Biology subjects

Guo, J.

Publications and source records attributed to Guo, J..

At least 325 records · Page 18Linked to original sources

bioRxiv2021

The Dynamic Continuum of Nanoscale Peptide Assemblies Facilitates Endocytosis and Endosomal Escape

Considerable number of works have reported alkaline phosphatase (ALP) enabled intracellular targeting by peptide assemblies, but little is known how these substrates of ALP enters cells. Here we show that the nanoscale assemblies of phosphopeptides, as a dynamic continuum, cluster ALP to enable caveolae mediated endocytosis (CME) and eventual endosomal escape. Specifically, fluorescent phosphopeptides, as substrates of tissue nonspecific alkaline phosphatase (TNAP), undergo enzyme catalyzed self-assembly to form nanofibers. As shown by live cell imaging, the nanoparticles of phosphopeptides, being incubated with HEK293 cells overexpressing red fluorescent protein-tagged TNAP (TNAP-RFP), cluster TNAP-RFP in lipid rafts to enable CME, further dephosphorylation of the phosphopeptides form the peptide nanofibers for endosomal escape inside cells. Inhibiting TNAP, cleaving the membrane anchored TNAP, or disrupting lipid rafts abolishes the endocytosis. Moreover, decreasing the formation of peptide nanofibers prevents the endosomal escape. As the first study establishing a dynamic continuum of supramolecular assemblies for cellular uptake, this work not only illustrates an effective design for enzyme responsive supramolecular therapeutics, but also provides mechanism insights for understanding the dynamics of cellular uptakes of proteins or exogenous peptide aggregates at nanoscale.

biochemistry↗

bioRxiv2021

Heat wave event facilitates defensive responses in invasive C3 plant Ambrosia artemisiifolia L. under elevated CO2 concentration to the detriment of insect herbivores

To predict and mitigate the effects of climate change on communities and ecosystems, the joint effects of extreme climatic events on species interactions need to be understood. Using the common ragweed (Ambrosia artemisiifolia L.)--leaf beetle (Ophraella communa) system, we investigated the effects of heat wave and elevated CO2 on common ragweed growth, secondary metabolism, and the consequent impacts on the beetle. The results showed that elevated CO2 and heat wave facilitated A. Artemisiifolia growth; further, A. artemisiifolia accumulated large amounts of defensive secondary metabolites. Being fed on A. artemisiifolia grown under elevated CO2 and heat wave conditions resulted in the poor performance of O. communa (high mortality, long development period, and low reproduction). Overall, under elevated CO2, heat wave improved the defensive ability of A. artemisiifolia against herbivores. This implies that heat wave event will relieve harm of A. artemisiifolia to human under elevated CO2. On the other hand, super adaptability to climatic changes may aggravate invasive plant distribution, posing a challenge to the control of invasive plants in the future.

ecology↗

bioRxiv2021

Niche signals regulate continuous transcriptional states in hematopoietic stem cells

Hematopoietic stem cells (HSCs) must ensure adequate blood cell production following distinct external stressors. A comprehensive understanding of in vivo heterogeneity and specificity of HSC responses to external stimuli is currently lacking. We performed single-cell RNA sequencing (scRNA-Seq) on functionally validated mouse HSCs and LSK (Lin-, c-Kit+, Sca1+) progenitors after in vivo perturbation of niche signals interferon, granulocyte-colony stimulating factor (G-CSF), and prostaglandin. We identified six HSC states that are characterized by enrichment but not exclusive expression of marker genes. Niche perturbations induce novel and rapid transitions between these HSC states. Differential expression analysis within each state revealed HSC- and LSK-specific molecular signatures for each perturbation. Chromatin analysis of unperturbed HSCs and LSKs by scATAC-Seq revealed HSC-specific, cell intrinsic predispositions to niche signals. We compiled a comprehensive resource of HSC- and progenitor-specific chromatin and transcriptional features that represent important determinants of regenerative potential during stress hematopoiesis.

developmental biology↗

bioRxiv2021

GFI1 cooperates with IKAROS/IKZF1 to activate gene expression in T-cell acute lymphoblastic leukemia

