LIO_LIBAY1082439 turns Pten-null prostate cancer from "cold" to T cell-inflamed C_LIO_LIBAY1082439 inhibits cancer cell-intrinsic immunosuppressive activity and Treg C_LIO_LIBAY1082439 promotes clonal expansion and immunity of tumor-associated CD8+ T cells C_LI

cancer biology↗

bioRxiv2020

CELL ATLAS OF THE CHICK RETINA: SINGLE CELL PROFILING IDENTIFIES 136 CELL TYPES

Retinal structure and function have been studied in many vertebrate orders, but molecular characterization has been largely confined to mammals. We used single-cell RNA sequencing (scRNA-seq) to generate a cell atlas of the chick retina. From [~]40,000 single cell transcriptomes, we identified 136 cell types distributed among the six classes conserved across vertebrates - photoreceptor, horizontal, bipolar, amacrine, retinal ganglion and glial cells. To match molecular profiles to morphology, we adapted a method for CRISPR-based integration of reporters into selectively expressed genes. For Muller glia, we found that transcriptionally distinct cells were regionally localized along the anterior-posterior, dorsal-ventral and central-peripheral retinal axes. We also identified immature photoreceptor, horizontal cell and oligodendrocyte types that persist into late embryonic stages. Finally, we analyzed relationships among chick, mouse and primate retinal cell classes and types. Taken together, our results provide a foundation for anatomical, physiological, evolutionary, and developmental studies of the avian visual system.

neuroscience↗

bioRxiv2020

Cevipabulin-tubulin complex reveals a novel agent binding site on α-tubulin and provides insights into microtubule dynamic instability

Microtubule, composed of {beta}-tubulin heterodimers, remains as one of the most popular anticancer targets for decades. To date, anti-microtubule drugs are mainly functionally divided into microtubule-destabilizing and microtubule-stabilizing agents while microtubule- or tubulin-degradation agents are rarely reported. Six known binding sites on tubulin dimer are identified with five sites on {beta}-tubulin and only one site on -tubulin (pironetin site), hinting compounds binding to -tubulin are less well characterized. Cevipabulin, a microtubule-active antitumor clinical candidate, is widely accepted as a microtubule-stabilizing agent by binding to the vinblastine site. Our X-ray crystallography study reveals that, in addition binding to the vinblastine site, cevipabulin also binds to a novel site on -tubulin (named the seventh site) which located near the nonexchangeable GTP. Interestingly, we find the binding of cevipabulin to the seventh site induces tubulin degradation. As the non-exchangeable GTP has structural role and is important for the stability of tubulin dimers, we propose and confirm the tubulin degradation mechanism as: Cevipabulin at the seventh site puts the T5 loop outward to make the non-exchangeable GTP exchangeable, which reduces the stability of tubulin and results in its destabilization and degradation. Our results confirm a novel agent binding site on -tubulin and shed light on the development of tubulin degraders as a new generation of anti-microtubule drugs targeting this novel site.

cell biology↗

bioRxiv2020

An effective, safe and cost-effective cell-based chimeric vaccine against SARS-CoV2

More than one hundred vaccines against SARS-CoV-2 have been developed and some of them have entered clinical trials, but the latest results revealed that these vaccines still face great challenges. Here, we developed a novel cell-based gp96-Ig-secreting chimeric vaccine which is composed of two viral antigens, the RBD of spike protein, and a truncated nucleocapsid protein that could induce epitope-specific cytotoxic T lymphocytes but low antibody response. Syrian hamsters immunized with the cell-based vaccine produced high level of SARS-CoV-2 specific NAbs and specific T cell immunity which could eliminate RBD-truncated N-expressing cells, without the induction of antibody against N protein and other observed toxicity. This study provides a proof of concept for clinical testing of this safe, effective and cost-effective vaccine against SARS-CoV2 infection.

immunology↗

bioRxiv2020

Interferons and viruses induce a novel primate-specific isoform dACE2 and not the SARS-CoV-2 receptor ACE2

