Biology subjects

Wang, N.

Publications and source records attributed to Wang, N..

At least 307 records · Page 17Linked to original sources

bioRxiv2020

Dopamine Release Neuroenergetics in Mouse Striatal Slices

Parkinsons disease (PD) is the second most common neurodegenerative disease. Dopamine (DA) neurons in the substantia nigra par compacta with axonal projections to the dorsal striatum (dSTR) degenerate in PD while in contrast, DA neurons in the ventral tegmental area with axonal projections to the ventral striatum including the nucleus accumbens (NAcc) shell, are largely spared. To understand the pathogenesis of PD, it is important to study the neuroenergetics of DA neurons. This study aims to uncover the relative contribution of glycolysis and oxidative phosphorylation (OxPhos) to evoked DA release in the striatum. We measured evoked DA release in mouse striatal brain slices by fast-scan cyclic voltammetry every 2 minutes. Blocking OxPhos caused a greater reduction in evoked DA release in the dSTR compared to the NAcc shell, and blocking glycolysis caused a greater reduction in evoked DA release in the NAcc shell than in the dSTR. Furthermore, when glycolysis was bypassed in favor of direct OxPhos, evoked DA release in the NAcc shell was decreased by [~]50% over 40 minutes whereas evoked DA release in the dSTR was largely unaffected. These results demonstrated that the dSTR relies primarily on OxPhos for energy production to maintain evoked DA release whereas the NAcc shell relies more on glycolysis. Using two-photon imaging, we consistently found that the oxidation level of the DA terminals was higher in the dSTR than in the NAcc shell. Together, these findings partially explain the specific vulnerability of DA terminals in the dSTR to degeneration in PD. Significant statementThe neuroenergetics of dopaminergic neuron is important to understand Parkinsons disease (PD), a neurodegenerative disorder associated with mitochondrial dysfunctions. However, the relative contributions of glycolysis and oxidative phosphorylation (OxPhos) to presynaptic energy demands in DA terminals are unclear. We addressed this question by measuring DA release in the dorsal striatum and nucleus accumbens (NAcc) shell of mouse brain using FSCV under reagents blocking different energy systems. We found that the NAcc shell relies on both glycolysis and OxPhos to maintain DA release while the dSTR relies heavily on OxPhos. We demonstrate the different neuroenergetics of DA terminals in these two brain areas, providing new fundamentally important insight into the specific vulnerability of DA terminals in the dSTR to degeneration in PD.

neuroscience↗

bioRxiv2020

Structural basis for neutralization of SARS-CoV-2 and SARS-CoV by a potent therapeutic antibody

The COVID-19 pandemic caused by the SARS-CoV-2 virus has resulted in an unprecedented public health crisis. There are no approved vaccines or therapeutics for treating COVID-19. Here we reported a humanized monoclonal antibody, H014, efficiently neutralizes SARS-CoV-2 and SARS-CoV pseudoviruses as well as authentic SARS-CoV-2 at nM level by engaging the S receptor binding domain (RBD). Importantly, H014 administration reduced SARS-CoV-2 titers in the infected lungs and prevented pulmonary pathology in hACE2 mouse model. Cryo-EM characterization of the SARS-CoV-2 S trimer in complex with the H014 Fab fragment unveiled a novel conformational epitope, which is only accessible when the RBD is in open conformation. Biochemical, cellular, virological and structural studies demonstrated that H014 prevents attachment of SARS-CoV-2 to its host cell receptors. Epitope analysis of available neutralizing antibodies against SARS-CoV and SARS-CoV-2 uncover broad cross-protective epitopes. Our results highlight a key role for antibody-based therapeutic interventions in the treatment of COVID-19. One sentence summaryA potent neutralizing antibody conferred protection against SARS-CoV-2 in an hACE2 humanized mouse model by sterically blocking the interaction of the virus with its receptor.

