Biology subjects

Lei, J.

Publications and source records attributed to Lei, J..

At least 73 records · Page 4Linked to original sources

bioRxiv2020

Impacted Spike Frequency Adaptation Associated with Reduction of KCNQ2/3 Promotes Seizure Activity in Temporal Lobe Epilepsy

Although numerous epilepsy-related genes have been identified by unbiased genome-wide screening based on samples from both animal models and patients, the druggable targets for temporal lobe epilepsy (TLE) are still limited. Meanwhile, a large number of candidate genes that might promote or inhibit seizure activities are waiting for further validation. In this study, we first analyzed two public databases and determined the significant down-regulations of two M-type potassium channel genes (KCNQ2/3) expressions in hippocampus samples from TLE patients. Then we reproduced the similar pathological changes in the pilocarpine mouse model of TLE and further detected the decrease of spike frequency adaptation driven by impacted M-currents on dentate gyrus granule neurons. Finally, we employed a small-scale simulation of dentate gyrus network to investigate potential functional consequences of disrupted neuronal excitability. We demonstrated that the impacted spike frequency adaptation of granule cells facilitated the epileptiform activity among the entire network, including prolonged seizure duration and reduced interictal intervals. Our results identify a new mechanism contributing to ictogenesis in TLE and suggest a novel target for the anti-epileptic drug discovery.

neuroscience↗

bioRxiv2020

Exponential-family embedding with application to cell developmental trajectories for single-cell RNA-seq data

Scientists often embed cells into a lower-dimensional space when studying single-cell RNA-seq data for improved downstream analyses such as developmental trajectory analyses, but the statistical properties of such non-linear embedding methods are often not well understood. In this article, we develop the eSVD (exponential-family SVD), a non-linear embedding method for both cells and genes jointly with respect to a random dot product model using exponential-family distributions. Our estimator uses alternating minimization, which enables us to have a computationally-efficient method, prove the identifiability conditions and consistency of our method, and provide statistically-principled procedures to tune our method. All these qualities help advance the single-cell embedding literature, and we provide extensive simulations to demonstrate that the eSVD is competitive compared to other embedding methods. We apply the eSVD via Gaussian distributions where the standard deviations are proportional to the means to analyze a single-cell dataset of oligodendrocytes in mouse brains (Marques et al., 2016). Using the eSVD estimated embedding, we then investigate the cell developmental trajectories of the oligodendrocytes. While previous results are not able to distinguish the trajectories among the mature oligodendrocyte cell types, our diagnostics and results demonstrate there are two major developmental trajectories that diverge at mature oligodendrocytes.

genomics↗

bioRxiv2020

Individual cell-based modeling of tumor cell plasticity-induced immune escape after CAR-T therapy

Chimeric antigen receptor (CAR) therapy targeting CD19 is an effective treatment for refractory B cell malignancies, especially B cell acute lymphoblastic leukemia (B-ALL). The majority of patients achieve a complete response following a single infusion of CD19-targeted CAR-modified T cells (CAR-19 T cells); however, many patients suffer relapse after therapy, and the underlying mechanism remains unclear. To better understand the mechanism of tumor relapse, we developed an individual cell based computational model for tumor cell plasticity and the heterogeneous responses to the CAR-T treatment. Model simulations reproduced the process of tumor relapse, and predicted that CAR-T stress-induced cell plasticity can lead to tumor relapse in B-ALL. Model predictions were verified by applying the second-generation CAR-T cells to mice injected with NALM-6-GL leukemic cells, in which 60% of the mice relapse within 3 months, and relapsed tumors retained CD19 expression but exhibited a subpopulation of cells with CD34 transcription. These findings lead to a mechanism of tumor replace by which CAR-T treatment induced tumor cells to transition to hematopoietic stem-like cells (HSLCs) and myeloid-like cells and hence escape of CAR-T targeting. The computational model framework was successfully developed to recapitulate the individual evolutionary dynamics, which could predict clinical survival outcomes in B-ALL patients after CAR-T therapy.

