Biology subjects

Yan, X.

Publications and source records attributed to Yan, X..

250 records · Page 14Linked to original sources

bioRxiv2019

Single Cell RNA-seq reveals ectopic and aberrant lung resident cell populations in Idiopathic Pulmonary Fibrosis

We provide a single cell atlas of Idiopathic Pulmonary Fibrosis (IPF), a fatal interstitial lung disease, focusing on resident lung cell populations. By profiling 312,928 cells from 32 IPF, 29 healthy control and 18 chronic obstructive pulmonary disease (COPD) lungs, we demonstrate that IPF is characterized by changes in discrete subpopulations of cells in the three major parenchymal compartments: the epithelium, endothelium and stroma. Among epithelial cells, we identify a novel population of IPF enriched aberrant basaloid cells that co-express basal epithelial markers, mesenchymal markers, senescence markers, developmental transcription factors and are located at the edge of myofibroblast foci in the IPF lung. Among vascular endothelial cells in the in IPF lung parenchyma we identify an expanded cell population transcriptomically identical to vascular endothelial cells normally restricted to the bronchial circulation. We confirm the presence of both populations by immunohistochemistry and independent datasets. Among stromal cells we identify fibroblasts and myofibroblasts in both control and IPF lungs and leverage manifold-based algorithms diffusion maps and diffusion pseudotime to infer the origins of the activated IPF myofibroblast. Our work provides a comprehensive catalogue of the aberrant cellular transcriptional programs in IPF, demonstrates a new framework for analyzing complex disease with scRNAseq, and provides the largest lung disease single-cell atlas to date.

genomics↗

bioRxiv2019

RecO impedes RecG-SSB binding to impair the strand annealing recombination pathway in E.coli

Faithful duplication of genomic DNA relies not only on the fidelity of DNA replication itself, but also on fully functional DNA repair and homologous recombination machinery. We report a molecular mechanism responsible for deciding homologous recombinational repair pathways during replication dictated by binding of RecO and RecG to SSB in E.coli. Using a RecG-yfp fusion protein, we found that RecG-yfp foci appeared only in the {Delta}recG, {Delta}recO and {Delta}recA, {Delta}recO double mutants. Surprisingly, foci were not observed in wild-type {Delta}recG, or double mutants where recG and either recF or, separately recR were deleted. In addition, formation of RecG-yfp foci in the {Delta}recO::kanR required wildtype ssb, as ssb-113 could not substitute. This suggests that RecG and RecO binding to SSB is competitive. We also found that the UV resistance of recO alone mutant increased to certain extent by supplementing RecG. In an ssb-113 mutant, RecO and RecG worked following a different pattern. Both RecO and RecG were able to participate in repairing UV damages when grown at permissive temperature, while they could also be involved in making DNA double strand breaks when grown at nonpermissive temperature. So, our results suggested that differential binding of RecG and RecO to SSB in a DNA replication fork in Escherichia coli.may be involved in determining whether the SDSA or DSBR pathway of homologous recombinational repair is used.\n\nAuthor summarySingle strand DNA binding proteins (SSB) stabilize DNA holoenzyme and prevent single strand DNA from folding into non-B DNA structures in a DNA replication fork. It has also been revealed that SSB can also act as a platform for some proteins working in DNA repair and recombination to access DNA molecules when DNA replication fork needs to be reestablished. In Escherichia coli, several proteins working primarily in DNA repair and recombination were found to participate in DNA replication fork resumption by physically interacting with SSB, including RecO and RecG etc. However the hierarchy of these proteins interacting with SSB in Escherichia coli has not been well defined. In this study, we demonstrated a differential binding of RecO and RecG to SSB in DNA replication was used to establish a RecO-dependent pathway of replication fork repair by abolishing a RecG-dependent replication fork repair. We also show that, RecG and RecO could randomly participate in DNA replication repair in the absence of a functional SSB, which may be responsible for the generation of DNA double strand breaks in an ssb-113 mutant in Escherichia coli.

molecular biology↗

bioRxiv2019

Effect of different forage-to-concentrate ratios on the structure of rumen bacteria and its relationship with nutrition levels and real-time methane production in sheep