biophysics↗

bioRxiv2020

Structure-based Design of Prefusion-stabilized SARS-CoV-2 Spikes

The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has led to accelerated efforts to develop therapeutics, diagnostics, and vaccines to mitigate this public health emergency. A key target of these efforts is the spike (S) protein, a large trimeric class I fusion protein that is metastable and difficult to produce recombinantly in large quantities. Here, we designed and expressed over 100 structure-guided spike variants based upon a previously determined cryo-EM structure of the prefusion SARS-CoV-2 spike. Biochemical, biophysical and structural characterization of these variants identified numerous individual substitutions that increased protein yields and stability. The best variant, HexaPro, has six beneficial proline substitutions leading to [~]10-fold higher expression than its parental construct and is able to withstand heat stress, storage at room temperature, and multiple freeze-thaws. A 3.2 [A]-resolution cryo-EM structure of HexaPro confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for SARS-CoV-2.

microbiology↗

bioRxiv2020

An efficient gene excision system in maize

Use of the morphogenic genes Baby Boom (Bbm) and Wuschel2 (Wus2), along with new ternary constructs, has increased the genotype range and the type of explants that can be used for maize transformation. In addition, altering the ectopic expression pattern for Bbm/Wus2 has resulted in rapid maize transformation methods that are faster and applicable to a broader range of inbreds. However, expression of Bbm/Wus2 can compromise the quality of regenerated plants, leading to sterility. We reasoned excising morphogenic genes after transformation but before regeneration would increase production of fertile T0 plants. We developed a method that uses an inducible site-specific recombinase (Cre) to excise morphogenic genes. The use of developmentally regulated promoters, such as Ole, Glb1, End2 and Ltp2, to drive Cre enabled excision of morphogenic genes in early embryo development and produced excised events at a rate of 25%-100%. A different strategy utilizing an excision-activated selectable marker produced excised events at a rate of 53.3%-68.4%; however, the transformation frequency was lower (12.9%-49.9%). The use of inducible heat shock promoters (e.g. Hsp17.7, Hsp26) to express Cre, along with improvements in tissue culture conditions and construct design, resulted in high frequencies of T0 transformation (29%-69%), excision (50%-97%), usable quality events (3.6%-14%), and few escapes (non-transgenic; 14%-17%) in three elite maize inbreds. Transgenic events produced by this method are free of morphogenic and marker genes.

plant biology↗

bioRxiv2020

Nutrient restriction, inducer of yeast meiosis, induces mammalian meiosis

From yeasts to mammals, the molecular machinery and chromosome structures carrying out meiosis are frequently conserved. However, the signal to initiate meiosis appears divergent: while nutrient restriction induces meiosis in the yeast system, retinoic acid (RA), a chordate morphogen, is necessary but not sufficient to induce meiotic initiation in mammalian germ cells via its target, Stra8. Here, using cultured mouse male germline stem cells without the support of gonadal somatic cells, we show that nutrient restriction in combination with RA robustly induces Spo11-dependent meiotic DNA double strand breaks (DSBs) and Stra8-dependent meiotic gene programs recapitulating those of early meiosis in vivo. Moreover, a distinct network of 11 nutrient restriction-upregulated transcription factor genes was identified, whose expression does not require RA and is associated with early meiosis in vivo. Thus, our study proposes a conserved model, in which nutrient restriction induces meiotic initiation by upregulating transcriptional factors for meiotic gene programs, and provides an in vitro platform to derive haploid gametes in culture. One Sentence Summarynutrient restriction synergizes with retinoic acid to induce mammalian meiotic initiation

developmental biology↗

bioRxiv2020

Broad sarbecovirus neutralizing antibodies define a key site of vulnerability on the SARS-CoV-2 spike protein

Broadly protective vaccines against known and pre-emergent coronaviruses are urgently needed. Critical to their development is a deeper understanding of cross-neutralizing antibody responses induced by natural human coronavirus (HCoV) infections. Here, we mined the memory B cell repertoire of a convalescent SARS donor and identified 200 SARS-CoV-2 binding antibodies that target multiple conserved sites on the spike (S) protein. A large proportion of the antibodies display high levels of somatic hypermutation and cross-react with circulating HCoVs, suggesting recall of pre-existing memory B cells (MBCs) elicited by prior HCoV infections. Several antibodies potently cross-neutralize SARS-CoV, SARS-CoV-2, and the bat SARS-like virus WIV1 by blocking receptor attachment and inducing S1 shedding. These antibodies represent promising candidates for therapeutic intervention and reveal a new target for the rational design of pan-sarbecovirus vaccines.