cancer biology↗

bioRxiv2020

Using single-cell entropy to describe the dynamics of reprogramming and differentiation of induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) provide a great model to study the process of stem cell reprogramming and differentiation. Single-cell RNA sequencing (scRNA-seq) enables us to investigate the reprogramming process at single-cell level. Here, we introduce single-cell entropy (scEntropy) as a macroscopic variable to quantify the cellular transcriptome from scRNA-seq data during reprogramming and differentiation of iPSCs. scEntropy measures the relative order parameter of genomic transcriptions at single cell level during the process of cell fate changes, which show increase tendency during differentiation, and decrease upon reprogramming. Hence, scEntropy provides an intrinsic measurement of the cell state, and can be served as a pseudo-time of the stem cell differentiation process. Moreover, based on the evolutionary dynamics of scEntropy, we construct a phenomenological Fokker-Planck equation model and the corresponding stochastic differential equation for the process of cell state transitions during pluripotent stem cell differentiation. These equations provide further insights to infer the processes of cell fates changes and stem cell differentiation. This study is the first to introduce the novel concept of scEntropy to quantify the biological process of iPSC, and suggests that the scEntropy can provide a suitable macroscopic variable for single cells to describe cell fate transition during differentiation and reprogramming of stem cells.

cell biology↗

bioRxiv2020

Molecular architecture of the luminal ring of the Xenopus laevis nuclear pore complex

Nuclear pore complex (NPC) mediates the flow of substances between the nucleus and cytoplasm in eukaryotic cells. Here we report the cryo-electron tomography (cryo-ET) structure of the luminal ring (LR) of the NPC from Xenopus laevis oocyte. The observed key structural features of the LR are independently confirmed by single-particle cryo-electron microscopy (cryo-EM) analysis. The LR comprises eight butterfly-shaped subunits, each containing two symmetric wings. Each wing consists of four elongated, tubular protomers. Within the LR subunit, the eight protomers form a Finger domain, which directly contacts the fusion between the inner and outer nuclear membranes, and a Grid domain, which serves as a rigid base for the Finger domain. Two neighbouring LR subunits interact with each other through the lateral edges of their wings to constitute a Bumper domain, which displays two major conformations and appears to cushion neighbouring NPCs. Our study reveals previously unknown features of the LR and potentially explains the elastic property of the NPC.

biophysics↗

bioRxiv2020

Structure of the Cytoplasmic Ring of the Xenopus laevis Nuclear Pore Complex

Nuclear pore complex (NPC) exhibits structural plasticity and has only been characterized at local resolutions of up to 15 [A] for the cytoplasmic ring (CR). Here we present a single-particle cryo-electron microscopy (cryo-EM) structure of the CR from Xenopus laevis NPC at average resolutions of 5.5-7.9 [A], with local resolutions reaching 4.5 [A]. Improved resolutions allow identification and placement of secondary structural elements in the majority of the CR components. The two Y complexes in each CR subunit interact with each other and associate with those from flanking subunits, forming a circular scaffold. Within each CR subunit, the Nup358-containing region wraps around the stems of both Y complexes, likely stabilizing the scaffold. Nup205 connects the short arms of the two Y complexes and associates with the stem of a neighbouring Y complex. The Nup214-containing region uses an extended coiled-coil to link Nup85 of the two Y complexes and protrudes into the axial pore of the NPC. These previously uncharacterized structural features reveal insights into NPC assembly.

biophysics↗

bioRxiv2020

Computational simulations reveal the binding dynamics between human ACE2 and the receptor binding domain of SARS-CoV-2 spike protein

A novel coronavirus (the SARS-CoV-2) has been identified in January 2020 as the causal pathogen for COVID-19 pneumonia, an outbreak started near the end of 2019 in Wuhan, China. The SARS-CoV-2 was found to be closely related to the SARS-CoV, based on the genomic analysis. The Angiotensin converting enzyme 2 protein (ACE2) utilized by the SARS-CoV as a receptor was found to facilitate the infection of SARS-CoV-2 as well, initiated by the binding of the spike protein to the human ACE2. Using homology modeling and molecular dynamics (MD) simulation methods, we report here the detailed structure of the ACE2 in complex with the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. The predicted model is highly consistent with the experimentally determined complex structures. Plausible binding modes between human ACE2 and the RBD were revealed from all-atom MD simulations. The simulation data further revealed critical residues at the complex interface and provided more details about the interactions between the SARS-CoV-2 RBD and human ACE2. Two mutants mimicking rat ACE2 were modeled to study the mutation effects on RBD binding to ACE2. The simulations showed that the N-terminal helix and the K353 of the human ACE2 alter the binding modes of the CoV2-RBD to the ACE2.

