Biology subjects

Zhu, Y.

Publications and source records attributed to Zhu, Y..

At least 577 records · Page 32Linked to original sources

bioRxiv2022

A Four-Step Enzymatic Cascade for Efficient Production of L-Phenylglycine from Biobased L-Phenylalanine

Enantiopure amino acids are of particular interest in the agrochemical and pharmaceutical industries. Here, we reported a multi-enzyme cascade for efficient production of L-phenylglycine (L-Phg) from biobased L-phenylalanine (L-Phe). We first attempted to engineer Escherichia coli for expressing L-amino acid deaminase (LAAD) from Proteus mirabilis, hydroxymandelate synthase (HmaS) from Amycolatopsis orientalis, (S)-mandelate dehydrogenase (SMDH) from Pseudomonas putida, the endogenous aminotransferase (AT) encoded by ilvE and L-glutamate dehydrogenase (GluDH) from E. coli. However, 10 mM L-Phe only afforded the synthesis of 7.21 {+/-} 0.15 mM L-Phg. The accumulation of benzoylformic acid suggested that the transamination step might be rate-limiting. We next used leucine dehydrogenase (LeuDH) from Bacillus cereus to bypass the use of L-glutamate as amine donor, and 40 mM L-Phe gave 39.97 {+/-} 3.84 mM (6.04 {+/-} 0.58 g/L) L-Phg, reaching 99.9% conversion. In summary, this work demonstrated a concise four-step enzymatic cascade for the L-Phg synthesis from biobased L-Phe, with a potential for future industrial applications. Graphical abstracta concise four-step enzymatic cascade for the L-phenylglycine synthesis from biobased L-phenylalanine was devised. 40 mM L-phenylalanine afforded the synthesis of 39.97 {+/-} 3.84 mM (6.04 {+/-} 0.58 g/L) L-phenylglycine, reaching 99.9% conversion. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/476296v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@4f4aa0org.highwire.dtl.DTLVardef@1ac5bdeorg.highwire.dtl.DTLVardef@c372a2org.highwire.dtl.DTLVardef@19afa7b_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2022

Single-nucleus RNA-seq of normal-appearing brain regions in relapsing-remitting vs. secondary progressive multiple sclerosis

Multiple sclerosis (MS) is an immune-mediated demyelinating disease that alters central nervous system (CNS) functions. Relapsing-remitting MS (RRMS) is the most common form, which can transform into secondary-progressive MS (SPMS) that is associated with progressive neurodegeneration. Single-nucleus RNA sequencing (snRNA-seq) of MS lesions identified disease-related transcriptomic alterations; however, their relationship to non-lesioned MS brain regions has not been reported and which could identify prodromal or other disease susceptibility signatures. Here, snRNA-seq was used to generate high-quality RRMS vs. SPMS datasets of 33,197 nuclei from 8 normal-appearing MS brains, which revealed divergent cell type-specific changes. Notably, SPMS brains downregulated astrocytic sphingosine kinases (SPHK1/2) - the enzymes required to phosphorylate and activate the MS drug, fingolimod. This reduction was modeled with astrocyte-specific Sphk1/2 null mice in which fingolimod lost activity, supporting functionality of observed transcriptomic changes. These data provide an initial resource for studies of single cells from non-lesioned RRMS and SPMS brains.

neuroscience↗

bioRxiv2022

Hypoxia Dynamically Regulates DBC1 Ubiquitination and Stability by SIAH2 and OTUD5 in Breast Cancer Progression

DBC1 has been characterized as a key regulator of physiological and pathophysiological activities, such as DNA damage, senescence and tumorigenesis. However, the mechanism by which the functional stability of DBC1 is regulated has yet to be elucidated. Here, we report that the ubiquitination-mediated degradation of DBC1 is dynamically regulated by the E3 ubiquitin ligase SIAH2 and deubiquitinase OTUD5 under hypoxic stress. Mechanistically, hypoxia promoted the competitive binding of SIAH2 with OTUD5 to DBC1, resulting in the ubiquitination and subsequent degradation of DBC1 through the ubiquitin-proteasome pathway. Siah2 knockout inhibited tumor cell proliferation and migration, which could be rescued by double knockout of Siah2/DBC1. Human tissue microarray analysis further revealed that the SIAH2/DBC1 axis was responsible for tumor progression under hypoxic stress. These findings define a key role of the hypoxia-mediated SIAH2-DBC1 pathway in the progression of human breast cancer and provide novel insights into the metastatic mechanism of breast cancer.

cell biology↗

bioRxiv2022

MvPPT: a highly efficient and sensitive pathogenicity prediction tool for missense variants

