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1.
J Ethnopharmacol ; 246: 112165, 2020 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-31445133

RESUMO

Blood-brain barrier (BBB) is a barrier which maintains the material exchange balance of brain microenvironment and could be destroyed by chronic stress (CS). Glucocorticoids (GCs) can mimic the chronic stress induced damage to BBB. GCs induced BBB trauma models in vitro and in vivo to explore the effects of the traditional medicine Xiao-Yao-San (XYS). In this research, we found CS could injure the BBB to change the biochemical index, which could be reversed by XYS in vitro. The abilities of cell proliferation, invasion, and the expression of tight junction related genes (Occludin, Claudin, JAM-1 and ZO-1) were suppressed by CS and the trauma could be reversed by XYS partly. It was showed that GRs interacted with Occludin directly and inhibited Occluding expression. In rats BBB trauma model, the GC content was deceased and BBB permeability was repaired by XYS. The expression of Occludin, Claudin, JAM-1 and ZO-1 were increased in the treatment of XYS. In our research, it shown that XYS affect the content of the GC and GR which interacted with Occludin directly for the first time. In addition, we also found that XYS could reduce BBB injury induced by CS via GR in BBB model in vitro. Therefore, it proves that XYS is a potential BBB repair medicine and may help to elucidate mechanism of brain pathology.

2.
J Gen Virol ; 2019 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-31702540

RESUMO

To gain insights into the role of the head-stalk linker region in the fusion triggering, we constructed mutants by deleting or substituting the linker region (115-NGAANNSG-122) of Newcastle disease virus (NDV) haemagglutinin-neuraminidase (HN) with the corresponding sequences of other paramyxoviruses. The results showed that these HN mutants exhibited different levels of fusion-triggering activity, but most of them maintained comparable levels with wide-type HN in both receptor recognition and neuraminidase activity. We tried to figure out reasons for fusion alteration through assessing the expression and the oligomeric state of HN mutants. Moreover, four mutants with significant fusion changes were introduced into the headless HN stem (HN1-123) to intensively investigate the role of the linker region in fusion triggering. Consequently, the stability of HN oligomers and the structural integrity of the 4 helical-bundle of stalk have complicated influences on the alteration of fusion-triggering activities for different mutants. These data suggested that the head-stalk linker could regulate the fusion triggering at both full-length and headless HN levels.

3.
Virus Res ; 275: 197791, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31628980

RESUMO

Human parainfluenza virus type 3 (HPIV3) fuses the viral envelope with the host cell membrane through the concerted action of the fusion (F) protein and the hemagglutinin-neuraminidase (HN). Upon HN binding to sialic acid (SA), the F protein in a metastable prefusion form is activated to undergo a series of structural rearrangements into a stable postfusion form to actuate the fusion between membranes. Various domains of F protein of some other paramyxoviruses, including HPIV3, have been reported to be differently functional. However, it is not yet clear what roles HRB linker plays. To clarify the roles that HRB linker might play in the F-mediated membrane fusion process, here we examined the effects of mutations introduced into the HRB linker of HPIV3 F protein. Six Single amino acid mutants, three chimeric mutants, and one deletion mutant were obtained and analyzed for membrane fusion activity and cell surface expression. The results showed that the membrane fusion activity of mutants changed to varying degrees in comparison with wild-type (wt) F, and some mutants even forfeited fusogenicity absolutely. It is indicated that the HRB linker domain plays an important role in the F-mediated membrane fusion process.