Emission from ruminants has become the largest source of anthropogenic emission of methane in China. The structure of the rumen flora has a significant effect on methane production. To establish a more accurate prediction model for methane production, the rumen flora should be one of the most important parameters. The objective of the present study was to investigate the relationship among changes in rumen flora, nutrient levels, and methane production in sheep fed with the diets of different forage-to-concentration ratios, as well as to screen for significantly different dominant genera. Nine rumen-cannulated hybrid sheep were separated into three groups and fed three diets with forage-to-concentration ratios of 50:50, 70:30, and 90:10. Three proportions of the diets were fed according to a 3 x 3 incomplete Latin square, design during three periods of 15 d each. The ruminal fluid was collected for real-time qPCR, high-throughput sequencing and in vitro rumen fermentation in a new real-time fermentation system wit. Twenty-two genera were screened, the abundance of which varied linearly with forage-to-concentration ratios and methane production. In addition, during the 12-hour in vitro fermentation, the appearance of peak concentration was delayed by 26-27 min with the different structure of rumen bacteria. The fiber-degrading bacteria were positively correlated with this phenomenon, but starch-degrading and protein-degrading bacteria were negative correlated. These results would facilitate macro-control of rumen microorganisms and better management of diets for improved nutrition in ruminants. In addition, our findings would help in screening bacterial genera that are highly correlated with methane production.

microbiology↗

bioRxiv2019

A 3-hydroxy-3-methylglutaryl-CoA synthase-based probe for the discovery of the acyltransferase-less type I polyketide synthases

Acyltransferase (AT)-less type I polyketide synthases (PKSs) produce complex natural products due to the presence of many unique tailoring enzymes. The 3-hydroxy-3-methylglutaryl coenzyme A synthases (HCSs) are responsible for {beta}-alkylation of the growing polyketide intermediates in AT-less type I PKSs. In this study, we discovered a large group of HCSs, closely associated with the characterized and orphan AT-less type I PKSs through in silico genome mining, sequence and genome neighborhood network analysis. Using HCS-based probes, the survey of 1207 inhouse strains and 18 soil samples from different geological locations revealed the vast diversity of HCS-containing AT-less type I PKSs. The presence of HCSs in many AT-less type I PKSs suggests their co-evolutionary relationship. Our study should inspire future efforts to discover new polyketide natural products from AT-less type I PKSs.

microbiology↗

bioRxiv2018

Single-cell RNA-seq reveals that glioblastoma recapitulates normal brain development

SummaryCancer stem cells are critical for cancer initiation, development, and resistance to treatments. Our understanding of these processes, and how they relate to glioblastoma heterogeneity, is limited. To overcome these limitations, we performed single-cell RNA-sequencing on 38 296 glioblastoma cells and 22 637 normal human fetal brain cells. Using an unbiased approach, we mapped the lineage hierarchy of the developing human brain and compared the transcriptome of each cancer cell to this roadmap. We discovered a conserved neural trilineage cancer hierarchy with glial progenitor-like cells at the apex. We also found that this progenitor population contains the majority of cancers cycling cells and is the origin of heterogeneity. Finally, we show that this hierarchal map can be used to identify therapeutic targets specific to progenitor cancer stem cells. Our analyses show that normal brain development reconciles glioblastoma development, unravels the origin of glioblastoma heterogeneity, and helps to identify cancer stem cell-specific targets.

cancer biology↗

bioRxiv2018

Comparative whole-genome analysis reveals genetic adaptation of the invasive pinewood nematode

Genetic adaptation to new environments is essential for invasive species. To explore the genetic underpinnings of invasiveness of a dangerous invasive species, the pinewood nematode (PWN) Bursaphelenchus xylophilus, we analysed the genome-wide variations of a large cohort of 55 strains isolated from both the native and introduced regions. Comparative analysis showed abundant genetic diversity existing in the nematode, especially in the native populations. Phylogenetic relationships and principal component analysis indicate a dominant invasive population/group (DIG) existing in China and expansion beyond, with few genomic variations. Putative origin and migration paths at a global scale were traced by targeted analysis of rDNA sequences. A progressive loss of genetic diversity was observed along spread routes. We focused on variations with a low frequency allele (<50%) in the native USA population but fixation in DIG, and a total of 25,992 single nuclear polymorphisms (SNPs) were screened out. We found that a clear majority of these fixation alleles originated from standing variation. Functional annotation of these SNP-harboured genes showed that adaptation-related genes are abundant, such as genes that encode for chemoreceptors, proteases, detoxification enzymes, and proteins involved in signal transduction and in response to stresses and stimuli. Some genes under positive selection were predicted. Our results suggest that adaptability to new environments plays essentially roles in PWN invasiveness. Genetic drift, mutation and strong selection drive the nematode to rapidly evolve in adaptation to new environments, which including local pine hosts, vector beetles, commensal microflora and other new environmental factors, during invasion process.