Next generation sequencing technologies both boost the discovery of variants in the human genome and exacerbate the challenges of pathogenic variant identification. In this study, we developed mvPPT (Pathogenicity Prediction Tool for missense variants), a highly sensitive and accurate missense variant classifier based on gradient boosting. MvPPT adopts high-confidence training sets with a wide spectrum of variant profiles, and extracts three categories of features, including scores from existing prediction tools, allele, amino acid and genotype frequencies, and genomic context. Compared with established predictors, mvPPT achieved superior performance in all test sets, regardless of data source. In addition, our study also provides guidance for training set and feature selection strategies, as well as reveals highly relevant features, which may further provide biological insights of variant pathogenicity.

bioinformatics↗

bioRxiv2021

Homologous or Heterologous Booster of Inactivated Vaccine Reduces SARS-CoV-2 Omicron Variant Escape from Neutralizing Antibodies

The massive and rapid transmission of SARS-CoV-2 has led to the emergence of several viral variants of concern (VOCs), with the most recent one, B.1.1.529 (Omicron), which accumulated a large number of spike mutations, raising the specter that this newly identified variant may escape from the currently available vaccines and therapeutic antibodies. Using VSV-based pseudovirus, we found that Omicron variant is markedly resistant to neutralization of sera form convalescents or individuals vaccinated by two doses of inactivated whole-virion vaccines (BBIBP-CorV). However, a homologous inactivated vaccine booster or a heterologous booster with protein subunit vaccine (ZF2001) significantly increased neutralization titers to both WT and Omicron variant. Moreover, at day 14 post the third dose, neutralizing antibody titer reduction for Omicron was less than that for convalescents or individuals who had only two doses of the vaccine, indicating that a homologous or heterologous booster can reduce the Omicron escape from neutralizing. In addition, we tested a panel of 17 SARS-CoV-2 monoclonal antibodies (mAbs). Omicron resists 7 of 8 authorized/approved mAbs, as well as most of the other mAbs targeting distinct epitopes on RBD and NTD. Taken together, our results suggest the urgency to push forward the booster vaccination to combat the emerging SARS-CoV-2 variants.

microbiology↗

bioRxiv2021

Broad neutralization of SARS-CoV-2 variants by an inhalable bispecific single-domain antibody

The effectiveness of SARS-CoV-2 vaccines and therapeutic antibodies has been limited by the continuous emergence of viral variants, and by the restricted diffusion of antibodies from circulation into the sites of respiratory virus infection. Here, we report the identification of two highly conserved regions on Omicron variant RBD recognized by broadly neutralizing antibodies. Based on this finding, we generated a bispecific single-domain antibody that was able to simultaneously and synergistically bind these two regions on a single Omicron variant RBD as revealed by Cryo-EM structures. This inhalable antibody exhibited exquisite neutralization breadth and therapeutic efficacy in mouse models of SARS-CoV-2 infections. The structures also deciphered an uncommon cryptic epitope within the spike trimeric interface that may have implications for the design of broadly protective SARS-CoV-2 vaccines and therapeutics.

immunology↗

bioRxiv2021

Models for Evaluation of Biological Age Based on Multi-omics

Aging is a complex process of systemic degeneration at multiple cellular and tissue levels that has a complex mechanism. Individuals age at different rates, and there is a high degree of aging heterogeneity. Therefore, it is inaccurate to judge an individuals degree of aging by their chronological age. We performed transcriptome-focused multi-omics analyses (including transcriptomics, DNA methylation, proteomics, cytokine and telomere analysis, and single-cell transcriptome sequencing) on 139 healthy individuals aged 23 to 88 years. We systematically analyzed linear and non-linear changes in gene expression throughout aging. Genes with similar expression trajectories were enriched in similar biological pathways, including the PI3K-AKT signaling pathway and the inflammatory response. Through DNA methylation detection, we found that the expression of the top genes correlated with age was affected by methylation in the gene promoter region. These genes had no significant correlation with the expression of downstream proteins, but they were enriched in PI3K-AKT-associated proteins. Single-cell transcriptome sequencing showed that the expression of these genes did not significantly change in different cell subtypes, which proves that the gene expression changes were caused by internal age-related cellular changes rather than cell composition changes. We designed a transcriptome age clock and a methylation age clock using a set of 787 genes. Our models can accurately predict age with a mean absolute error (MAE) of 5.203 and 3.28, respectively, which is better than previously established aging models. The accuracy of our model was further verified by the detection of telomeres, which can identify accelerated aging of individuals.