4.
Biomed Pharmacother ; 120: 109519, 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31629951

RESUMO

XIAOPI formula is a national approved drug prescribed to patients with high breast cancer risk. Previously we demonstrated that XIAOPI formula could inhibit breast cancer metastasis via suppressing CXCL1 expression, and postulated that "autophagy in cancer" might be one of its most core anti-cancer mechanisms. However, whether XIAOPI formula could be simultaneously applied with chemodrugs and their synergistic mechanisms are still remained unknown. In the present study, XIAOPI formula at non-cytotoxic doses could synergistically enhance the chemosensitivity of breast cancer cells MDA-MB-231 and MCF-7. We found that rapamycin-induced autophagy could reduce the chemosensitivity of breast cancer cells to XIAOPI formula, and the autophagy suppression and chemosensitizing activity of this formula was CXCL1-dependent. The evidence came from that XIAOPI formula was associated with a lower expression of CXCL1 combined with either rapamycin or taxol alone. Besides, the inhibitory effect of XIAOPI formula on the LC3-II and ABCG2 signals was weakened following CXCL1 over-expression, whereas P62 upregulation induced by XIAOPI formula was re-declined. A high throughput - qPCR (HT-qPCR) assay identified HMGB1 as the main autophagic target of XIAOPI formula in chemosensitizing breast cancer. and furhter validation suggested XIAOPI formula exerted chemosensitivity mainly via CXCL1/HMGB1 autophagic axis. Finally, we generated both mice and zebrafish xenotransplantation models bearing MDA-MB-231 breast cancer cells, and found that XIAOPI formula safely enhanced in vivo taxol chemosensitivity on breast cancer. Taken together, XIAOPI formula is a potential adjuvant drug via inhibiting CXCL1/HMGB1-mediated autophagy for breast cancer treatment with good safety.

5.
Nanoscale ; 11(37): 17407-17414, 2019 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-31528935

RESUMO

Tamm plasmonic (TP) structures, consisting of a metallic film and a distributed Bragg reflector (DBR), can exhibit pronounced light confinement allowing for enhanced absorption in the metallic film at the wavelength of the TP resonance. This wavelength dependent absorption can be converted into an electrical signal through the internal photoemission of energetic hot-electrons from the metallic film. Here, by replacing the metallic film at the top of a TP structure with a hot-electron device in a metal-semiconductor-ITO (M-S-ITO) configuration, for the first time, we experimentally demonstrate a wavelength-selective photoresponse around the telecommunication wavelength of 1550 nm. The M-S-ITO junction is deliberately designed to have a low energy barrier and asymmetrical hot-electron generation, in order to guarantee a measurable net photocurrent even for sub-bandgap incident light with a photon energy of 0.8 eV (1550 nm). Due to the excitation of TPs between the metallic film in the M-S-ITO structure and the underlying DBR, the fabricated TP coupled hot-electron photodetector exhibits a sharp reflectance dip with a bandwidth of 43 nm at a wavelength of 1581 nm. The photoresponse matches the absorptance spectrum, with a maximum value of 8.26 nA mW-1 at the absorptance peak wavelength that decreases by more than 80% when the illumination wavelength is varied by only 52 nm (from 1581 to 1529 nm), thus realizing a high modulation wavelength-selective photodetector. This study demonstrates a high-performance, lithography-free, and wavelength-selective hot-electron near-infrared photodetector using an M-S-ITO-DBR planar structure.

6.
Oxid Med Cell Longev ; 2019: 8781690, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31531187

RESUMO

Targeting aberrant metabolism is a promising strategy for inhibiting cancer growth and metastasis. Research is now geared towards investigating the inhibition of glycolysis for anticancer drug development. Betulinic acid (BA) has demonstrated potent anticancer activities in multiple malignancies. However, its regulatory effects on glycolysis and the underlying molecular mechanisms are still unclear. BA inhibited invasion and migration of highly aggressive breast cancer cells. Moreover, BA could suppress aerobic glycolysis of breast cancer cells presenting as a reduction of lactate production, quiescent energy phenotype transition, and downregulation of aerobic glycolysis-related proteins. In this study, glucose-regulated protein 78 (GRP78) was also identified as the molecular target of BA in inhibiting aerobic glycolysis. BA treatment led to GRP78 overexpression, and GRP78 knockdown abrogated the inhibitory effect of BA on glycolysis. Further studies demonstrated that overexpressed GRP78 activated the endoplasmic reticulum (ER) stress sensor PERK. Subsequent phosphorylation of eIF2α led to the inhibition of ß-catenin expression, which resulted in the inhibition of c-Myc-mediated glycolysis. Coimmunoprecipitation assay revealed that BA interrupted the binding between GRP78 and PERK, thereby initiating the glycolysis inhibition cascade. Finally, the lung colonization model validated that BA inhibited breast cancer metastasis in vivo, as well as suppressed the expression of aerobic glycolysis-related proteins. In conclusion, our study not only provided a promising drug for aerobic glycolysis inhibition but also revealed that GRP78 is a novel molecular link between glycolytic metabolism and ER stress during tumor metastasis.