immunology↗

bioRxiv2020

Rapid development of an inactivated vaccine for SARS-CoV-2

The COVID-19 pandemic caused by SARS-CoV-2 has brought about an unprecedented crisis, taking a heavy toll on human health, lives as well as the global economy. There are no SARS-CoV-2-specific treatments or vaccines available due to the novelty of this virus. Hence, rapid development of effective vaccines against SARS-CoV-2 is urgently needed. Here we developed a pilot-scale production of a purified inactivated SARS-CoV-2 virus vaccine candidate (PiCoVacc), which induced SARS-CoV-2-specific neutralizing antibodies in mice, rats and non-human primates. These antibodies potently neutralized 10 representative SARS-CoV-2 strains, indicative of a possible broader neutralizing ability against SARS-CoV-2 strains circulating worldwide. Immunization with two different doses (3g or 6 g per dose) provided partial or complete protection in macaques against SARS-CoV-2 challenge, respectively, without any antibody-dependent enhancement of infection. Systematic evaluation of PiCoVacc via monitoring clinical signs, hematological and biochemical index, and histophathological analysis in macaques suggests that it is safe. These data support the rapid clinical development of SARS-CoV-2 vaccines for humans. One Sentence SummaryA purified inactivated SARS-CoV-2 virus vaccine candidate (PiCoVacc) confers complete protection in non-human primates against SARS-CoV-2 strains circulating worldwide by eliciting potent humoral responses devoid of immunopathology

microbiology↗

bioRxiv2020

Citrus vascular proteomics highlights the role of peroxidases and serine proteases during Huanglongbing disease progression

Huanglongbing (HLB) is the most devastating and widespread citrus disease. All commercial citrus varieties are susceptible to the HLB-associated bacterium, Candidatus Liberibacter asiaticus (CLas), which resides in the phloem. The phloem is part of the plant vascular system and is involved in sugar transport. To investigate the plant response to CLas, we enriched for proteins surrounding the phloem in an HLB susceptible sweet orange variety, Washington navel (Citrus sinensis (L) Osbeck). Quantitative proteomics revealed global changes in the citrus proteome after CLas inoculation. Plant metabolism and translation were suppressed, while defense-related proteins such as peroxidases, proteases and protease inhibitors were induced in the vasculature. Transcript accumulation and enzymatic activity of plant peroxidases in CLas infected sweet orange varieties under greenhouse and field conditions were assessed. While peroxidase transcript accumulation was induced in CLas infected sweet orange varieties, peroxidase enzymatic activity varied. Specific serine proteases were upregulated in Washington navel in the presence of CLas based on quantitative proteomics. Subsequent activity-based protein profiling revealed increased activity of two serine proteases, and reduced activity of one protease in two C. sinensis sweet orange varieties under greenhouse and field conditions. The observations in the current study highlight global reprogramming of the citrus vascular proteome and differential regulation of enzyme classes in response to CLas infection. These results open an avenue for further investigation of diverse responses to HLB across different environmental conditions and citrus genotypes.

plant biology↗

bioRxiv2020

A Novel Non-hypermucoviscous ST11 Hypervirulent Carbapenem-resistant Klebsiella pneumoniae Lacking Classical Virulence Factors

