Biology subjects

Zhang, M.

Publications and source records attributed to Zhang, M..

At least 613 records · Page 34Linked to original sources

bioRxiv2021

In-cell structures of a conserved supramolecular array at the mitochondria-cytoskeleton interface in mammalian sperm

Mitochondria-cytoskeleton interactions modulate cellular physiology by regulating mitochondrial transport, positioning, and immobilization. However, there is very little structural information defining mitochondria-cytoskeleton interfaces in any cell type. Here, we use cryo-focused ion beam milling-enabled cryo-electron tomography to image mammalian sperm, where mitochondria wrap around the ciliary cytoskeleton. We find that mitochondria are tethered to their neighbors through inter-mitochondrial linkers and are anchored to the cytoskeleton through ordered arrays on the outer mitochondrial membrane. We use subtomogram averaging to resolve in-cell structures of these arrays from three mammalian species, revealing they are conserved across species despite variations in mitochondrial dimensions and cristae organization. We find that the arrays consist of boat-shaped particles anchored on a network of membrane pores whose arrangement and dimensions are consistent with voltage dependent anion channels. Proteomics and in-cell cross-linking mass spectrometry suggest that the conserved arrays are composed of glycerol kinase-like proteins. Ordered supramolecular assemblies may serve to stabilize similar contact sites in other cell types where mitochondria need to be immobilized in specific subcellular environments, such as in muscles and neurons.

cell biology↗

bioRxiv2021

Discovery of a molecular glue that enhances UPRmt to restore proteostasis via TRKA-GRB2-EVI1-CRLS1 axis

Lowering proteotoxicity is a potentially powerful approach for the treatment of neurological disorders, such as Parkinsons disease. The unfolded protein response (UPR) is a major mechanism that preserves the network maintaining cellular proteostasis. In the present study, we developed the screening strategy to discover compounds that significantly enhanced the activation of mitochondrial UPR (UPRmt) through increasing cardiolipin content. We identified that ginsenoside Rg3 (Rg3) increased cardiolipin depending on cardiolipin synthase 1 (CRLS1) in both worms and in human neural cells. Using LiP-SMap (limited proteolysis-mass spectrometry) strategy, we identified GRB2 (growth factor receptor bound protein 2) as a direct target of Rg3 in human neural cells. Rg3 enhances the binding between GRB2 and TRKA, that transduces signals via phosphrorylation of ERK. We provide bioinformatic and experimental evidence that EVI1, the critical oncogenic transcriptional regulator in leukemia, binds to CRLS1 promoter region and stimulated CRLS1 expression and subsequently increased cardiolipin content in the presence of Rg3. In a Parkinsons disease mouse model, Rg3 restores motor function by protecting nigral dopaminergic neurons dependent on Grb2. Our data recapitulate the TRKA-GRB2-EVI1-CRLS1 axis in maintaining proteostasis in Parkinsons disease via UPRmt.

pharmacology and toxicology↗

bioRxiv2021

An integrated strategy reveals complex glycosylation of erythropoietin using top-down and bottom-up mass spectrometry

The characterization of glycoproteins, like erythropoietin, is challenging due to the structural micro- and macro-heterogeneity of the protein glycosylation. This study presents an in-depth strategy for glycosylation analysis of a first-generation erythropoietin (epoetin beta), including a developed top-down mass spectrometric workflow for N-glycan analysis, bottom-up mass spectrometric methods for site-specific N-glycosylation and a LC-MS approach for O-glycan identification. Permethylated N-glycans, peptides and enriched glycopeptides of erythropoietin were analyzed by nanoLC-MS/MS and de-N-glycosylated erythropoietin was measured by LC-MS, enabling the qualitative and quantitative analysis of glycosylation and different glycan modifications (e.g., phosphorylation and O-acetylation). Extending the coverage of our newly developed Python script to phosphorylated N-glycans enabled the identification of 140 N-glycan compositions (237 N-glycan structures) from erythropoietin. The site-specificity of N-glycans was revealed at glycopeptide level by pGlyco software using different proteases. In total, 215 N-glycan compositions were identified from N-glycan and glycopeptide analysis. Moreover, LC-MS analysis of de-N-glycosylated erythropoietin species identified two different O-glycan compositions, based on the mass shifts between non-O-glycosylated and O-glycosylated species. This integrated strategy allows the in-depth glycosylation analysis of a therapeutic glycoprotein to understand its pharmacological properties and improving the manufacturing processes.