7.
Aging (Albany NY) ; 11(18): 7553-7569, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31532756

RESUMO

Long non-coding RNA taurine up-regulated gene 1 (TUG1) emerges as new players in gene regulation in several cancers; however, its mechanism of action in non-small cell lung cancer (NSCLC) has not been well-studied. Herein, we determined expression pattern of TUG1 in NSCLC and further identified its effect on the chemosensitivity of NSCLC. Low expression of TUG1 was found in NSCLC tissues obtained from non-responders to platinum-based chemotherapy and reflected poor overall survival. TUG1 overexpression was shown to inhibit cell proliferation, migration, invasion, but facilitate apoptosis and autophagy in NSCLC cells resistant to cisplatin (DDP). Smaller size of tumor xenografts of DDP resistant NSCLC cells in the presence of TUG1 demonstrated enhancement of chemosensitivity by TUG1 in vivo. High expression of miR-221 and low expression of PTEN were determined in cancer tissues obtained from non-responders to platinum-based chemotherapy and reflected poor overall survival. TUG1 inhibited miR-221 that targeted PTEN, as evidenced by an elevated expression of PTEN in the presence of miR-221 or the absence of TUG1. Our present study reveals a model of enhancement of chemosensitivity that consists of TUG1, miR-221 and PTEN. Modulation of their levels may offer a new approach for improving anti-tumor efficacy for chemotherapeutic agents in NSCLC.

8.
J Am Chem Soc ; 141(43): 17189-17197, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31539231

RESUMO

Synthetic catalytic DNA circuits are important signal amplification tools for molecular programming due to their robust and modular properties. In catalytic circuits, the reactant recycling operation is essential to facilitate continuous processes. Therefore, it is desirable to develop new methods for the recycling of reactants and to improve the recyclability in entropy-driven DNA circuit reactions. Here, we describe the implementation of a nicking-assisted recycling strategy for reactants in entropy-driven DNA circuits, in which duplex DNA waste products are able to revert into active components that could participate in the next reaction cycle. Both a single-layered circuit and multiple two-layered circuits of different designs were constructed and analyzed. During the reaction, the single-layered catalytic circuit can consume excess fuel DNA strands without depleting the gate components. The recycling of the two-layered circuits occurs during the fuel DNA digestion but not during the release of the downstream trigger. This strategy provides a simple yet versatile method for creating more efficient entropy-driven DNA circuits for molecular programming and synthetic biology.

9.
ACS Appl Mater Interfaces ; 11(40): 37256-37262, 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31496216

RESUMO

Facet engineering of anatase TiO2 by controlling the {001} exposure ratio has been the focus of numerous investigations to optimize photocatalytic activity. In particular, an introduction of fluoride ions during the crystal growth has been demonstrated to be very effective and decisive in realizing the facet exposure of the crystals. However, a key role of fluoride ions in stabilizing {001} exposure and improving subsequent photocatalytic activity of anatase TiO2 remains unclear up to date. Herein, a controlled thickness of anatase TiO2 nanosheets has been realized by introducing different amounts of ethanol into a HF acid-assisted hydrothermal reaction. The thinnest nanosheets with a thickness of ∼2.9 nm were evaluated to have the highest H2 production rate of 41.04 mmol·h-1·g-1 under ultraviolet light irradiation, and the corresponding quantum efficiency was determined to be 41.6% (λ = 365 nm). Moreover, it is proved for the first time that fluoride ions are bonded with Ti vacancies on {001} facets, and such defects are crucial for stabilizing the ultrathin nanosheets and improving their electron-hole separation, therefore leading to a highly efficient photocatalytic activity. The findings offer an opportunity to engineer facets and functionality of anatase TiO2 by controlling surface defects.