genomics↗

bioRxiv2021

Interactions of SARS-CoV-2 protein E with cell junctions and polarity PDZ-containing proteins

The C-terminus of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein E contains a PBM (PDZ binding motif) targeting PDZ (PSD-95/Dlg/ZO-1) domains identical to the PBM of SARS-CoV. The latter is involved in the pathogenicity of the virus. Recently, we identified ten human PDZ-containing proteins showing significant interactions with SARS-CoV-2 protein E PBM. We selected several of them involved in cellular junctions and cell polarity (TJP1, PARD3, MLLT4, LNX2) and MPP5/Pals1 previously shown to interact with SARS-CoV E PBM. Targeting cellular junctions and polarity components is a common strategy by viruses to hijack cell machinery to their advantage. In this study, we showed that these host PDZ domains TJP1, PARD3, MLLT4, LNX2 and MPP5/PALS1 interact in a PBM-dependent manner in vitro and colocalize with the full-length E protein in cellulo, sequestrating the PDZ domains to the Golgi compartment. We solved three crystal structures of complexes between human LNX2, MLLT4 and MPP5 PDZs and SARS-CoV-2 E PBM highlighting its binding preferences for several cellular targets. Finally, we showed different affinities for the PDZ domains with the original SARS-CoV-2 C-terminal sequence containing the PBM and the one of the beta variant that contains a mutation close to the PBM. The acquired mutations in E protein localized near the PBM might have important effects both on the structure and the ion-channel activity of the E protein and on the host machinery targeted by the variants during the infection.

biochemistry↗

bioRxiv2021

A human monoclonal antibody potently pan-neutralizes SARS-CoV-2 VOCs by targeting RBD invariant sites

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of novel corona virus disease (COVID-19). The neutralizing monoclonal antibodies (mAbs) targeting the receptor binding domain (RBD) of SARS-CoV-2 are among the most promising strategies to prevent and treat COVID-19. However, SARS-CoV-2 variants of concern (VOCs) profoundly reduced the efficacies of most of mAbs and vaccines approved for clinical use. Herein, we demonstrated mAb 35B5 efficiently neutralizes both wild-type (WT) SARS-CoV-2 and VOCs, including B.1.617.2 (delta) variant, in vitro and in vivo. Cryo-electron microscopy (cryo-EM) revealed that 35B5 neutralizes SARS-CoV-2 by targeting a unique epitope that avoids the prevailing mutation sites on RBD identified in circulating VOCs, providing the molecular basis for its pan-neutralizing efficacy. The 35B5-binding epitope could also be exploited for the rational design of a universal SARS-CoV-2 vaccine.

immunology↗

bioRxiv2021

The Flowering Repressor SVP recruits the TOPLESS co-repressor to control flowering in chrysanthemum and Arabidopsis

Plant flowering time is a consequence of the perception of environmental and endogenous signals. The MCM1-AGAMOUSDEFICIENS-SRF-box (MADS-box) gene SHORT VEGETATIVE PHASE (SVP) is a pivotal repressor that negatively regulates the floral transition during the vegetative phase. The transcriptional corepressor TOPLESS (TPL) plays critical roles in many aspects of plant life. An interaction first identified between the second LXLXLX motif (LRLGLP) of CmSVP with CmTPL1-2, which can repress the expression of a key flowering factor CmFTL3 by binding its promotor CArG element in chrysanthemum. Genetic analysis suggested that the CmSVP-CmTPL1-2 transcriptional complex is a prerequisite for SVP to act as a floral repressor, which reduces CmFTL3 transcriptional activity. CmSVP rescued the phenotype of the svp-31 mutant in Arabidopsis, and overexpression of AtSVP or CmSVP in the Arabidopsis dominant negative mutation tpl-1 led to a loss-of-function in late flowering, which confirmed the highly conserved function of SVP in the two completely different species. Thus, we have validated a conserved machinery wherein SVP relies on TPL to inhibit flowering through the direct regulation of FT, which is more meaningful for the evolution of species and could be translated to high-quality cultivation and breeding of crops.

plant biology↗

bioRxiv2021

Real-time Image Denoising of Mixed Poisson-Gaussian Noise in Fluorescence Microscopy Images using ImageJ