Growth factor independence-1 (GFI1) is a transcriptional repressor and master regulator of normal and malignant hematopoiesis. Repression by GFI1 is attributable to recruitment of LSD1-containing protein complexes via its SNAG domain. However, the full complement of GFI1 partners in transcriptional control is not known. We show that in T-ALL cells, GFI1 and IKAROS are transcriptional partners that co-occupy regulatory regions of hallmark T cell development genes. Transcriptional profiling reveals a subset of genes directly transactivated through the GFI1--IKAROS partnership. Among these is NOTCH3, a key factor in T-ALL pathogenesis. Surprisingly, NOTCH3 transactivation by GFI1 and IKAROS requires the GFI1 SNAG domain but occurs independent of SNAG--LSD1 binding. GFI1 variants deficient in LSD1 binding fail to transactivate NOTCH3, but conversely, small molecules that disrupt the SNAG--LSD1 interaction while leaving the SNAG primary structure intact stimulate NOTCH3 expression. These results identify a non-canonical transcriptional control mechanism in T-ALL which supports GFI1-mediated transactivation in partnership with IKAROS and suggest competition between LSD1-containing repressive complexes and others favoring transactivation.

biochemistry↗

bioRxiv2021

De novo purine nucleotide biosynthesis mediated by amidophosphoribosyl transferase is required for conidiation and essential for the successful host colonization of Magnaporthe oryzae

Amidophosphoribosyl transferase catalyzes the first step of the purine nucleotide biosynthesis by converting 5-phosphoribosyl-1-pyrophosphate into 5-phosphoribosyl-1-amine. In this study, we identified and characterized the functions of MoAde4, an ortholog of yeast Ade4 in the rice blast fungus. MoAde4 is a 537-amino acid protein containing the GATase_6 and pribosyltran domains. Quantitative real-time PCR analysis showed MoADE4 transcripts were highly expressed during conidiation, early-infection, and late-infection stages of the fungus. Disruption of MoADE4 gene resulted in {Delta}Moade4 mutant exhibiting adenine, adenosine, and hypoxanthine auxotrophy on MM. Conidia quantification assays showed {Delta}Moade4 mutant was significantly reduced in sporulation. The conidia of {Delta}Moade4 mutant could still form appressoria but mostly failed to penetrate the rice cuticle. Pathogenicity test showed {Delta}Moade4 was completely nonpathogenic on rice and barley leaves which was attributed by failure of its infectious hyphae to colonize the host cells. The {Delta}Moade4 was defective in induction of strong host immunity and had its purine transporter genes repressed during in planta infection. Addition of exogenous adenine partially rescued conidiation and pathogenicity defects of the {Delta}Moade4 mutant on the barley and rice leaves. Localization assays showed that MoAde4 is located in the cytoplasm. Taken together, our results demonstrate that purine biosynthesis orchestrated by MoAde4 is required for fungal development, conidiation, more importantly, we found it to be essential for fungal pathogenicity not because of the appressorial formation, but appressorium penetration and host colonization during the plant infection of M. oryzae. Thus this findings suggests that purine biosynthesis could act as an important target for combating recalcitrant plant fungal pathogens.

molecular biology↗

bioRxiv2021

A phosphorylation of RIPK3 kinase initiates an intracellular apoptotic pathway that promotes corpus luteum regression

Receptor-interacting serine/threonine-protein kinase 3 (RIPK3) normally signals to necroptosis by phosphorylating MLKL. We report here that when the cellular RIPK3 chaperone Hsp90/CDC37 level is low, RIPK3 also signals to apoptosis. The apoptotic function of RIPK3 requires phosphorylation of the serine 165/threonine 166 sites on its kinase activation loop, resulting in inactivation of RIPK3 kinase activity while gaining the ability to recruit RIPK1, FADD, and caspase-8 to form a cytosolic caspase-activating complex, thereby triggering apoptosis. We found that PGF2 induces RIPK3 expression in luteal granulosa cells in the ovary to cause luteal regression through this RIPK3-mediated apoptosis pathway. Mice carrying homozygous phosphorylation-resistant RIPK3 S165A/T166A knockin mutations failed to respond to PGF2 but retained pro-necroptotic function, whereas mice with phospho-mimicking S165D/T166E homozygous knockin mutation underwent spontaneous apoptosis in multiple RIPK3-expressing tissues and died shortly after birth. Thus, RIPK3 signals to either necroptosis or apoptosis depending on its serine 165/threonine 166 phosphorylation status.

cell biology↗

bioRxiv2021

RNAi screening reveals requirement for PDGFRβ in JEV infection.