10.
PLoS One ; 14(8): e0221677, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31454372

RESUMO

BACKGROUND: Natural-focal diseases are serious diseases that endanger human health. They threaten about 100 million people in Shandong Province, and cause illness in thousands of people each year. However, information on the epidemiological characteristics of natural-focal diseases in Shandong Province has been limited. The purpose of the study was to describe and analyze the epidemiological characteristics of natural-focal diseases in Shandong Province, 2009-2017. METHODS: We describe the incidence and distribution of four natural-focal diseases in Shandong Province using surveillance data from 2009-2017. RESULTS: From 2009-2017, 11123 cases of four natural-focal diseases including 257 deaths were reported in Shandong Province, China. The four natural-focal diseases were severe fever with thrombocytopenia syndrome (SFTS), human granulocytic anaplasmosis (HGA), typhus, and scrub typhus. The high-risk groups of the four diseases were farmers and the elderly. The incidence rate of scrub typhus was significantly higher in females. However, this difference was not seen in the other three diseases. The four diseases were mainly clustered in the middle-southern part of Shandong Province and the Shandong Peninsula. The annual incidence of SFTS and scrub typhus increased, typhus was relatively stable, and HGA declined. However, the range of SFTS expanded, while HGA shrunk, and typhus and scrub typhus were unchanged. The epidemic period of SFTS and HGA was from May to October, typhus was from October to November, and scrub typhus was from September to November. The fatality rates of SFTS, typhus, scrub typhus, and HGA were 9.19%, 0%, 0.01%, and 2.24%, respectively. CONCLUSIONS: Our study described and analyzed the prevalence of natural-focal diseases in Shandong Province, and confirmed that age was closely related to the SFTS fatality rate. This study may help to improve the understanding of the prevalence of natural-focal diseases in Shandong Province in recent years and to better develop accurate prevention and control strategies for natural-focal diseases.

11.
MBio ; 10(4)2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31455649

RESUMO

Rediscovery of the ancient evolutionary relationship between archaea and eukaryotes has revitalized interest in archaeal cell biology. Key to the understanding of archaeal cells is the surface layer (S-layer), which is commonly found in Archaea but whose in vivo function is unknown. Here, we investigate the architecture and cellular roles of the S-layer in the hyperthermophilic crenarchaeon Sulfolobus islandicus Electron micrographs of mutant cells lacking slaA or both slaA and slaB confirm the absence of the outermost layer (SlaA), whereas cells with intact or partially or completely detached SlaA are observed for the ΔslaB mutant. We experimentally identify a novel S-layer-associated protein, M164_1049, which does not functionally replace its homolog SlaB but likely assists SlaB to stabilize SlaA. Mutants deficient in the SlaA outer layer form large cell aggregates, and individual cell size varies, increasing significantly up to six times the diameter of wild-type cells. We show that the ΔslaA mutant cells exhibit more sensitivity to hyperosmotic stress but are not reduced to wild-type cell size. The ΔslaA mutant contains aberrant chromosome copy numbers not seen in wild-type cells, in which the cell cycle is tightly regulated. Together, these data suggest that the lack of SlaA results in either cell fusion or irregularities in cell division. Our studies show the key physiological and cellular functions of the S-layer in this archaeal cell.IMPORTANCE The S-layer is considered to be the sole component of the cell wall in Sulfolobales, a taxonomic group within the Crenarchaeota whose cellular features have been suggested to have a close relationship to the last archaea-eukaryote common ancestor. In this study, we genetically dissect how the two previously characterized S-layer genes as well as a newly identified S-layer-associated protein-encoding gene contribute to the S-layer architecture in Sulfolobus We provide genetic evidence for the first time showing that the slaA gene is a key cell morphology determinant and may play a role in Sulfolobus cell division or/and cell fusion.