AO_SCPLOWBSTRACTC_SCPLOWFluorescence microscopy imaging speed is fundamentally limited by the measurement signal-to-noise ratio (SNR). To improve image SNR for a given image acquisition rate, computational denoising techniques can be used to suppress noise. However, common techniques to estimate a denoised image from a single frame are either computationally expensive or rely on simple noise statistical models. These models assume Poisson or Gaussian noise statistics, which are not appropriate for many fluorescence microscopy applications that contain quantum shot noise and electronic Johnson-Nyquist noise, therefore a mixture of Poisson and Gaussian noise. In this paper, we show convolutional neural networks (CNNs) trained on mixed Poisson and Gaussian noise images to overcome the limitations of existing image denoising methods. The trained CNN is presented as an open-source ImageJ plugin that performs real-time image denoising (within tens of milliseconds) with superior performance (SNR improvement) compared to the conventional fluorescence microscopy denoising methods. The method is validated on external datasets with out-of-distribution noise, contrast, structure, and imaging modalities from the training data and consistently achieves high performance (> 8 dB) denoising in less time than other fluorescence microscopy denoising methods.

neuroscience↗

bioRxiv2021

A novel cryo-electron microscopy support film based on 2D crystal of HFBI protein

Cryo-electron microscopy (cryo-EM) has become the most powerful tool to resolve the high-resolution structures of biomacromolecules in solution. However, the air-water interface induced preferred orientation, dissociation or denaturation of biomacromolecules during cryo-vitrification is still a major limitation factor for many specimens. To solve this bottleneck, we developed a new type of cryo-EM support film using the 2D crystal of hydrophobin I (HFBI) protein. The HFBI-film utilizes its hydrophilic side to adsorb protein particles via electrostatic interactions and keep air-water interface away, allowing thin-enough ice and high-quality data collection. The particle orientation distribution can be optimized by changing the buffer pH. We, for the first time, solved the cryo-EM structure of catalase (2.28-[A]) that exhibited strong preferred orientation using conventional cryo-vitrification protocol. We further proved the HFBI-film is suitable to solve the high-resolution structures of small proteins including aldolase (150 kDa, 3.34-[A]) and hemoglobin (64 kDa, 3.6-[A]). Our work implied that the HFBI-film will have a wide application in the future to increase the successful rate and efficiency of cryo-EM.

biophysics↗

bioRxiv2021

Determining the architecture of nuclear ring of Xenopus laevis nuclear pore complex using integrated approaches

The nuclear pore complexes (NPCs) are large protein assemblies as a physical gate to regulate nucleocytoplasmic transport. Here, using integrated approaches including cryo-electron microscopy, hybrid homology modeling and cell experiment, we determined the architecture of the nuclear ring (NR) from Xenopus laevis oocytes NPC at subnanometer resolution. In addition to the improvement of the Y complex model, eight copies of Nup205 and ELYS were assigned in NR. Nup205 connects the inner and outer Y complexes and contributes to the assembly and stability of the NR. By interacting with both the inner Nup160 and the nuclear envelope (NE), the N-terminal {beta}-propeller and -solenoid domains of ELYS were found to be essential for accurate assembly of the NPC on the NE.

molecular biology↗

bioRxiv2021

8 A structure of the nuclear ring of the Xenopus laevis nuclear pore complex solved by cryo-EM and AI

The nuclear pore complex (NPC), one of the largest protein complexes in eukaryotes, serves as a physical gate to regulate nucleocytoplasmic transport. Here, we determined the 8 [A] resolution cryo-electron microscopic (cryo-EM) structure of the nuclear ring (NR) from the Xenopus laevis NPC, with local resolutions reaching 4.9 [A]. With the aid of AlphaFold2, we managed to build a pseudoatomic model of the NR, including the Y complexes and flanking components. In this most comprehensive and accurate model to date, the almost complete Y complex structure exhibits much tighter interaction in the hub region. Each NR asymmetric subunit contains two copies of Y complexes, one copy of Nup205 that connects the Y complexes to the neighbouring complex, one copy of ELYS that stabilizes the long arm region of the inner Y complex, and one copy of newly identified Nup93 that forms a bridge across the stems of Y complexes. These in-depth structural features represent a great advance in understanding the assembly of NPCs.

molecular biology↗

bioRxiv2021

8 A structure of the cytoplasmic ring of the Xenopus laevis nuclear pore complex solved by cryo-EM and AI

As one of the largest protein complexes in eukaryotes, the nuclear pore complex (NPC) forms a conduit regulating nucleocytoplasmic transport. Here, we determined 8 [A] resolution cryo-electron microscopic (cryo-EM) structure of the cytoplasmic ring (CR) from the Xenopus laevis NPC. With the aid of AlphaFold2, we managed to build a most comprehensive and accurate pseudoatomic model of the CR to date, including the Y complexes and flanking components of Nup358, Nup214 complexes, Nup205 and Nup93. Comparing with previously reported CR model, the Y complex structure in our model exhibits much tighter interactions in the hub region mediated by -solenoid domain in Nup160 C-terminus. Five copies of Nup358 are identified in each CR subunit to provide rich interactions with other Nups in stem regions of Y complexes. Two copies of Nup214 complexes lay in a parallel pattern and attach to the short arm region of Y complexes towards the central channel of NPC. Besides, the structural details of two copies of Nup205 on the side of the short arm region and one copy of Nup93 on the stem region of Y complexes in each CR subunit are also revealed. These in-depth novel structural features represent a great advance in understanding the assembly of NPCs.