Mosquito-borne Japanese encephalitis virus (JEV) causes serious illness worldwide and is associated with high morbidity and mortality. To identify potential host therapeutic targets, a high-throughput receptor tyrosine kinase small interfering RNA library screening was performed with recombinant JEV particles. Platelet-derived growth factor receptor beta (PDGFR{beta}) was identified as a hit after two rounds of screening. Knockdown of PDGFR{beta} blocked JEV infection, and trans-complementation of PDGFR{beta} could partly restore its infectivity. The PDGFR{beta} inhibitor imatinib, which has been approved for the treatment of malignant metastatic cancer, protected mice against JEV-induced lethality by decreasing the viral load in the brain, while abrogating the histopathological changes associated with JEV infection. These findings demonstrated that PDGFR{beta} is important in viral infection and provided evidence for the potential to develop imatinib as a therapeutic intervention against JEV infection.

microbiology↗

bioRxiv2021

Nuclear m6A reader Ythdc1 regulates the scaffold function of LINE1 in mouse ESCs

N6-methyladenosine (m6A) on chromosome-associated regulatory RNAs (carRNAs), including repeat RNAs, play important roles in tuning the chromatin state and transcription1. Among diverse RNA-chromatin interacting modes, the nuclear RNA scaffold is considered important for trans-interactions2,3 but has not yet been connected with m6A yet. Here, we found that Ythdc1 played indispensable roles in the embryonic stem cell (ESC) self-renewal and differentiation potency, and these roles highly depended on its m6A-binding ability. Ythdc1 deficiency in ESCs resulted in decreased rRNA synthesis and the activation of 2-cell (2C) embryo-specific transcriptional program, and these observations recapitulated the transcriptome defects induced by dysfunction of the long interspersed nuclear element-1 (LINE1)-scaffold, which were unrelated to the direct targeting of Ythdc1. A detailed analysis revealed that Ythdc1 recognized m6A on LINE1 and was physically involved in the formation of the LINE1-Nucleolin partnership and the chromatin recruitment of Kap1. In summary, our study reveals a new link between m6A and the RNA scaffold and thus provides a new regulatory model for the crosstalk between RNA and the chromatin epigenome.

cell biology↗

bioRxiv2021

Glial activation in prion diseases is strictly nonautonomous and requires neuronal PrPSc

Although prion infections cause cognitive impairment and neuronal death, transcriptional and translational profiling shows progressive derangement within glia but surprisingly little changes within neurons. Here we expressed PrPC selectively in neurons, astrocytes or oligodendrocytes of mice. After prion infection, both astrocyte and neuron-restricted PrPC expression led to copious brain accumulation of PrPSc. As expected, neuron-restricted expression was associated with typical prion disease. However, mice with astrocyte-restricted PrPC expression experienced a normal life span, did not develop clinical disease, and did not show astro- or microgliosis. Besides confirming that PrPSc is innocuous to PrPC-deficient neurons, these results show that astrocyte-born PrPSc does not activate the extreme neuroinflammation that accompanies the onset of prion disease and precedes any molecular changes of neurons. This points to a nonautonomous mechanism by which prion-infected neurons instruct astrocytes and microglia to acquire a specific cellular state that, in turn, drives neural dysfunction.

neuroscience↗

bioRxiv2020

A small increase in CHEK1 activity leads to the arrest of the first zygotic division in human