biophysics↗

bioRxiv2019

Structural basis for pore blockade of the human cardiac sodium channel Nav1.5 by tetrodotoxin and quinidine

Among the nine subtypes of human voltage-gated Na+ (Nav) channels, tetrodotoxin (TTX)-resistant Nav1.5 is the primary one in heart. More than 400 point mutations have been identified in Nav1.5 that are associated with cardiac disorders exemplified by type 3 long QT syndrome and Brugada syndrome, making Nav1.5 a common target for class I antiarrythmic drugs. Here we present the cryo-EM structures of Nav1.5 bound to quinidine and TTX with resolutions at 3.2 [A] and 3.3 [A], respectively, for the pore domain. The structures allow mapping of 278 disease-related mutations in the resolved region, establishing the framework for mechanistic investigation of these pathogenic mutations. Quinidine is positioned right below the selectivity filter and coordinated by residues from repeats I, III, and IV. Pore blockade is achieved through both direct obstruction of the ion permeation path and induced rotation of a Tyr residue that leads to intracellular gate closure. Structural comparison of Nav1.5 with our previously reported Nav1.2/1.4/1.7 reveals the molecular determinant for the different susceptibility to TTX by TTX-resistant and -sensitive Nav subtypes, and highlights the functional significance of the underexplored and least conserved extracellular loops.

biophysics↗

bioRxiv2019

Structural variation, functional differentiation and expression characteristics of the AP2/ERF gene family and its response to cold stress and methyl jasmonate in Panax ginseng C.A. Meyer

The APETALA2/Ethylene Responsive Factor (AP2/ERF) gene family has been shown to play a crucial role in plant growth and development, stress responses and secondary metabolite biosynthesis. Nevertheless, little is known about the gene family in ginseng (Panax ginseng), an important traditional medicinal herb in Asia and North America. Here, we report the systematic analysis of the gene family present in ginseng using several transcriptomic databases. A total of 189 putative AP2/ERF genes, defined as PgERF001 through PgERF189. The 93 PgERF genes that have the complete AP2 domain in their open reading frames were classified into five subfamilies, DREB, ERF, AP2, RAV and Soloist. The DREB subfamily and ERF subfamily were further clustered four and six groups, respectively, compared to the 12 groups of these subfamilies found in Arabidopsis. Gene ontology categorized these 397 transcripts of the 189 PgERF genes into eight functional subcategories, suggesting their functional differentiation and they have been especially enriched for the nucleic acid binding transcription factor activity subcategory. The expression activity and networks of the 397 PgERF transcripts have substantially diversified across tissues, developmental stages and genotypes. Then, the expression change of six PgERF genes randomly selected from DREB subfamily, i.e., PgERF073, PgERF079, PgERF110, PgERF115, PgERF120 and PgERF128 responding to cold stress suggesting that DREB subfamily genes played an important role in cold resistance of ginseng. Finally, we studied the responses of the PgERF genes to methyl jasmonate (MeJA). 288 (72.5%) of the 397 PgERF gene transcripts responded to the MeJA treatment, with 136 up-regulated and 152 down-regulated, indicating that most members of the PgERF gene family are responsive to MeJA. These results provide resources and knowledge necessary for family-wide functional analysis of the PgERF genes in ginseng and related species.

genomics↗

bioRxiv2019

Conformational Dynamics of Nonenveloped Circovirus Capsid to the Host Cell Receptor

Circovirus, comprising one capsid protein, is the smallest nonenveloped virus and induces lymphopenia. Circovirus can be used to explore the cell adhesion mechanism of nonenveloped viruses. We developed a single-molecule fluorescence resonance energy transfer (smFRET) assay to directly visualize the capsids conformational feature. The capsid underwent reversible dynamic transformation between three conformations. The cell surface receptor heparan sulfate (HS) altered the dynamic equilibrium of the capsid to the high-FRET state, revealing the HS binding region. Neutralizing antibodies restricted capsid transition to a low-FRET state, masking the HS binding domain. The lack of positively charged amino acids in the HS binding site reduced cell surface affinity and attenuated virus infectivity via conformational changes. These intrinsic characteristics of the capsid suggested that conformational dynamics is critical for the structural changes occurring upon cell surface receptor binding, supporting a dynamics-based mechanism of receptor binding.\n\nImportanceViral proteins were commom working as ligand to interacte with cell surface glycosaminoglycan receptors to achieve the virus attachment, during which the conformational dynamics of the protein ligand are also vital for the binding properties. In this study, PCV2 capsid and heparin sulfate were used to study the protein conformational dynamics of nonenveloped and icosahedral circovirus capsid during triggering to cell surface receptor. we demonstrated the PCV2 capsid could acts as a dynamic machine, spontaneously adopting multiple conformations with reversible interconversion and intrinsic conformational features could be regulated by glycosaminoglycan receptors and neutralizing antibodies. These increased our understanding of the mechanism by which nonenveloped virus attach to cells.