genomics↗

bioRxiv2018

Effects of the anesthetic MS-222 on silver pomfret (Pampus argenteus) juveniles under aquaculture treatment stresses

The silver pomfret (Pampus argenteus) is a major economically important marine fish in China. However, P. argenteus is sensitive to many stress factors and susceptible to injury. This problem could be resolved using anesthesia. We determined the lowest effective dose (LED) of tricaine methanesulfonate (MS-222) and assessed the longest safe deep anesthesia time and effect after aquaculture treatment stresses. P. argenteus juveniles were exposed to six concentrations of MS-222 (10, 25, 50, 75, 100, and 125 mg L-1); LED was established at 75 mg L-1. The juveniles were exposed to different deep anesthesia times (4, 7, 10, 12, and 15 min) at 75 mg L-1; the longest safe deep anesthesia time under LED was 10 min. Finally, the juveniles were randomly divided into four groups: control group (CG), draining group (DG, drain), anesthetic group (AG, drain + MS-222 + aquaculture treatment); and non-anesthetic group (NAG, drain + aquaculture treatment). Plasma cortisol levels in the NAG, AG, DG, and CG groups were 38.739 {+/-} 1.065 (highest), 25.083 {+/-} 0.587, 28.644 {+/-} 0.612, and 22.620 {+/-} 0.836 ng mL-1 (lowest). The AG group showed significant differences in superoxide dismutase, catalase, and malondialdehyde activities, except for glutathione. HSP70, HSP90, GR1, and GR2 mRNA levels in the NAG group increased sharply in response to stressors. GR1 and GR2 mRNA levels in the AG group also increased significantly, whereas HSP70 and HSP90 mRNA levels showed no significant differences. Thus, MS-222 can reduce oxidative damage, stress reaction, and resistance to aquaculture treatment stresses in P. argenteus.

animal behavior and cognition↗

bioRxiv2018

Magnaporthe oryzae SMO1 encodes a Ras GTPase-activating protein required for spore morphology, appressorium function and rice blast disease

The pathogenic life cycle of the rice blast fungus Magnaporthe oryzae involves a series of morphogenetic changes, essential for its ability to cause disease. The smo mutation was identified more than twenty-five years ago and affects the shape and development of diverse cell types in M. oryzae, including conidia, appressoria and asci. All attempts to clone the SMO1 gene by map-based cloning and/or complementation, have failed over many years. Here, we report the identification of SMO1 by a combination of bulk segregant analysis and comparative genome analysis. SMO1 encodes a GTPase-activating protein (GAP), which regulates Ras signalling during infection-related development. Targeted deletion of SMO1 results in abnormal, non-adherent conidia, impaired in their production of spore tip mucilage. Smo1 mutants also develop smaller appressoria, with a severely reduced capacity to infect rice plants. SMO1 is necessary for organisation of microtubules and for septin-dependent remodelling of the F-actin cytoskeleton at the appressorium pore. Smo1 physically interacts with components of the Ras2 signaling complex, and a range of other signalling and cytoskeletal components, including the four core septins. SMO1 is therefore necessary for regulation of RAS activation required for conidial morphogenesis and septin-mediated plant infection.

microbiology↗

bioRxiv2018

Mother centrioles are dispensable for deuterosome formation and function during basal body amplification