biochemistry↗

bioRxiv2021

Methylation of KRAS at lysine 182 and 184 by SETD7 promotes KRAS degradation

Oncogenic KRAS mutations are considered to be a key driver for initiation and progression in non-small-cell lung cancer (NSCLC). However, how post-translational modifications (PTMs) of KRAS, especially methylation, modify KRAS activity and downstream signals remain largely unclear. Here, we showed that SET domain containing histone lysine methyltransferase 7 (SETD7) interacts with KRAS and methylates KRAS at lysine 182 and 184. SETD7-mediated methylation of KRAS led to degradation of KRAS and attenuation of the RAS/MEK/ERK signaling cascade, endowing SETD7 with a potent tumor-suppressive role in NSCLC, both in vitro and in vivo. Mechanistically, RABGEF1, a ubiquitin E3 ligase of KRAS, was recruited and promoted KRAS degradation in a K182/K184 methylation-dependent manner. Notably, low SETD7 or RABGEF1 expression was associated with poor prognosis in lung adenocarcinoma patients. Taken together, our results establish a novel connection between lysine methylation and KRAS protein stability, in addition to elucidating a tumor-suppressive function of SETD7 that operates via modulation of oncogenic RAS signaling.

cancer biology↗

bioRxiv2021

Molecular mechanism underlying the non-essentiality of Bub1 for the fidelity of chromosome segregation in human cells

The multi-task protein kinase Bub1 has long been considered important for chromosome alignment and spindle assembly checkpoint signaling during mitosis. However, recent studies provide surprising evidence that Bub1 may not be essential in human cells, with the underlying mechanism unknown. Here we show that Bub1 plays a redundant role with the non-essential CENP-U complex in recruiting Polo-like kinase 1 (Plk1) to the kinetochore. While disrupting either pathway of Plk1 recruitment does not affect the accuracy of whole chromosome segregation, loss of both pathways leads to a strong reduction in the kinetochore accumulation of Plk1 under a threshold level required for proper chromosome alignment and segregation. Thus, parallel recruitment of Plk1 to kinetochores by Bub1 and the CENP-U complex ensures high fidelity of mitotic chromosome segregation. This study may have implications for targeted treatment of cancer cells harboring mutations in either Bub1 or the CENP-U complex.

cell biology↗

bioRxiv2021

Expression and Manufacturing of Protein Therapeutics in Spirulina

Arthrospira platensis (commonly known as spirulina) is a photosynthetic cyanobacterium1. It is a highly nutritious food that has been consumed for decades in the US, and even longer by indigenous cultures2. Its widespread use as a safe food source and proven scalability have driven frequent attempts to convert it into a biomanufacturing platform. But these were repeatedly frustrated by spirulinas genetic intractability. We report here efficient and versatile genetic engineering methodology for spirulina that allows stable expression of bioactive protein therapeutics at high levels. We further describe large-scale, indoor cultivation and downstream processing methods appropriate for the manufacturing of biopharmaceuticals in spirulina. The potential of the platform is illustrated by pre-clinical development and human testing of an orally delivered antibody therapeutic against campylobacter, a major cause of infant mortality in the developing world and a growing antibiotic resistance threat3,4. This integrated development and manufacturing platform blends the safety of food-based biotechnology with the ease of genetic manipulation, rapid growth rates and high productivity characteristic of microbial platforms. These features combine for exceptionally low-cost production of biopharmaceuticals to address medical needs that are unfeasible with current biotechnology platforms.

molecular biology↗

bioRxiv2021

Myocardial BDNF regulates cardiac bioenergetics through the transcription factor Yin Yang 1