12.
Chem Commun (Camb) ; 55(75): 11231-11234, 2019 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-31469133

RESUMO

A Co-based mixed-ligand metal-organic framework (MOF), ZJU-109, is reported by utilizing 6-(4-pyridyl)-terephthalic acid (H2pta) and 4,4'-bis(imidazolyl)biphenyl (4,4'-bimbp) as double linkers. By tuning the d-PET process between ligands 4,4'-bimbp and H2pta, a fluorescent turn-on sensor for Fe3+ with a detection limit as low as 0.053 µM has been demonstrated.

13.
Biomed Pharmacother ; 118: 109225, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31325705

RESUMO

Lung cancer remains the leading cause of cancer associated deaths worldwide. Recent efforts have been focused on combinational and nanoparticulate therapies that can efficiently deliver multiple therapeutics. Herein, we reported cetuximab (CET) functionalized, paclitaxel (PTX) and 5-Demethylnobiletin (DMN) co-loaded nanostructured lipid carriers (NLCs) (CET-PTX/DMN-NLCs). The morphology, particle size, zeta potential, stability and drug release were tested. Cellular uptake, cell viability, synergistic effects and in vivo anti-tumor effects were evaluated on human lung adenocarcinoma cells (A549 cells), human embryonic lung cells (MRC-5 cells) and A549 paclitaxel-resistant cells bearing mice models. NLCs had sizes of around 130 nm and zeta potentials of +20-30 mV. The release of drugs from NLCs was relatively fast at the first 12 h and then became slow until completion of sustained release behavior. Cells uptake of CET-PTX/DMN-NLCs (65.8%) was remarkably higher than that of PTX/DMN-NLCs (35.5%) in A549 cells. The combination treatment with PTX and DMN synergistically decreases the viability of cells than the single PTX-NLCs and DMN-NLCs. CET-PTX/DMN-NLCs exhibited the most remarkable in vivo tumor inhibition efficiency, which suspended the tumor growth from 1010.23 to 211.18 mm3 at the end of the study. The highest tumor accumulation amount and low toxicity made CET-PTX/DMN-NLCs a promising system for the synergistic combination therapy of lung cancer.

14.
Infect Genet Evol ; 75: 103958, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31299322

RESUMO

The avian infectious disease, Newcastle disease (ND), caused by Newcastle disease virus (NDV) can cause severe economic losses to poultry whether vaccinated or not in many countries. In this study, a strain of NDV isolated from an outbreak in China was subjected to biological, phylogenetic and genetic characterization. The results showed that the mean death time (MDT) was 52.4 h and the intracerebral pathogenicity indices (ICPI) value was 1.95. In addition, amino acid sequencing result showed that it had a sequence 112R-R-Q-R-R↓F117 at fusion protein cleaving site (FPCS) indicating a velogenic strain. And its genome length is 15,192 nucleotide (nt) with the conserved complementary 3' leader and 5' trailer regions encoding six genes, 3'-NP-P-M-F-HN-L-5'. Based on phylogenetic analyses for hyper-variable region and complete genome of F gene, the strain studied here can be clustered into genotype IX, Class II, which has little evolution distance with strains of genotype III, being considered as a transitional strain in the evolution history of NDV. The rescue of infectious cDNA is proceeded in 9-day-old embryonated SPF chicken eggs. Despite the death of the first generation, the allantoic fluid harvested from the first generation lost its pathogenicity after passage. And we found the phenomenon happened due to the antibody appearing in the allantoic fluid. These findings offer our understanding of circulating strains of NDV in China and lay scientific foundations for making more efficient vaccines for Newcastle disease.