The first mitotic division in mammalian zygotes is unique. The fertilized egg reactivates its cell cycle, and the maternal and paternal genomes start to reprogram to become totipotent. The first division is very sensitive to a range of perturbations, particularly the DNA damage, leading to the embryo's failure to enter the first mitosis. We discovered that a point mutation in the human CHEK1 gene resulted in an Arginine 442 to Glutamine change at the C-terminus of the CHEK1 protein. CHEK1 R442Q mutation caused the zygote to arrest just before the first division. Heterozygote individuals appeared to be healthy except that the female carriers are infertile. Expressing the corresponding mouse mutant Chk1 protein in zygotes also caused arrest before the first mitosis. Treating Chk1 R442Q mouse zygotes with low concentrations of CHEK1 inhibitor enabled the embryos to overcome the cell cycle arrest and resume normal development. Our results revealed an unexpected zygote mitotic checkpoint, which is extremely sensitive to the CHEK1 kinase activity. The fine-tuning of the DNA damage checkpoint permits the arrested one-cell embryos to overcome the first mitotic block and develop into healthy animals. These findings have important implications in assisted human reproduction.

developmental biology↗

bioRxiv2020

Functional Characterization of a Lassa Virus Fusion Inhibitors Adaptive Mutant

Lassa virus (LASV) glycoprotein complex (GPC) contains retained stable-signal peptide (SSP), GP1, and GP2. SSP interacts with GP2 and provides an interface targeted by numerous fusion inhibitors. Serially passaging of LASV with inhibitors allowed some adaptive mutants to be obtained of which most had mutations located in the transmembrane (TM) domain of GP2. In the current study, we focused on the F446L mutant, which is reported to confer resistance to ST-series inhibitors. We found that F446L conferred cross-resistance to structurally distinct inhibitors. Furthermore, F446L increased the fusion activities of LASV and Mopeia virus GPC, elevating the pH threshold for fusion of LASV and promoting fusion of MOPV at neutral pH. F446L exerted little effect on the pseudotype viral growth profile or thermostability. By introducing other residues to the conserved F446 locus, it was found that this site was less compatible with a similar tyrosine residue and was intolerable to charged residues. These results help characterize the fusion inhibitor target located in the TM domain of GP2, which should be useful for drug and vaccine design. IMPORTANCEThe LASV SSP-GP2 interface provides an Achilles heel that is targeted by numerous inhibitors. However, the emergence of resistant viruses is a major concern for direct antiviral drugs. In this study, we investigated the F446L mutant located in the GPC TM domain to determine the relationship between drug resistance, membrane fusion activity, viral growth kinetics, and thermostability. These results will be helpful in monitoring drug-resistant variants, as well as the advancement of drug and vaccine design.

microbiology↗

bioRxiv2020

Mechanism through which retrocyclin targets flavivirus multiplication

Currently, there are no approved drugs for the treatment of flavivirus infection. Accordingly, we tested the inhibitory effects of the novel {theta}-defensin retrocyclin-101 (RC-101) against flavivirus infection, and investigated the mechanism underlying the potential inhibitory effects. First, RC-101 robustly inhibited both Japanese encephalitis virus (JEV) and Zika virus (ZIKV) infections. RC-101 exerted inhibitory effects on the entry and replication stages. Results also indicated that the non-structural protein NS2B-NS3 serine protease might serve as a potential viral target. Further, RC-101 inhibited protease activity at the micromolar level. We also demonstrated that with respect to the glycoprotein E protein of flavivirus, the DE loop of domain III, which is the receptor-binding domain of the E protein, might serve as another viral target of RC-101. Moreover, a JEV DE mutant exhibited resistance to RC-101, which was associated with deceased binding affinity of RC-101 to DIII. These findings provide a basis for the development of RC-101 as a potential candidate for the treatment of flavivirus infection. ImportanceRC has been reported to have a broad-spectrum antimicrobial activity. In this study, we firstly report that RC-101 could inhibit ZIKV and JEV infections. Moreover, both the NS2B-NS3 serine protease and the DE loop in the E glycoprotein might serve as the viral targets of RC-101.

microbiology↗

bioRxiv2020

A LAMP-based microfluidic chip for rapid detection of pathogen in Cryptococcal meningitis