Mammalian epithelial cells use a pair of mother centrioles (MCs) and numerous deuterosomes as platforms for efficient basal body assembly during multiciliogenesis. How deuterosomes form and function, however, remain controversial. They are proposed to either arise spontaneously followed by maturation into larger ones with increased procentriole-producing capacity or be assembled solely on the young MC, nucleate procentrioles under the MCs guidance, and released as procentriole-occupied \"halos\". Here we show that both MCs are dispensable for deuterosome formation in multiciliate cells. In both mouse tracheal epithelial and ependymal cells (mTECs and mEPCs), discrete deuterosomes in the cytoplasm were initially procentriole-free and then grew into halos. More importantly, eliminating the young MC or both MCs in proliferating precursor cells through shRNA-mediated depletion of Plk4, a kinase essential to procentriole assembly, did not abolish deuterosome formation when these cells were induced to differentiate into mEPCs. The average deuterosome numbers per cell only reduced by 21% as compared to control mEPCs. Therefore, MC is not essential to the assembly of both deuterosomes and deuterosome-mediated procentrioles.

cell biology↗

bioRxiv2018

NudCL2 is an Hsp90 cochaperone to regulate sister chromatid cohesion by stabilizing cohesin subunits

Sister chromatid cohesion plays a key role in ensuring precise chromosome segregation during mitosis, which is mediated by the multisubunit complex cohesin. However, the molecular regulation of cohesin subunits stability remains unclear. Here, we show that NudCL2 (NudC-like protein 2) is essential for the stability of cohesin subunits by regulating Hsp90 ATPase activity in mammalian cells. Depletion of NudCL2 induces mitotic defects and premature sister chromatid separation and destabilizes cohesin subunits that interact with NudCL2. Similar defects are also observed upon inhibition of Hsp90 ATPase activity. Interestingly, ectopic expression of Hsp90 efficiently rescues the protein instability and functional deficiency of cohesin induced by NudCL2 depletion, but not vice versa. Moreover, NudCL2 not only binds to Hsp90, but also significantly modulates Hsp90 ATPase activity and promotes the chaperone function of Hsp90. Taken together, these data suggest that NudCL2 is a previously undescribed Hsp90 cochaperone to modulate sister chromatid cohesion by stabilizing cohesin subunits, providing a hitherto unrecognized mechanism that is crucial for faithful chromosome segregation during mitosis.

cell biology↗

bioRxiv2018

CRISPR-Cas9 ribonucleoprotein-mediated co-editing and counterselection in the rice blast fungus

The rice blast fungus Magnaporthe oryzae is the most serious pathogen of cultivated rice and a significant threat to global food security. To accelerate targeted mutation and specific gene editing in this species, we have developed a rapid plasmid-free CRISPR-Cas9-based gene editing method. It has previously been reported in M. oryzae that transformation with plasmids expressing Cas9 can generate specific mutations using sgRNAs, directing the endonuclease to specific genes. We show, however, that expression of Cas9 is highly toxic to M. oryzae, rendering this approach impractical. We demonstrate that using purified Cas9 pre-complexed to RNA guides to form ribonucleoproteins (RNPs), provides an alternative and very effective gene editing procedure. When used in combination with oligonucleotide or PCR-generated donor DNAs, generation of strains with specific base pair edits, in-locus gene replacements, or multiple gene edits, is very rapid and straightforward. Additionally, we report a novel counterselection strategy which allows creation of precisely edited fungal strains that contain no foreign DNA and are completely isogenic to the wild type. Together, these developments represent a scalable improvement in the precision and speed of genetic manipulation in M. oryzae and are likely to be broadly applicable to other fungal species.

microbiology↗

bioRxiv2018

Timing Determines Tuning: a Rapid Spatiotemporal Transformation in Superior Colliculus Neurons During Reactive Gaze Shifts.