Circulating Brain-derived Neurotrophic Factor (BDNF) is markedly decreased in heart failure patients. Both BDNF and its receptor, Tropomyosin Related Kinase Receptor (TrkB), are expressed in cardiomyocytes, however the role of myocardial BDNF signaling in cardiac pathophysiology is poorly understood. We found that cardiac-specific TrkB knockout (cTrkB KO) mice displayed a blunted adaptive cardiac response to exercise, with attenuated upregulation of transcription factor networks controlling mitochondrial biogenesis/metabolism, including Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1). The cTrkB KO mice developed an exacerbated heart failure progression with transaortic constriction. The downregulation of PGC-1 in cTrkB KO mice exposed to exercise or TAC resulted in decreased cardiac energetics. We further unraveled that BDNF induces PGC-1 upregulation and bioenergetics through a novel signaling pathway, the pleiotropic transcription factor Yin Yang 1 (YY1). Taken together, our findings suggest that myocardial BDNF plays a critical role in regulating cellular energetics in the cardiac stress response.

molecular biology↗

bioRxiv2021

Single particle combinatorial multiplexed liposome fusion mediated by DNA

Combinatorial high throughput methodologies are central for both screening and discovery in synthetic biochemistry and biomedical sciences. They are, however, often reliant on large scale analyses and thus limited by long running time and excessive materials cost. We herein present Single PARticle Combinatorial multiplexed Liposome fusion mediated by DNA (SPARCLD), for the parallelized, multi-step and non-deterministic fusion of individual zeptoliter nanocontainers. We observed directly the efficient (>93%), and leakage free stochastic fusion sequences for arrays of surface tethered target liposomes with six freely diffusing populations of cargo liposomes, each functionalized with individual lipidated ssDNA (LiNA) and fluorescent barcoded by distinct ratio of chromophores. The stochastic fusion results in distinct permutation of fusion sequences for each autonomous nanocontainer. Real-time TIRF imaging allowed the direct observation of >16000 fusions and 566 distinct fusion sequences accurately classified using machine learning. The high-density arrays of surface tethered target nanocontainers [~]42,000 containers per mm2 offers entire combinatorial multiplex screens using only picograms of material.

biophysics↗

bioRxiv2021

Brassinosteroid signaling component SlBES1 promotes tomato fruit softening through transcriptional repression of PMEU1

Firmness is one of the most important factors that affect postharvest properties of tomato fruit. However, the regulatory mechanism underlying firmness formation in tomato fruit is poorly understood. Here, we report a novel role of SlBES1, a transcriptional factor (TF) mediating brassinosteroid (BR) signaling, in tomato fruit softening. We first found that SlBES1 promotes fruit softening during tomato fruit ripening and postharvest storage. RNA-seq analysis suggested that PMEU1, which encodes a pectin de-methylesterification protein, might participate in SlBES1-mediated fruit softening. Biochemical and immunofluorescence assays in SlBES1 transgenic fruits indicated that SlBES1 inhibited PMEU1-related pectin de-methylesterification. Further molecular and genetic evidence verified that SlBES1 directly binds to the E-box in the promoter of PMEU1 to repress its expression, leading to the softening of the tomato fruits. Loss-of-function SlBES1 mutant generated by CRISPR/cas9 showed firmer fruits and longer shelf life during postharvest storage without the color, size and nutritional quality alteration. Collectively, our results indicated the potential of manipulating SlBES1 to regulate fruit firmness via transcriptional inhibition of PMEU1 without negative consequence on visual and nutrition quality.

plant biology↗

bioRxiv2021

Spatio-temporal evolution of urban thermal environment and its driving factors: Case study of Nanjing, China

In recent years, with the rapid urbanization, the urban underlying surface has changed dramatically. Various urban eco-environmental problems have emerged in the world, among which the urban heat island effect has become one of the most obvious urban eco-environmental problems. In this study, Nanjing, China is chosen as the study area. Based on the Landsat8 remote sensing image data in Nanjing from 2014 to 2018, the land surface temperature is retrieved, the spatiotemporal variation track and characteristics of the thermal environment pattern are systematically depicted, and its driving factors are revealed. The results show that: in the past five years, the spatial pattern of heat field in Nanjing has changed from scattered distribution in the periphery of the city to centralized in the center of the city, and the heat island intensity has increased year by year. The changes of administrative divisions, the layout of transportation trunk lines, the transfer of industrial centers, and ecological construction projects are important driving factors for the evolution of land surface thermal environment pattern of these regions. The research results will provide scientific and technological support for similar cities with typical heat island effect in the world to make urban planning and development decisions, and to govern and improve urban ecological environment.