Cryptococcal meningitis (CM) is a global threat with significant attributable morbidity and mortality. Information on integrated detection for CM diagnosis is still limited. This is mainly due to the presence of a large polysaccharide capsule and the tough cell wall of Cryptococcus, which makes it difficult to extract nucleic acids on the chip. In this study, we developed a LAMP-based microfluidic chip for rapid detection of pathogen in CM. We adopted 4 duplicate filtration membrane structures to improve target capture and simplify the enrichment process, and combined lyticase digestion and thermal alkaline lysisto optimize the nucleic acid extraction of Cryptococcus on the chip, and selected a portable UVA flashlight to shine the LAMP products to obtain the visual detection results which could be observed by the naked eye. This microfluidic chip, integrating sample Cryptococcus enrichment, nucleic acid extraction and LAMP detection unit, streamlined the operation process and reduced the exposure risk of directly handling cryptococcal samples. It did not require any additional instruments and demonstrated a rapid, reliable, as well as high-efficiency approach. It truly realized the "sample-to-answer" application and could be easily used for clinical cryptococcal prediagnosis.

microbiology↗

bioRxiv2020

Genomic Insights into the Demographic History of Southern Chinese

Southern China is the birthplace of rice-cultivating agriculture, different language families, and human migrations that facilitated these cultural diffusions. The fine-scale demographic history in situ, however, remains unclear. To comprehensively cover the genetic diversity in East and Southeast Asia, we generated genome-wide SNP data from 211 present-day Southern Chinese and co-analyzed them with more than 1,200 ancient and modern genomes. We discover that the previously described Southern East Asian or Yangtze River Farmer lineage is monophyletic but not homogeneous, comprising four regionally differentiated sub-ancestries. These ancestries are respectively responsible for the transmission of Austronesian, Kra-Dai, Hmong-Mien, and Austroasiatic languages and their original homelands successively distributed from East to West in Southern China. Multiple phylogenetic analyses support that the earliest living branching among East Asian-related populations is First Americans ([~]27,700 BP), followed by the pre-LGM differentiation between Northern and Southern East Asians ([~]23,400 BP) and the pre-Neolithic split between Coastal and Inland Southern East Asians ([~]16,400 BP). In North China, distinct coastal and inland routes of south-to-north gene flow had established by the Holocene, and further migration and admixture formed the genetic profile of Sinitic speakers by [~]4,000 BP. Four subsequent massive migrations finalized the complete genetic structure of present-day Southern Chinese. First, a southward Sinitic migration and the admixture with Kra-Dai speakers formed the Sinitic Cline. Second, a bi-directional admixture between Hmong-Mien and Kra-Dai speakers gave rise to the Hmong-Mien Cline in the interior of South China between [~]2,000 and [~]1,000 BP. Third, a southwestward migration of Kra-Dai speakers in recent [~]2,000 years impacted the genetic profile for the majority of Mainland Southeast Asians. Finally, an admixture between Tibeto-Burman incomers and indigenous Austroasiatic speakers formed the Tibeto-Burman speakers in Southeast Asia by [~]2,000 BP.

genomics↗

bioRxiv2020

In situ 3D comparison of Chlorella pyrenoidosa with nuclear-irradiated mutagenic strains by using focused ion beam milling and cryo-electron tomography

Microalgae are highly efficient photosynthetic organisms that hold enormous potential as sources of renewable energy. In particular, Chlorella pyrenoidosa displays a rapid growth rate, high tolerance to light, and high lipid content, making it especially valuable for applications such as flue gas CO2 fixation, biofuel production, and nutritional extracts. In order to unveil its full potential, it is necessary to characterize its subcellular architecture. Here, we achieved three-dimensional (3D) visualization of the architectures of C. pyrenoidosa cells, by combining focused ion beam scanning electron microscopy (FIB/SEM), cryo-FIB milling, and cryo-electron tomography (cryo-ET). These high-resolution images bring to light intricate features of intact organelles, including thylakoid membranes, pyrenoid, starch granules, mitochondria, nucleus, lipid droplets and vacuoles, as well as the fine architectures within the chloroplast, including the concave-convex pyrenoid, plastoglobules, thylakoid tips, and convergence zones. Significantly, comparative analysis of wild-type and nuclear-irradiated mutagenic strains determined that cell volume and surface area of mutant cells have increased substantially to 2.2-fold and 1.7-fold, respectively, consistent with up-regulation of the enzyme Rubisco and enhanced photosynthetic metabolic processes. Moreover, quantitative analysis established that the thylakoid membrane width in mutant cells increased to 1.3-fold, while the membrane gap decreased to 0.8-fold, possibly contributing to the higher biomass growth rate of mutant cells. Our work reveals the first 3D subcellular architectures of C. pyrenoidosa cell and provides a structural framework for unlocking the higher growth rate in microalgae relevant to a wide range of industrial applications.