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes COVID-19, utilizes angiotensin-converting enzyme 2 (ACE2) for entry into target cells. ACE2 has been proposed as an interferon-stimulated gene (ISG). Thus, interferon-induced variability in ACE2 expression levels could be important for susceptibility to COVID-19 or its outcomes. Here, we report the discovery of a novel, primate-specific isoform of ACE2, which we designate as deltaACE2 (dACE2). We demonstrate that dACE2, but not ACE2, is an ISG. In vitro, dACE2, which lacks 356 N-terminal amino acids, was non-functional in binding the SARS-CoV-2 spike protein and as a carboxypeptidase. Our results reconcile current knowledge on ACE2 expression and suggest that the ISG-type induction of dACE2 in IFN-high conditions created by treatments, inflammatory tumor microenvironment, or viral co-infections is unlikely to affect the cellular entry of SARS-CoV-2 and promote infection.

genetics↗

bioRxiv2020

Characterizing transcriptional regulatory sequences in coronaviruses and their role in recombination

Novel coronaviruses, including SARS-CoV-2, SARS, and MERS, often originate from recombination events. The mechanism of recombination in RNA viruses is template switching. Coronavirus transcription also involves template switching at specific regions, called transcriptional regulatory sequences (TRS). It is hypothesized but not yet verified that TRS sites are prone to recombination events. Here, we developed a tool called SuPER to systematically identify TRS in coronavirus genomes and then investigated whether recombination is more common at TRS. We ran SuPER on 506 coronavirus genomes and identified 465 TRS-L and 3509 TRS-B. We found that the TRS-L core sequence (CS) and the secondary structure of the leader sequence are generally conserved within coronavirus genera but different between genera. By examining the location of recombination breakpoints with respect to TRS-B CS, we observed that recombination hotspots are more frequently co-located with TRS-B sites than expected.

genomics↗

bioRxiv2020

MOLECULAR IDENTIFICATION OF SIXTY-THREE AMACRINE CELL TYPES COMPLETES A MOUSE RETINAL CELL ATLAS

Amacrine cells (ACs) are a diverse class of interneurons that modulate input from photoreceptors to retinal ganglion cells (RGCs), rendering each RGC type selectively sensitive to particular visual features, which are then relayed to the brain. While many AC types have been identified morphologically and physiologically, they have not been comprehensively classified or molecularly characterized. We used high-throughput single-cell RNA sequencing (scRNA-seq) to profile >32,000 ACs from mouse retina, and applied computational methods to identify 63 AC types. We identified molecular markers for each type, and used them to characterize the morphology of multiple types. We show that they include nearly all previously known AC types as well as many that had not been described. Consistent with previous studies, most of the AC types express markers for the canonical inhibitory neurotransmitters GABA or glycine, but several express neither or both. In addition, many express one or more neuropeptides, and two express glutamatergic markers. We also explored transcriptomic relationships among AC types and identified transcription factors expressed by individual or multiple closely related types. Noteworthy among these were Meis2 and Tcf4, expressed by most GABAergic and most glycinergic types, respectively. Together, these results provide a foundation for developmental and functional studies of ACs, as well as means for genetically accessing them. Along with previous molecular, physiological and morphological analyses, they establish the existence of at least 130 neuronal types and nearly 140 cell types in mouse retina. SIGNIFICANCE STATEMENTThe mouse retina is a leading model for analyzing the development, structure, function and pathology of neural circuits. A complete molecular atlas of retinal cell types provides an important foundation for these studies. We used high-throughput single-cell RNA sequencing (scRNA-seq) to characterize the most heterogeneous class of retinal interneurons, amacrine cells, identifying 63 distinct types. The atlas includes types identified previously as well as many novel types. We provide evidence for use of multiple neurotransmitters and neuropeptides and identify transcription factors expressed by groups of closely related types. Combining these results with those obtained previously, we proposed that the mouse retina contains 130 neuronal types, and is therefore comparable in complexity to other regions of the brain.

neuroscience↗

bioRxiv2020

Assessment of Operant Learning and Memory in Mice Born through Intracytoplasmic Sperm Injection