BackgroundHypervirulent Klebsiella pneumoniae lacking classical virulence factors is uncommon, and the virulence mechanisms of this organism are not understood. MethodsFollowing a retrospective study of carbapenem-resistant K. pneumoniae based on core genome multilocus sequence typing (cgMLST), isolates that caused high mortality were investigated with a genome-wide association study (GWAS), proteome analysis and an animal model. ResultsThe sublineage of sequence type 11 (ST11) K. pneumoniae, which belongs to complex type 3176 (CT3176) and K-locus 47 (KL47), was highlighted due to the high mortality of infected patients. GWAS analysis showed that ampR was associated with the CT3176 isolates. In a mouse model, the mortality of ampR-carrying isolates was comparable to that of the typical hypervirulent isolate GM2. Even during the first 24 hours of infection, the bacterial load and pathological changes of the ampR-carrying isolates in the lungs were more severe than those of GM2. The ampR complement mutant was able to enhance the virulence of the KL47 isolate but not the virulence of KL1. Proteome analysis showed that the expression of WcaJ in the ampR+ isolates was significantly higher than that in the ampR- isolates, and this result was also confirmed by transcription tests and capsule staining. It is suggested that the enhancement of the initial stage of capsule synthesis may be the cause of the high virulence of these non-hypermucoviscous ST11 carbapenem-resistant K. pneumoniae isolates. ConclusionsNon-hypermucoviscous ST11 hypervirulent carbapenem-resistant K. pneumoniae warrants continued surveillance and investigation.

microbiology↗

bioRxiv2020

Tracing the origin of a new organ by inferring the genetic basis of rumen evolution

The rumen is the hallmark organ of ruminants and hosts a diverse ecosystem of microorganisms that facilitates efficient digestion of plant fibers. We used 897 transcriptomes from three Cetartiodactyla lineages: ruminants, camels and cetaceans, as well as data from ruminant comparative genomics and functional assays to explore the genetic basis of rumen origin and evolution. Comparative analyses reveal that the rumen and the first-chamber stomachs of camels and cetaceans shared a common tissue origin from the esophagus. The rumen recruited genes from other tissues/organs and up-regulated many esophagus genes to aquire functional innovations involving epithelium absorption, improvement of the ketone body metabolism and regulation of microbial community. These innovations involve such genetic changes as ruminant-specific conserved elements, newly evolved genes and positively selected genes. Our in vitro experiements validate the functions of one enhancer, one positively selected gene and two newly evolved antibacterial genes. Our study provides novel insights into the origin and evolution of a complex organ.

evolutionary biology↗

bioRxiv2020

Cryo-EM Structure of the 2019-nCoV Spike in the Prefusion Conformation

The outbreak of a novel betacoronavirus (2019-nCov) represents a pandemic threat that has been declared a public health emergency of international concern. The CoV spike (S) glycoprotein is a key target for urgently needed vaccines, therapeutic antibodies, and diagnostics. To facilitate medical countermeasure (MCM) development we determined a 3.5 [A]-resolution cryo-EM structure of the 2019-nCoV S trimer in the prefusion conformation. The predominant state of the trimer has one of the three receptor-binding domains (RBDs) rotated up in a receptor-accessible conformation. We also show biophysical and structural evidence that the 2019-nCoV S binds ACE2 with higher affinity than SARS-CoV S. Additionally we tested several published SARS-CoV RBD-specific monoclonal antibodies and found that they do not have appreciable binding to nCoV-2019 S, suggesting antibody cross-reactivity may be limited between the two virus RBDs. The atomic-resolution structure of 2019-nCoV S should enable rapid development and evaluation of MCMs to address the ongoing public health crisis.

microbiology↗

bioRxiv2020

Therapeutic Drugs Targeting 2019-nCoV Main Protease by High-Throughput Screening

2019 Novel Coronavirus (2019-nCoV) is a virus identified as the cause of the outbreak of pneumonia first detected in Wuhan, China. Investigations on the transmissibility, severity, and other features associated with this virus are ongoing. Currently, there is no vaccine or therapeutic antibody to prevent the infection, and more time is required to develop an effective immune strategy against the pathogen. In contrast, specific inhibitors targeting the key protease involved in replication and proliferation of the virus are the most effective means to alleviate the epidemic. The main protease of SARS-CoV is essential for the life cycle of the virus, which showed 96.1% of similarity with the main proteaseof 2019-nCoV, is considered to be an attractive target for drug development. In this study, we have identified 4 small molecular drugs with high binding capacity with SARS-CoV main protease by high-throughput screening based on the 8,000 clinical drug libraries, all these drugs have been widely used in clinical applications with guaranteed safety, which may serve as promising candidates to treat the infection of 2019-nCoV.