Gaze saccades -rapid shifts of the eyes and head toward a goal-- have provided fundamental insights into the neural control of movement. For example, it has been shown that the superior colliculus (SC) transforms a visual target (T) code to future gaze (G) location commands after a memory delay. However, this transformation has not been observed in reactive saccades made directly to a stimulus, so its contribution to normal gaze behavior is unclear. Here, we tested this using a quantitative measure of the spatial continuum between T and G coding based on variable gaze errors. We demonstrate that a rapid T-G transformation occurs between SC visual and motor responses during reactive saccades, even within visuomotor cells, with a continuous spatiotemporal shift in coding occurring in cell types (visual, visuomotor, motor). We further show that the primary determinant of this spatial code was not the intrinsic visual-motor index of different cells or populations, but rather the timing of the response in all cells. These results suggest that the SC provides a rapid spatiotemporal transformation for normal gaze saccades, that its motor responses contribute to variable gaze errors, and that those errors arise from a noisy spatiotemporal transformation involving all SC neurons.\n\nSignificance StatementOculomotor studies have demonstrated visuomotor transformations in structures like the superior colliculus with the use of trained behavioral manipulations, like the memory delay and antisaccades tasks, but it is not known how this happens during normal saccades. Here, using a spatiotemporal model fitting method based on endogenous gaze errors in reactive gaze saccades, we show that the superior colliculus provides a rapid spatiotemporal transformation from target to gaze coding that involves visual, visuomotor, and motor neurons. This technique demonstrates that SC spatial codes are not fixed, and may provide a quantitative biomarker for assessing the health of sensorimotor transformations.

neuroscience↗

bioRxiv2018

Comparative Analysis of UL16 Mutants Derived from Multiple Strains of HSV-2 and HSV-1 Reveals Species-Specific Requirements for the UL16 Protein

Orthologs of the herpes simplex virus (HSV) UL16 gene are conserved throughout the Herpesviridae. Because of this conservation, one might expect that these proteins perform similar functions for all herpesviruses. Previous studies on a UL16 null mutant derived from HSV-2 strain 186 revealed a roughly 100-fold replication defect and a critical role for UL16 in the nuclear egress of capsids. These findings were in stark contrast to what has been observed with UL16 mutants of HSV-1 and pseudorabies virus where roughly 10-fold replication deficiencies were reported that were accompanied by defects in the secondary envelopment of cytoplasmic capsids. One possible explanation for this discrepancy is that the HSV-2 186 strain is not representative of the HSV-2 species. To address this possibility, multiple UL16 null mutants were constructed in multiple HSV-2 and HSV-1 strains by CRISPR/Cas9 mutagenesis and their phenotypes characterized side-by-side. This analysis showed that all the HSV-2 UL16 mutants had 50 to 100-fold replication deficiencies that were accompanied by defects in the nuclear egress of capsids as well as defects in the secondary envelopment of cytoplasmic capsids. By contrast, most HSV-1 UL16 mutants had 10-fold replication deficiencies that were accompanied by defects in secondary envelopment of cytoplasmic capsids. These findings indicated that UL16 has HSV species-specific functions. Interestingly, HSV-1 UL16 could promote the nuclear egress of HSV-2 UL16 null strains, suggesting that, unlike HSV-1, HSV-2 lacks an activity that can compensate for nuclear egress in the absence of UL16.\n\nImportanceHSV-2 and HSV-1 are important human pathogens that cause distinct diseases in their hosts. A complete understanding of the morphogenesis of these viruses is expected to reveal vulnerabilities that can be exploited in the treatment of HSV disease. UL16 is a virion structural component that is conserved throughout the Herpesviridae and functions in virus morphogenesis, however, previous studies have suggested different roles for UL16 in the morphogenesis of HSV-2 and HSV-1. This study sought to resolve this apparent discrepancy by analyzing multiple UL16 mutant viruses derived from multiple strains of HSV-2 and HSV-1. The data indicate that UL16 has HSV species-specific functions insofar as HSV-2 has a requirement for UL16 in the escape of capsids from the nucleus whereas both HSV-2 and HSV-1 require UL16 for final envelopment of capsids at cytoplasmic membranes.

microbiology↗

bioRxiv2017

Use of a Neural Circuit Probe to Validate in silico Predictions of Inhibitory Connections

Understanding how neuronal signals propagate in local network is an important step in understanding information processing. As a result, spike trains recorded with Multi-electrode Arrays (MEAs) have been widely used to study behaviors of neural connections. Studying the dynamics of neuronal networks requires the identification of both excitatory and inhibitory connections. The detection of excitatory relationships can robustly be inferred by characterizing the statistical relationships of neural spike trains. However, the identification of inhibitory relationships is more difficult: distinguishing endogenous low firing rates from active inhibition is not obvious. In this paper, we propose an in silico interventional procedure that makes predictions about the effect of stimulating or inhibiting single neurons on other neurons, and thereby gives the ability to accurately identify inhibitory causal relationships. To experimentally test these predictions, we have developed a Neural Circuit Probe (NCP) that delivers drugs transiently and reversibly on individually identified neurons to assess their contributions to the neural circuit behavior. With the help of NCP, three inhibitory connections identified by our in silico modeling were validated through real interventional experiments. Together, these methods provide a basis for mapping complete neural circuits.