15.
Chem Commun (Camb) ; 55(51): 7378-7381, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-31173001

RESUMO

We propose synthetic DNA/RNA transcription circuits based on specific aptamer recognition. By mimicking transcription factor regulation, combined with specific enzyme/DNA aptamer binding, multiple biomolecules including DNA, RNA, polymerase, restriction enzymes and methylase were used as regulators. In addition, multi-level cascading networks and methylation-switch circuits were also established. This regulation strategy has the potential to expand the toolkit of in vitro synthetic biology.


Assuntos
Aptâmeros de Nucleotídeos/química , Fatores de Transcrição/química , DNA/química , Enzimas de Restrição do DNA/química , DNA Polimerase Dirigida por DNA/química , RNA Polimerases Dirigidas por DNA/química , Metilação , RNA/química , Transcrição Genética
16.
Free Radic Res ; 53(7): 800-814, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31223046

RESUMO

Acute kidney injury (AKI) is a major kidney disease associated with poor clinical outcomes. Oxidative stress is predominantly involved in the pathogenesis of AKI. Autophagy and the Keap1-Nrf2 signalling pathway are both involved in the oxidative-stress response. However, the cross talk between these two pathways in AKI remains unknown. Here, we found that autophagy is upregulated during cisplatin-induced AKI. In contrast with previous studies, we observed a marked increase in p62. We also found that p62 knockdown reduces autophagosome formation and the expression of LC3II. To explore the cross talk between p62 and the Keap1-Nrf2 signalling pathway, HK-2 cells were transfected with siRNA targeting Nrf2, and we found that Nrf2 knockdown significantly reduced cisplatin-induced p62 expression. Moreover, p62 knockdown significantly decreased the protein expression of Nrf2, as well as Heme Oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase l (NQO1), whereas the expression of kelch-like ECH-associated protein 1 (Keap1) was upregulated. These results indicate that p62 creates a positive feedback loop in the Keap1-Nrf2 signalling pathway. Finally, we examined the role of p62 in cell protection during cisplatin-induced oxidative stress, and we found that p62 silencing in HK-2 cells increases apoptosis and reactive oxygen species (ROS) levels, which further indicates the protective role of p62 under oxidative stress and suggests that the cytoprotection 62 mediated is in part by regulating autophagic activity or the Keap1-Nrf2 signalling pathway. Taken together, our results have demonstrated a reciprocal regulation of p62, autophagy and the Keap1-Nrf2 signalling pathway under oxidative stress, which may be a potential therapeutic target against AKI.

17.
Sci Rep ; 9(1): 6952, 2019 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-31061431

RESUMO

Persistent hyperglycemia is causally associated with pancreatic ß-cell dysfunction and loss of pancreatic insulin. Glucose normally enhances ß-cell excitability through inhibition of KATP channels, opening of voltage-dependent calcium channels, increased [Ca2+]i, which triggers insulin secretion. Glucose-dependent excitability is lost in islets from KATP-knockout (KATP-KO) mice, in which ß-cells are permanently hyperexcited, [Ca2+]i, is chronically elevated and insulin is constantly secreted. Mouse models of human neonatal diabetes in which KATP gain-of-function mutations are expressed in ß-cells (KATP-GOF) also lose the link between glucose metabolism and excitation-induced insulin secretion, but in this case KATP-GOF ß-cells are chronically underexcited, with permanently low [Ca2+]i and lack of glucose-dependent insulin secretion. We used KATP-GOF and KATP-KO islets to examine the role of altered-excitability in glucotoxicity. Wild-type islets showed rapid loss of insulin content when chronically incubated in high-glucose, an effect that was reversed by subsequently switching to low glucose media. In contrast, hyperexcitable KATP-KO islets lost insulin content in both low- and high-glucose, while underexcitable KATP-GOF islets maintained insulin content in both conditions. Loss of insulin content in chronic excitability was replicated by pharmacological inhibition of KATP by glibenclamide, The effects of hyperexcitable and underexcitable islets on glucotoxicity observed in in vivo animal models are directly opposite to the effects observed in vitro: we clearly demonstrate here that in vitro, hyperexcitability is detrimental to islets whereas underexcitability is protective.