pharmacology and toxicology↗

bioRxiv2020

Efficient long fragment editing technique enables rapid construction of genetically stable bacterial strains

Bacteria are versatile living systems that enhance our understanding of nature and enable biosynthesis of valuable chemicals. Long fragment editing techniques are of great importance for accelerating bacterial genome engineering to obtain desirable and genetically stable strains. However, the existing genome editing methods cannot meet the needs of engineers. We herein report an efficient long fragment editing method for large-scale and scarless genome engineering in Escherichia coli. The method enabled us to insert DNA fragments up to 12 kb into the genome and to delete DNA fragments up to 186.7 kb from the genome, with positive rates over 95%. We applied this method for E. coli genome simplification, resulting in 12 individual deletion mutants and four cumulative deletion mutants. The simplest genome lost a total of 370.6 kb of DNA sequence containing 364 open reading frames. Additionally, we applied this technique to metabolic engineering and obtained a genetically stable plasmid-independent isobutanol production strain that produced 1.3 g/L isobutanol via shake-flask fermentation. These results suggest that the method is a powerful genome engineering tool, highlighting its potential to be applied in synthetic biology and metabolic engineering.

bioengineering↗

bioRxiv2019

An upstream open reading frame represses translation of chicken PPARγ transcript variant 1

Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) is a master regulator of adipogenesis. The PPAR{gamma} gene produces various transcripts with different 5'-untranslated regions (5' UTRs) because of alternative promoter usage and splicing. The 5' UTR plays important roles in posttranscriptional gene regulation. However, to date, the regulatory role and underlying mechanism of 5' UTRs in the posttranscriptional regulation of PPAR{gamma} expression remain largely unclear. In this study, we investigated the effects of 5' UTRs on posttranscriptional regulation using reporter assays. Our results showed that the five PPAR{gamma} 5' UTRs exerted different effects on reporter gene activity. Bioinformatics analysis showed that chicken PPAR{gamma} transcript 1 (PPAR{gamma}1) possessed an upstream open reading frame (uORF) in its 5' UTR. Mutation analysis showed that a mutation in the uORF led to increased Renilla luciferase activity and PPAR{gamma} protein expression, but decreased Renilla luciferase and PPAR{gamma}1 mRNA expression. mRNA stability analysis using real-time RT-PCR showed that the uORF mutation did not interfere with mRNA stability, but promoter activity analysis of the cloned 5' UTR showed that the uORF mutation reduced promoter activity. Furthermore, in vitro transcription/translation assays demonstrated that the uORF mutation markedly increased the translation of PPAR{gamma}1 mRNA. Collectively, our results indicate that the uORF represses the translation of chicken PPAR{gamma}1 mRNA.

molecular biology↗

bioRxiv2019

Reversed paired-gRNA plasmid cloning strategy for efficient genome editing in Escherichia coli

A growing number of CRISPR-Cas9 associated applications require co-expression of two distinct gRNAs. However, coexpressing paired gRNAs under the driving of independent but identical promoters in the same direction triggers plasmid instability, due to the presence of direct repeats (DRs). In this study, deletion between DRs occurred with high frequencies during plasmid construction and duplication processes, when three DRs-involved paired-gRNA plasmids cloning strategies were tested. This recombination phenomenon was RecA-independent, in agreement with the replication slippage model. To completely eliminate the DRs-induced plasmid instability, a reversed paired-gRNA plasmids (RPGPs) cloning strategy was developed by converting DRs to the more stable invert repeats (IRs). Using RPGPs, we achieved a rapid deletion of chromosome fragments up to 100 kb with high efficiency of 83.33% in Escherichia coli. This study provides general solutions to construct stable plasmids containing short DRs, which can improve the performances of CRISPR systems that relied on paired gRNAs, and also facilitate other applications involving repeated genetic parts.