neuroscience↗

bioRxiv2017

The Novel Monocomponent FAD-dependent Monooxygenase HpaM Catalyzes the 2-Decarboxylative Hydroxylation of 5-Hydroxypicolinic Acid in Alcaligenes faecalis JQ135

5-hydroxypicolinic acid (5HPA) is a natural pyridine derivative that can be microbially degraded. However, the physiological, biochemical, and genetic foundation of the microbial catabolism of 5HPA remains unknown. In this study, a gene cluster hpa (which is involved in degradation of 5HPA in Alcaligenes faecalis JQ135) was cloned and HpaM was identified as a novel monocomponent FAD-dependent monooxygenase. HpaM shared a sequence only 31% similarity with the most related protein 6-hydroxynicotinate 3-monooxygenase (NicC) of Pseudomonas putida KT2440. hpaM was heterologously expressed in E. coli BL21(DE3), and the recombinant HpaM was purified via Ni-affinity chromatography. HpaM catalyzed the 2-decarboxylative hydroxylation of 5-HPA, thus generating 2,5-dihydroxypyridine (2,5-DPH). Monooxygenase activity was only detected in the presence of FAD and NADH, but not of FMN and NADPH. The apparent Km values of HpaM toward 5HPA and NADH were 45.4 and 37.8 , respectively. Results of gene deletion and complementation showed that hpaM was essential for 5HPA degradation in Alcaligenes faecalis JQ135.\n\nImportancePyridine derivatives are ubiquitous in nature and important chemical materials that are currently widely used in agriculture, pharmaceutical, and chemical industries. Thus, the microbial degradation and transformation mechanisms of pyridine derivatives received considerable attention. Decarboxylative hydroxylation was an important degradation process in pyridine derivatives, and previously reported decarboxylative hydroxylations happened in the C3 of the pyridine ring. In this study, we cloned the gene cluster hpa, which is responsible for 5HPA degradation in Alcaligenes faecalis JQ135, thus identifying a novel monocomponent FAD-dependent monooxygenase HpaM. Unlike 3-decarboxylative monooxygenases, HpaM catalyzed decarboxylative hydroxylation in the C2 of the pyridine ring in 5-hydroxypicolinic acid. These findings deepen our understanding of the molecular mechanism of microbial degradation of pyridine derivatives. Furthermore, HpaM offers potential for applications to transform useful pyridine derivatives.

microbiology↗

bioRxiv2017

Near-Atomic Resolution Structure Determination in Over-Focus with Volta Phase Plate by Cs-corrected Cryo-EM

Volta phase plate (VPP) is a recently developed transmission electron microscope (TEM) apparatus that can significantly enhance the image contrast of biological samples in cryo-electron microscopy (cryo-EM) therefore impose the possibility to solve structures of relatively small macromolecules at high resolution. In this work, we performed theoretical analysis and found that using phase plate on objective lens spherical aberration (Cs)-corrected TEM may gain some interesting optical properties, including the over-focus imaging of macromolecules. We subsequently evaluated the imaging strategy of frozen-hydrated apo-ferritin with VPP on a Cs-corrected TEM and obtained the structure of apo-ferritin at near atomic resolution from both under- and over-focused dataset, illustrating the feasibility and new potential of combining VPP with Cs-corrected TEM for high resolution cryo-EM.\n\nHighlightsThe successful combination of volta phase plate and Cs-corrector in single particle cryo-EM.\n\nNear-atomic structure determined from over-focused images by cryo-EM. VPP-Cs-corrector coupled EM provides interesting optical properties.\n\nIn BriefWe took the unique advantage of the optical system by combining the volta phase plate and Cs-corrector in a modern TEM to collect high resolution micrographs of frozen-hydrated apo-ferritin in over-focus imaging conditions and determined the structure of apo-ferritin at 3.0 Angstrom resolution.

biophysics↗