microbiology↗

bioRxiv2019

Molecular basis for allosteric regulation of the type 2 ryanodine receptor channel gating by key modulators

The type-2 ryanodine receptor (RyR2) is responsible for releasing Ca2+ from the sarcoplasmic reticulum of cardiomyocytes, subsequently leading to muscle contraction. Here, we report four cryo-EM structures of porcine RyR2 bound to distinct modulators that collectively provide mechanistic insight into RyR2 regulation. Ca2+ alone induces a contraction of the Central domain that facilitates the dilation of S6 bundle, but is insufficient to open the pore. The small molecule agonist PCB95 helps Ca2+ to overcome the barrier for opening. FKBP12.6 induces a relaxation of the Central domain that decouples it from the S6 bundle, stabilizing RyR2 in a closed state. Caffeine locks the Central domain in a constitutively contracted state, while further addition of ATP opens the channel by strengthening the coupling between the U-motif and S6. Our study marks an important step towards mechanistic understanding of the complicated regulation of this key channel whose aberrant activity engenders life-threatening cardiac disorders.

biochemistry↗

bioRxiv2019

Single-cell entropy to quantify the cellular transcriptome from single-cell RNA-seq data

We present the use of single-cell entropy (scEntropy) to measure the order of the cellular transcriptome profile from single-cell RNA-seq data, which leads to a method of unsupervised cell type classification through scEntropy followed by the Gaussian mixture model (scEGMM). scEntropy is straightforward in defining an intrinsic transcriptional state of a cell. scEGMM is a coherent method of cell type classification that includes no parameters and no clustering; however, it is comparable to existing machine learning-based methods in benchmarking studies and facilitates biological interpretation.

bioinformatics↗

bioRxiv2019

A general mathematical framework for understanding the behavior of heterogeneous stem cell regeneration

Stem cell heterogeneity is essential for the homeostasis in tissue development. This paper established a general formulation for understanding the dynamics of stem cell regeneration with cell heterogeneity and random transitions of epigenetic states. The model generalizes the classical G0 cell cycle model, and incorporates the epigenetic states of stem cells that are represented by a continuous multidimensional variable and the kinetic rates of cell behaviors, including proliferation, differentiation, and apoptosis, that are dependent on their epigenetic states. Moreover, the random transition of epigenetic states is represented by an inheritance probability that can be described as a conditional beta distribution. This model can be extended to investigate gene mutation-induced tumor development. The proposed formula is a generalized formula that helps us to understand various dynamic processes of stem cell regeneration, including tissue development, degeneration, and abnormal growth.

systems biology↗

bioRxiv2019

Honey bee trypanosomatid parasite dynamically changes the transcriptome during the infection of honey bee and modifies the host physiology

Although there are many honey bee pathogens/parasites, it is still not understood how they change their gene expression to adapt to the host environment or how the host simultaneously responds to pathogen/parasite infection by modifying its own gene expression. Such interactions must lead to changes in the physiological states of both host and parasite. To address this question, we studied a trypanosomatid, Lotmaria passim, which can be cultured in medium and inhabit the honey bee hindgut. We found that L. passim dynamically modifies the expression of mRNAs associated with protein translation and the electron transport chain to adapt to the anaerobic and nutritionally poor honey bee hindgut at early stages of infection, and to become dormant at late stages of infection. Meanwhile, several genes are continuously up- or down-regulated during infection, including GP63 as well as genes coding for host cell signaling pathway modulators (up-regulated), and those involved in detoxification of radical oxygen species as well as flagellar formation (down-regulated). L. passim infection only slightly increases honey bee mortality and does not affect the number of microorganisms in the gut microbiota; but it induces honey bee innate immune response. Upon infection, the host appears to be in poor nutritional status, indicated by the increase in the levels of mRNAs for take-out and facilitated trehalose transporter and the decrease of vitellogenin mRNA level. Simultaneous gene expression profiling of L. passim and honey bee during infection provided insight into how both parasite and host modify their gene expressions. This study presents one of the best models to understand host-parasite interactions at the molecular and cellular levels in honey bee.