microbiology↗

bioRxiv2019

MicroRNA-148a Regulates Low-Density Lipoprotein Metabolism by Repressing the (Pro)renin Receptor

High plasma LDL cholesterol (LDL-c) concentration is a major risk factor for atherosclerosis. Hepatic LDLR regulates LDL metabolism, and thereby plasma LDL-c concentration. Recently, we identified the (pro)renin receptor [(P)RR] as a novel regulator of LDL metabolism, which regulates LDLR degradation and hence its protein abundance and activity. In silicon analysis suggests that the (P)RR is a target of miR-148a. In this study we determined whether miR-148a could regulate LDL metabolism by regulating (P)RR expression in HepG2 and Huh7 cells. We found that miR-148a suppressed (P)RR expression by binding to the 3-untranslated regions (3-UTR) of (P)RR mRNA. Mutating the binding sites for miR-148a in the 3-UTR of (P)RR mRNA abolished the inhibitory effects of miR-148a on (P)RR expression. In line with our recent findings, reduced (P)RR expression resulted in decreased cellular LDL uptake, likely as a consequence of decreased LDLR protein abundance. Overexpressing the (P)RR prevented miR-148a-induced reduction in LDLR abundance and cellular LDL uptake. Our study supports a new concept that miR-148a is a regulator of (P)RR expression. By reducing (P)RR abundance, miR-148a decreases LDLR protein abundance and consequently cellular LDL uptake.

molecular biology↗

bioRxiv2019

Chromatin organization in early land plants reveals an ancestral association between H3K27me3, transposons, and constitutive heterochromatin

Genome packaging by nucleosomes is a hallmark of eukaryotes. Histones and the pathways that deposit, remove, and read histone modifications are deeply conserved. Yet, we lack information regarding chromatin landscapes in extant representatives of ancestors of the main groups of eukaryotes and our knowledge of the evolution of chromatin related processes is limited. We used the bryophyte Marchantia polymorpha, which diverged from vascular plants 400 Mya, to obtain a whole chromosome genome assembly and explore the chromatin landscape and three-dimensional organization of the genome of early land plants. Based on genomic profiles of ten chromatin marks, we conclude that the relationship between active marks and gene expression is conserved across land plants. In contrast, we observed distinctive features of transposons and repeats in Marchantia compared with flowering plants. Silenced transposons and repeats did not accumulate around centromeres, and a significant proportion of transposons were marked by H3K27me3, which is otherwise dedicated to the transcriptional repression of protein coding genes in flowering plants. Chromatin compartmentalization analyses of Hi-C data revealed that chromatin regions belonging to repressed heterochromatin were densely decorated with H3K27me3 but not H3K9 or DNA methylation as reported in flowering plants. We conclude that in early plants, H3K27me3 played an essential role in heterochromatin function, suggesting an ancestral role of this mark in transposon silencing.

molecular biology↗

bioRxiv2019

Potential function of CbuSPL and gene encoding its interacting protein during flowering in Catalpa bungei

\"Bairihua\", a variety of the Catalpa bungei, has a large amount of flowers and a long flowering period which make it an excellent material for flowering researches in trees. SPL is one of the hub genes that regulate both flowering transition and development. Here, a SPL homologues CbuSPL9 was cloned using degenerate primers with RACE. Expression studies during flowering transition in Bairihua and ectopic expression in Arabidopsis showed that CbuSPL9 was functional similarly with its Arabidopsis homologues. In the next step, we used Y2H to identify the proteins that could interact with CbuSPL9. HMGA, an architectural transcriptional factor, was identified and cloned for further research. BiFC and BLI showed that CbuSPL9 could form a heterodimer with CbuHMGA in the nucleus. The expression analysis showed that CbuHMGA had a similar expression trend to that of CbuSPL9 during flowering in \"Bairihua\". Intriguingly, ectopic expression of CbuHMGA in Arabidopsis would lead to aberrant flowers, but did not effect flowering time. Taken together, our results implied a novel pathway that ChuSPL9 regulated flowering development, but not flowering transition, with the participation of ChuHMGA. Further investments need to be done to verify the details of this pathway.

plant biology↗