Study questionAre there differences in operant learning and memory between mice born through intracytoplasmic sperm injection (ICSI) and naturally-conceived control (CTL) mice? Summary answerICSI females exhibited deficits in acquisition learning relative to CTL females, whereas ICSI males exhibited deficiency in discrimination learning and memory relative to CTL males during initial assessments. ICSI and CTL groups exhibited equally poor long-term retention of learned discrimination and memory performances at old age. What is known alreadySome human outcome studies have suggested that ICSI might be associated with an increased risk of certain cognitive disorders, but only one of two behavioral studies with ICSI mouse models have reported differences between ICSI and CTL females. No studies to date have investigated associative learning in ICSI mice. Study design, size, duration36 ICSI mice (18 male, 18 female) and 37 CTL mice (19 male, 18 female) aged 3-6 months were compared in a series of operant learning procedures that assessed acquisition of a new behavior, discrimination learning, and memory. 16 ICSI mice (9 male, 7 female) and 17 CTL mice (10 males, 7 females) received follow-up discrimination learning and memory assessments at 12 months of age (six months after the end of initial training) to evaluate retention and reacquisition of learned performances. Participants/materials, setting, methodsMice received daily operant learning sessions in experimental chambers in which all stimulus events and the recording of responses were automated. Food rewards were delivered for responding under different conditions of reinforcement, which varied by procedure. Subjects received a successive series of sessions of nose poke acquisition training, discrimination training, and the delayed non-matching-to-position (DNTMP) memory procedure. Mixed repeated measures ANOVAs in which the between-subjects factor was group (ICSI vs. CTL) and the within-subjects factor was repeated exposures to learning procedures (i.e., sessions) were used to analyze data. Main results and the role of chanceIn comparisons between all mice (i.e., males and females combined), CTL mice exhibited superior performance relative to ICSI in response acquisition (p = 0.03), discrimination (p = 0.001), and memory (p = 0.007). Sex-specific comparisons between the groups yielded evidence of sexual dimorphism. ICSI females exhibited a deficit in acquisition learning relative to CTL females (p < 0.001) but there was not a significant difference between CTL and ICSI males. In the discrimination and memory tasks, ICSI males exhibited deficits relative to CTL males (p = 0.002 and p = 0.02, respectively) but the differences between females in these tasks were not significant. There was no difference in discrimination or memory retention/re-acquisition assessments conducted with mice at 12 months of age. ICSI males and females weighed significantly more than CTL counterparts at all points during the experiment. Limitations, reasons for cautionThe study was not blinded. All learning assessments utilized food reward; other assessments of operant, Pavlovian, and nonassociative learning are needed to fully characterize learning in ICSI mice and speculate regarding the implications for cognitive function in humans conceived via ICSI. Wider implications of the findingsStudying learning and memory processes in mouse models has the potential to shed light on ICSI outcomes at the level of cognitive function. Future research should use multiple learning paradigms, assess both males and females, and investigate the effects of variables related to the ICSI procedure. Studying cognitive function in ICSI is an interdisciplinary endeavor and requires coordination between researchers at the genetic and psychological levels of analysis. Study funding/competing interest(s)This work was supported, in part, by grants from NIH (P30GM110767, HD071736 and HD085506 to WY), the Templeton Foundation (Grant ID: 61174 to WY), and a New Scholarly Endeavor Grant from the University of Nevada, Reno Office of Research and Innovation (to ML, YW, HZ, LH, and WY). The authors declare no competing interests.

genetics↗

bioRxiv2020

CELL ATLAS OF THE HUMAN FOVEA AND PERIPHERAL RETINA

Most irreversible blindness results from retinal disease. To advance our understanding of the etiology of blinding diseases, we used single-cell RNA-sequencing (scRNA-seq) to analyze the transcriptomes of [~]85,000 cells from the fovea and peripheral retina of seven adult human donors. Utilizing computational methods, we identified 58 cell types within 6 classes: photoreceptor, horizontal, bipolar, amacrine, retinal ganglion and non-neuronal cells. Nearly all types are shared between the two retinal regions, but there are notable differences in gene expression and proportions between foveal and peripheral cohorts of shared types. We then used the human retinal atlas to map expression of 636 genes implicated as causes of or risk factors for blinding diseases. Many are expressed in striking cell class-, type-, or region-specific patterns. Finally, we compared gene expression signatures of cell types between human and the cynomolgus macaque monkey, Macaca fascicularis. We show that over 90% of human types correspond transcriptomically to those previously identified in macaque, and that expression of disease-related genes is largely conserved between the two species. These results validate the use of the macaque for modeling blinding disease, and provide a foundation for investigating molecular mechanisms underlying visual processing.

neuroscience↗

bioRxiv2020

Cell Atlas of Aqueous Humor Outflow Pathways in Eyes of Humans and Four Model Species Provides Insights into Glaucoma Pathogenesis