18.
J Gen Virol ; 100(6): 958-967, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31140969

RESUMO

Newcastle disease (ND), which is caused by Newcastle disease virus (NDV), is a highly contagious disease in chickens and is a great threat to the poultry industry. Fusion of the viral and target cell membranes is a prerequisite for NDV's entry into host cells. This process is directly mediated by the fusion (F) protein. Although several domains of F are known to regulate membrane fusion activity, the roles of the DI-DII linker (residues 376-381) of the NDV F protein in membrane fusion still remain unclear. To investigate the roles of this linker in NDV F-induced cell-cell fusion, mutations were engineered into this linker by site-directed mutagenesis. These mutants were analysed with respect to cell surface expression and membrane fusion activity. Each of the mutated F proteins in this linker was expressed at the cell surface at a similar level to wild-type (WT) F. However, most of them resulted in significant alterations in fusion activity. In particular, the mutants G377S, A378D, L379A and T380P were able to independently mediate cell fusion in the absence of HN protein in BHK-21 cells. Taken together, the results indicated that the DI-DII linker region has an important effect on the fusion activity of NDV F and mutants in this region could alter the requirement for HN for the promotion of membrane fusion.

19.
Proc Natl Acad Sci U S A ; 116(22): 10658-10663, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31088971

RESUMO

Ribozymes synthesize proteins in a highly regulated local environment to minimize side reactions caused by various competing species. In contrast, it is challenging to prepare synthetic polypeptides from the polymerization of N-carboxyanhydrides (NCAs) in the presence of water and impurities, which induce monomer degradations and chain terminations, respectively. Inspired by natural protein synthesis, we herein report the preparation of well-defined polypeptides in the presence of competing species, by using a water/dichloromethane biphasic system with macroinitiators anchored at the interface. The impurities are extracted into the aqueous phase in situ, and the localized macroinitiators allow for NCA polymerization at a rate which outpaces water-induced side reactions. Our polymerization strategy streamlines the process from amino acids toward high molecular weight polypeptides with low dispersity by circumventing the tedious NCA purification and the demands for air-free conditions, enabling low-cost, large-scale production of polypeptides that has potential to change the paradigm of polypeptide-based biomaterials.

20.
Biosci Trends ; 13(3): 225-233, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31142702

RESUMO

Newcastle disease virus (NDV), an avian paramyxovirus, causes Newcastle disease (ND) which is a highly contagious and fatal viral disease affecting poultry and most species of birds. The fusion (F) protein of NDV mediates membrane fusion, which is essential to the processes of viral entry, replication, and dissemination. Although several domains of NDV F are known to have important effects on regulating the membrane fusion activity, the role of the region around domain III (DIII) and domain I (DI) still remains ill-defined. Site-directed mutagenesis was utilized to change the conserved amino acids at 269, 274, 277, 286, 287, 290, 295, and 297 to alanine in order to investigate the effects of these conserved amino acids around the DIII and DI linker region of the NDV F protein on fusion activity. It was found that five of these substitutions almost abolished fusion activity except for mutants I269A, Q286A, and N297A, which showed 57.1%, 161.1%, and 97.7% of the wt F level, respectively. Four (I274A, D277A, V287A, and P290A) of these five mutants likely result in interfering with folding or transporting of the molecule since these proteins were minimally expressed at the cell surface, formed aggregates, or not proteolytically cleaved. However, mutant L295A almost abolished fusion activity even with a similar level of cell surface expression. These data indicated that conserved amino acids around the DIII-DI linker region are critical for the folding of the F protein and have an important influence on fusion activity.

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