ecology↗

bioRxiv2021

Cysteine is a limiting factor for glioma proliferation and survival

Nutritional intervention is becoming more prevalent as adjuvant therapy for many cancers in view of the tumor dependence on external sources for some nutrients. We report the dependence of glioma cells on exogenous cysteine/cystine, despite this amino acid being nonessential. 13C-tracing and the analysis of cystathionine synthase and cystathioninase levels revealed the metabolic landscape attributable to cysteine deprivation, and the disconnection between the methionine cycle and the transsulfuration pathway. Therefore, we explored the nutritional deprivation in a mouse model of glioma. Animals subjected to a cysteine/cystine-free diet survived longer, with concomitant reductions in glutathione and cysteine plasma levels. At the end point, however, tumors displayed the ability to synthesize glutathione, although higher levels of oxidative stress were detected. We observed a compensation from the nutritional intervention revealed as the recovery of cysteine-related metabolites in plasma. Our study highlights a time window where cysteine deprivation can be exploited for additional therapeutic strategies.

cancer biology↗

bioRxiv2021

All-Flesh Tomato Regulated by Reduced Dosage of AFF Provides New Insights into Berry Fruit Evolution

The formation of locule gel is not only an important developmental process in tomato but also a typical characteristic of berry fruit. In this study, we collected a tomato material that produces all-flesh fruits (AFF), whose locule tissue remains in a solid state during fruit development. We built genetic populations to fine map the causal gene of AFF trait, investigate the function of AFF gene, and identified it as the causal locus conferring the locule gel formation. We determined the causal mutation as a 416-bp deletion that occurred in the promoter region of AFF, which reduces the expression dosage of AFF. The 416-bp deleted sequence has a high level of conservation among closely related Solanaceae species, as well as in the tomato population. The activity of the 416-bp deletion in down-regulating gene expression was further verified by the relative activity in a luciferase experiment. Furthermore, with the BC6 NIL materials, we reveal that the reduced expression dosage of AFF does not impact the normal development of seeds, while produces non-liquefied locule tissue, which is distinct from that of the normal tomatoes in terms of metabolic components. Based on these findings, we propose that the AFF gene is the core node in locule tissue liquefaction, whose function cannot be compensated by its paralogs TAG1, TAGL1, or TAGL11. Our findings provide clues to investigate fruit type differentiation among Solanaceae crops, and also contributes to the breeding application of all flesh fruit tomatoes for the tomato processing industry. One Sentence SummaryThe sequence deletion that occurred in the cis-regulatory region of AFF--the core node of locule tissue liquefaction determined here--reduced its expression dosage, and produced all flesh fruit tomato.

plant 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

Growth stage-dependent changes of the levels of keratin 1 and keratin 10 as well as the green autofluorescence of the skin of the back and the ears of C57BL/6 mice under basal conditions and after UVC irradiation

Our previous studies have indicated that oxidative stress and inflammation can dose-dependently induce increased skins green autofluorescence (AF) of mice, which results at least partially from keratin 1 (K1) cleavage. Increased green AF was also found in patients skin of several major diseases, which may become a novel biomarker for non-invasive diagnosis. We also found age-dependent changes of the skins green AF of natural populations. In this study we tested our hypothesis that there are growth stage-dependent changes of K1 and keratin 10 (K10) levels in the skin of mice, which may underlie the age-dependent changes of the skins green AF. We found that in the skin of both mices back and ears, there were growth stage-dependent changes of the levels of K1 and K10 as well as the basal green AF. The K1 and K10 levels in the backs skin were significantly different from those in the ears skin. There were also growth stage-dependent changes of the UVC-induced changes of K1 and K10 levels of both the ears and the back. Collectively, our study has provided first evidence showing growth stage-dependent and differential changes of the levels of K1 and K10 as well as skins green AF in the back and the ears of mice under basal conditions and after UVC irradiation. These findings are valuable for understanding the age-dependent changes of the skins green AF of natural populations, which are also important for establishing the keratins AF-based method for non-invasive diagnosis of diseases.