Increased intraocular pressure (IOP) represents a major risk factor for glaucoma, a prevalent eye disease characterized by death of retinal ganglion cells that carry information from the eye to the brain; lowering IOP is the only proven treatment strategy to delay disease progression. The main determinant of IOP is the equilibrium between production and drainage of aqueous humor, with compromised drainage generally viewed as the primary contributor to dangerous IOP elevations. Drainage occurs through two pathways in the anterior segment of the eye, called conventional and uveoscleral. To gain insights into the cell types that comprise these pathways, we used high-throughput single cell RNA sequencing (scRNA-seq). From [~]24,000 single cell transcriptomes, we identified 19 cell types with molecular markers for each and used histological methods to localize each type. We then performed similar analyses on four organisms used for experimental studies of IOP dynamics and glaucoma: cynomolgus macaque (Macaca fascicularis), rhesus macaque (Macaca mulatta), pig (Sus scrofa) and mouse (Mus musculus). Many human cell types had counterparts in these models, but differences in cell types and gene expression were evident. Finally, we identified the cell types that express genes implicated in glaucoma in all five species. Together, our results provide foundations for investigating the pathogenesis of glaucoma, and for using model systems to assess mechanisms and potential interventions.

cell biology↗

bioRxiv2020

IL4-driven microglia modulate stress resilience through BDNF-dependent neurogenesis

Adult neurogenesis in the dentate gyrus of the hippocampus is regulated by specific groups of microglia and is functionally implicated in behavioral responses to stress. However, the role of microglia in modulating hippocampal neurogenesis in stress responses remains poorly understood. Here we investigated the effects of IL4-driven Arg1+ microglia in the restoration of hippocampal neurogenesis and conferment of stress resilience. We found that low IL4 levels in the hippocampus of mice was associated with greater stress vulnerability and, conversely, overexpression of IL4 in the hippocampus induced a large number of Arg1+ microglia and ameliorated stress-induced depressive-like behaviors. Knockdown of microglial IL4 receptors in the hippocampus of mice exacerbated the stress-induced inflammatory response and abolished the antidepressant effects of IL4 overexpression. Enhancement or inhibition of IL4 signaling in hippocampal microglia modulated neurogenesis, and blockade of neurogenesis abolished the resilience to stress-induced depression. We further show that IL4-activated microglia is associated with upregulation of BDNF levels and neurogenesis. Taken together, our findings suggest that IL4-driven microglia in the hippocampus trigger BDNF-dependent neurogenesis in response to chronic stress, helping protect against depressive-like symptoms. These findings identify the modulation of a specific microglial phenotype as a treatment strategy for mood disorders. In BriefZhang et al. show that IL4-induced Arg1+ microglia restore hippocampal neurogenesis and promote resilience against stress in mice by increasing BDNF levels. Targeting microglia with immunomodulatory factors may be a strategy for treating mood disorders. HighlightsO_LIVulnerability to stress in mice is associated with reduced IL4 signaling in the hippocampus C_LIO_LIBrain-derived IL4 promotes adult hippocampal neurogenesis and stress resistance by driving Arg1+ microglia C_LIO_LIIL4-driven Arg1+ microglia enhance hippocampal neurogenesis via a BDNF-dependent pathway C_LI

neuroscience↗

bioRxiv2020

Nuclear Plasticity Increases Susceptibility to Damage During Confined Migration

Large nuclear deformations during migration through confined spaces have been associated with nuclear membrane rupture and DNA damage. However, the stresses associated with nuclear damage remain unclear. Here, using a quasi-static plane strain finite element model, we map evolution of nuclear shape and stresses during confined migration of a cell through a deformable matrix. Plastic deformation of the nucleus observed for a cell with stiff nucleus transiting through a stiffer matrix lowered nuclear stresses, but also led to kinking of the nuclear membrane. In line with model predictions, transwell migration experiments with fibrosarcoma cells showed that while nuclear softening increased invasiveness, nuclear stiffening led to plastic deformation and higher levels of DNA damage. In addition to highlighting the advantage of nuclear softening during confined migration, our results suggest that plastic deformations of the nucleus during transit through stiff tissues may lead to bending-induced nuclear membrane disruption and subsequent DNA damage.

biophysics↗