microbiology↗

bioRxiv2020

Enhanced ultrasound neuromodulation in vitro using nanobubble actuators

Ultrasound neuromodulation is a promising new method to manipulate brain activity noninvasively. Here, we detail a neurostimulation scheme using gas-filled nanostructures, gas vesicles (GVs), as actuators for improving the efficacy and precision of ultrasound stimuli. Sonicated primary neurons displayed dose-dependent, repeatable Ca2+ responses, closely synced to stimuli, and increased nuclear expression of the activation marker c-Fos only in the presence of GVs but not without. We identified mechanosensitive ion channels as important mediators of this effect, and neurons heterologously expressing the mechanosensitive MscL-G22S channel showed greater activation at lower acoustic pressure. This treatment scheme was also found not to induce significant cytotoxicity, apoptosis or membrane poration in treated cells. Altogether, we demonstrate a simple and effective method to achieve enhanced and more selective ultrasound neurostimulation. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

neuroscience↗

bioRxiv2020

Dissecting Mammalian Spermatogenesis Using Spatial Transcriptomics

Single-cell RNA sequencing has revealed extensive molecular diversity in gene programs governing mammalian spermatogenesis but fails to delineate their dynamics in the native context of seminiferous tubules -- the spatially-confined functional units of spermatogenesis. Here, we use Slide-seq, a novel spatial transcriptomics technology, to generate a comprehensive spatial atlas that captures the spatial gene expression patterns at near single-cell resolution in the mouse and human testis. By using Slide-seq data, we devise a computational framework that accurately localizes testicular cell types in individual seminiferous tubules. Unbiased spatial transcriptome analysis systematically identifies spatially patterned genes and gene programs, nominating genes with previously underappreciated but important functions in spermatogenesis. Using the human testicular spatial atlas, we identify two spatially segregated spermatogonial populations composed of stem cells at distinct transcriptional states. Finally, a comparison of the spatial atlas generated from the wild type and diabetic mouse testis reveals a disruption in the spatial cellular organization in diabetic seminiferous tubules.

genomics↗

bioRxiv2020

Effects of N-linked glycan of Lassa Virus Envelope Glycoprotein on the Immune Response

Lassa virus (LASV) belongs to the Mammarenavirus genus (family Arenaviridae) and causes severe hemorrhagic fever in humans. The glycoprotein precursor (GPC) contains eleven N-linked glycans that play essential roles in GPC functionalities such as cleavage, transport, receptor recognition, epitope shielding, and immune response. We used three mutagenesis strategies to abolish the individual glycan chains on the GPC and found that all three mutations led to cleavage inefficiency on the 2nd, 5th, and 8th glycosylation motifs. To evaluate N to Q mutagenesis for further research, it was found that deletion of the 2nd and 8th glycans completely inhibited the infectivity. We further investigated the role of glycans on GPC-mediated immune response by DNA immunization of mice. Deletion of the individual 1st, 3rd, 5th and 6th glycans significantly enhanced the proportion of effector CD4+ cells, whereas deletion of the 1st, 2nd, 3rd, 4th 5th, 6th, and 9th glycans enhanced the proportion of CD8+ effector T cells. Deletion of specific glycans improves the Th1-type immune response, and abolishment of glycan on GPC generally increases the antibody titer to the glycan-deficient GPC. However, the antibodies from either the mutant or WT GPC-immunized mice show little neutralization effect on wild-type LASV. The glycan residues on GPC provide an immune shield for the virus, and thus represent a target for the design and development of a vaccine. ImportanceAt present, there are no Food and Drug Administration-approved drugs or vaccines specific for LASV. Similar to other enveloped viruses with a heavy glycan shield, the N-linked glycans of LASV make it difficult for effector T cells and neutralization antibodies to access the glycoprotein epitope. In this study, we evaluated the effect of the individual glycan chains on GPC-mediated immune response, and found that deletion of the glycan improves the proportion of effector T cells, improving the Th1-type immune response, and increasing the antibody titer to the WT and mutant GPC, which may be beneficial to vaccine design and development.

microbiology↗