molecular biology↗

bioRxiv2021

Quantitative analysis of transcription start site selection in Saccharomyces cerevisiae determines contributions of DNA sequence and RNA Polymerase II activity

Transcription start site (TSS) selection is a key step in gene expression and occurs at many promoter positions over a wide range of efficiencies. Here, we develop a massively parallel reporter assay to quantitatively dissect contributions of promoter sequence, NTP substrate levels, and RNA polymerase II (Pol II) activity to TSS selection by "promoter scanning" in Saccharomyces cerevisiae (Pol II MAssively Systematic Transcript End Readout, "Pol II MASTER"). Using Pol II MASTER, we measure the efficiency of Pol II initiation at 1,000,000 individual TSS sequences in a defined promoter context. Pol II MASTER confirms proposed critical qualities of S. cerevisiae TSS -8, -1, and +1 positions quantitatively in a controlled promoter context. Pol II MASTER extends quantitative analysis to surrounding sequences and determines that they tune initiation over a wide range of efficiencies. These results enabled the development of a predictive model for initiation efficiency based on sequence. We show that genetic perturbation of Pol II catalytic activity alters initiation efficiency mostly independently of TSS sequence, but selectively modulates preference for initiating nucleotide. Intriguingly, we find that Pol II initiation efficiency is directly sensitive to GTP levels at the first five transcript positions and to CTP and UTP levels at the second position genome wide. These results suggest individual NTP levels can have transcript-specific effects on initiation, representing a cryptic layer of potential regulation at the level of Pol II biochemical properties. The results establish Pol II MASTER as a method for quantitative dissection of transcription initiation in eukaryotes.

molecular biology↗

bioRxiv2021

Nuclear progesterone receptor regulates blood coagulation facilitating ovulation in zebrafish

Ovulation is a dramatic remodeling process that includes rupture of blood capillaries and clotting, but coagulation is not thought to directly regulate this process. Herein, we report remarkable increases of coagulation factors V (f5, [~]3145-fold) and tissue factor (f3a, [~]120-fold) in zebrafish ovarian follicle cells during ovulation. This increase was mediated through the nuclear progestin receptor (Pgr), which is essential for ovulation in zebrafish, and was totally abolished in ovarian follicular cells from pgr-/- mutants. In addition, promoter activities of f5 and f3a were significantly enhanced by progestin (DHP) via Pgr. Similar regulation of human F5 promoter activity was induced via human PGRB, suggesting a conserved mechanism. Site-directed mutagenesis of the zebrafish f5 promoter further demonstrated a direct regulation of coagulation factors via progestin response elements. Moreover, a stark increase of erythrocytes occurred in capillaries meshed in wildtype preovulatory follicles but was absent in pgr-/- mutants. Interestingly, anticoagulants significantly inhibited ovulation both in vitro and in vivo, respectively. Furthermore, reduced fecundity was observed in f5+/- female zebrafish. Taken together, our study provides plausible evidence for steroid regulation of coagulation factors, and a new hypothesis for blood clotting triggered ovulation in vertebrates.

physiology↗

bioRxiv2021

Transcriptomics analysis reveals intracellular mutual regulation of coral-zooxanthella holobionts

Corals should make excellent models for cross-kingdom regulation research because of their natural animal-photobiont holobiont composition, yet a lack of studies and experimental data restricts their use. Here we integrate new full-length transcriptomes and small RNAs of four common reef-building corals with the published Symbiodinium C1 genome to gain deeper insight into mutual gene regulation in coral-zooxanthella holobionts. We show that zooxanthellae secrete miRNA to downregulate rejection from host coral cells, and that a potential correlation exists between miRNA diversity and physiological activity. Convergence of these holobionts biological functions in different species is also revealed, which implies the low gene impact on bottom ecological niche organisms. This work provides evidence for the early origin of cross-kingdom regulation as a mechanism of self-defense autotrophs can use against heterotrophs, sheds more light on coral-zooxanthella holobionts, and contributes valuable data for further coral research.

ecology↗