microbiology↗

bioRxiv2018

Honey bee parasitic mite contains the sensory organ expressing ionotropic receptors with conserved functions

Honey bee parasitic mites (Tropilaelaps mercedesae and Varroa destructor) detect temperature, humidity, and odor but the underlying sensory mechanisms are poorly understood. To uncover how T. mercedesae responds to environmental stimuli inside a hive, we identified the sensilla-rich sensory organ on the foreleg tarsus. The organ contained four types of sensilla, which may respond to different stimuli based on their morphology. We found the forelegs were enriched with mRNAs encoding sensory proteins such as ionotropic receptors (IRs) and gustatory receptors (GRs), as well as proteins involved in ciliary transport. We also found that T. mercedesae and Drosophila melanogaster IR25a and IR93a are functionally equivalent. These results demonstrate that the structures and physiological functions of ancient IRs have been conserved during arthropod evolution. Our study provides insight into the sensory mechanisms of honey bee parasitic mites, as well as potential targets for methods to control the most serious honey bee pest.

physiology↗

bioRxiv2018

Structures of the Human Spliceosomes Before and After Release of the Ligated Exon

Pre-mRNA splicing is executed by the spliceosome, which has eight major functional states each with distinct composition. Five of these eight human spliceosomal complexes, all preceding exon ligation, have been structurally characterized. In this study, we report the cryo-electron microscopy structures of the human post-catalytic spliceosome (P complex) and intron lariat spliceosome (ILS) at average resolutions of 3.0 and 2.9 [A], respectively. In the P complex, the ligated exon remains anchored to loop I of U5 small nuclear RNA, and the 3-splice site is recognized by the junction between the 5-splice site and the branch point sequence. The ATPase/helicase Prp22, along with the ligated exon and eight other proteins, are dissociated in the P-to-ILS transition. Intriguingly, the ILS complex exists in two distinct conformations, one with the ATPase/helicase Prp43 and one without. Comparison of these three late-stage human spliceosomes reveals mechanistic insights into exon release and spliceosome disassembly.

biochemistry↗

bioRxiv2018

Structures of the Catalytically Activated Yeast Spliceosome Reveal the Mechanism of Branching

Pre-mRNA splicing is executed by the spliceosome. Structural characterization of the catalytically activated complex (B*) is pivotal for mechanistic understanding of catalysis of the branching reaction by the spliceosome. In this study, we assembled the B* complex on two different pre-mRNAs from Saccharomyces cerevisiae and determined the cryo-EM structures of four distinct B complexes at overall resolutions of 2.9-3.8 [A]. The duplex between U2 snRNA and the branch point sequence (BPS) is located 13-20 [A] away from the 5-splice site (5SS) in the B* complexes that are devoid of the step I splicing factors Yju2 and Cwc25. Recruitment of Yju2 into the active site brings the U2/BPS duplex into the vicinity of 5SS, ready for branching. In the absence of Cwc25, the nucleophile from BPS is positioned about 4 [A] away from, and remains to be activated by, the catalytic metal M2. This analysis reveals the functional mechanism of Yju2 and Cwc25 in branching. These four structures constitute compelling evidence for substrate-specific conformations of the spliceosome in a major functional state.

biophysics↗

bioRxiv2018

Single particle cryo-EM reconstruction of 52 kDa streptavidin at 3.2 Angstrom resolution

The fast development of single particle cryo-EM has made it more feasible to obtain the 3D structure of well-behaved macromolecules with molecular weight higher than 300 kDa at ~3 [A] resolution. It remains a challenge to obtain high resolution structure of molecules smaller than 100 kDa using single particle cryo-EM, mainly due to the low contrast of the molecules embedded in vitreous ice. In this work, we applied the Cs-corrector-VPP coupled cryo-EM to study 52 kDa streptavidin (SA) protein supported on a thin layer of graphene film and embedded in vitreous ice. We were able to solve both the apo-SA and biotin-bound SA at near-atomic resolution using single particle cryo-EM. We demonstrated that the method is capable to determine the structure of molecule as small as 39 kDa and potentially even smaller molecules. Furthermore, we found that using the graphene film to avoid the adsorption to the air-water interface is critical to maintain the proteins high-resolution structural information.

biophysics↗