developmental biology↗

bioRxiv2020

Isothermal Amplification of Nucleic Acids with Ladder-shape Melting Curve

A novel method termed isothermal amplification of nucleic acids with ladder-shape melting curve (LMCP) was developed in the study. In this method, one pair of primers or two pairs of nested primers and a thermostable DNA polymerase (large fragment) were employed to amplify the internal transcribed spacer (ITS) of Oryza sativa with ladder-shape melting curve. Our results demonstrated that the LMCP assay with nested primers was 50-fold more sensitive and one-hour faster than the LAMP assay with the same level of specificity. The LMCP method has the potential to be used for the prevention and control of the emerging epidemics.

biochemistry↗

bioRxiv2020

Coordinated regulation of ribosomes and proteasomes by PRMT1 in the maintenance of neural stemness of cancer cells and neural stem cells

Our studies suggest that neural stemness contributes to cell tumorigenicity. The basic cell physiological machineries and developmental programs, such as cell cycle, ribosomes, proteasomes, epigenetic factors, etc., which are upregulated in and promote cancers, are enriched in embryonic neural cells. How these machineries are coordinated is unknown. Here, we show that loss of neural stemness in cancer cells or neural stem cells leads to simultaneous downregulation of components of ribosomes and proteasomes, which are responsible for protein synthesis and degradation, respectively, and downregulation of major epigenetic factors. Inhibition of PRMT1 causes neuron-like differentiation, downregulation of a similar set of proteins, and alteration of subcellular localization of ribosome and proteasome components. PRMT1 interacts with these components, catalyzes arginine methylation of them and protects them from degradation, thereby maintaining a high level of expression of epigenetic factors that maintain neural stemness. PRMT1 inhibition results in repression of cell tumorigenicity. Therefore, PRMT1 coordinates ribosomes and proteasomes to match the needs for high protein production and protein homeostasis in cells with fast cell cycle and proliferation.

cell biology↗

bioRxiv2020

Sugarcane Nitrogen and Irrigation Level Prediction Based on UAV-Captured Multispectral Image at Elongating Stage

IntroductionSugarcane is the main industrial crop for sugar production; its growth status is closely related to fertilizer, water, and light input. Unmanned aerial vehicle (UAV)-based multispectral imagery is widely used for high-throughput phenotyping because it can rapidly predict crop vigor. This paper mainly studied the potential of multispectral images obtained by low-altitude UAV systems in predicting canopy nitrogen (N) content and irrigation level for sugarcane. MethodsAn experiment was carried out on sugarcane fields with three irrigation levels and five nitrogen levels. A multispectral image at a height of 40 m was acquired during the elongation stage, and the canopy nitrogen content was determined as the ground truth. N prediction models, including partial least square (PLS), backpropagation neural network (BPNN), and extreme learning machine (ELM) models, were established based on different variables. A support vector machine (SVM) model was used to recognize the irrigation level. ResultsThe PLS model based on band reflectance and five vegetation indices had better accuracy (R=0.7693, root mean square error (RMSE)=0.1109) than the BPNN and ELM models. Some spectral information from the multispectral image had obviously different features among the different irrigation levels, and the SVM algorithm was used for irrigation level classification. The classification accuracy reached 77.8%. ConclusionLow-altitude multispectral images could provide effective information for N prediction and water irrigation level recognition.

plant biology↗

bioRxiv2020

A Systematic and Consistent Assay for High-throughput Characterization of Stalk Quality in Sugarcane by Near-infrared Spectroscopy

Stalk quality improvement is deemed a promising strategy to enhance sugarcane production. However, the lack of efficient approaches for a systematic evaluation of sugarcane germplasm limited stalk quality improvement. In this study, 628 sugarcane samples were employed to take a high-throughput assay for determining the sugarcane stalk quality. Based on the high-performance anion chromatography method, large sugarcane stalk quality variations were detected in biomass composition and the corresponding fundamental ratio values. Online and offline Near-infrared Spectroscopy (NIRS) modeling strategies were applied for multiple purpose calibration. Consequently, 25 equations were generated with the excellent determination coefficient (R2) and ratio performance deviation (RPD) values. Notably, for some observations, RPD values as high as 6.3 were observed that indicated their exceptional performance potential and prediction capacity. Hence, this study provides a feasible way for high-throughput assessment of stalk quality, permitting large-scale screening of optimal sugarcane germplasm.

plant biology↗