Amino Residues Research Articles

How Candida antarctica lipase B can be activated in natural deep eutectic solvents: experimental and molecular dynamics studies

AbstractBACKGROUNDNatural deep eutectic solvents (NADESs), a class of green solvents which completely accords to 12 principles of green chemistry, have proven to have great potential applications in enzymatic reactions. Despite strong interest, the role of NADESs in these processes, and the molecular interaction between enzymes and NADESs, still remain ambiguous. In the present study, the stability and activity of Candida Antarctica lipase B (CALB) were studied, and the mechanism by which CALB was activated in NADESs was explored systematically from both molecular‐ and macroscopic‐scale perspectives.RESULTSThe results suggested that the activity of CALB in all NADESs was significantly higher than that of the control (ethanol). Moreover, the stability of CALB increased to 115.48 ± 1.36% and 108.54 ± 1.26% in betaine‐glycerin (B‐Gly) and choline chloride‐glycerol (C‐Gly), but decreased to 91.69 ± 3.26% and 92.31 ± 3.36% in betaine‐xylitol (B‐X) and choline chloride‐xylitol (C‐X), respectively. The results of circular dichroism (CD), fluorescence spectroscopy and molecular dynamics studies (MD) indicated that there was no significant change in the secondary structure of CALB. Furthermore, the results of MD provide some information supporting that CALB was stabilized by the hydrogen(H)‐bonding interaction between surface amino residues of CALB and NADESs and was activated via the H‐bonding interaction between substrate and NADESs in the acyl‐binding pocket.CONCLUSIONThe mechanism by which CALB was activated and stabilized via the H‐bonding interactions in NADESs was revealed in the present study. This provides a scientific basis from which to further explore the potential application of NADESs in enzymatic reactions in food engineering and health‐related fields. © 2019 Society of Chemical Industry

D-Alanine Is Reduced by Ocular Hypertension in the Rat Retina

ABSTRACTPurpose: The purpose of this study was to investigate the physiological changes of amino acids in the rat retina caused by ocular hypertension.Methods: Adult Wistar rats were used as an experimental model of ocular hypertension. Retinas were hydrolyzed with HCl at 108°C to isolate amino acids. Residual amino acids were measured by reverse-phase high-performance liquid chromatography and the total volume of residual amino acids and the ratio of D- and L-amino acids were analyzed. Free D- and L-alanine levels were also measured using two-dimensional HPLC.Results: The amount of retinal alanine decreased in ocular hypertension compared with the control (p < .05, Student’s t-test); the amounts of other amino acids did not differ between the two conditions.The D/L ratio of alanine was higher than that of other amino acids. Ocular hypertension reduced the D/L ratio of retinal alanine, while that of other amino acids was unchanged. Ocular hypertension increased the D/L ratio of free alanine.Conclusions: Ocular hypertension reduced the D/L ratio of retinal alanine, presumably due in large part to alanine peptides.

Genome-wide identification and analyses of bHLH family genes inBrassica napus

The bHLH (basic/helix-loop-helix) protein is one of the largest transcription factors (TFs) that is essential in regulating plant growth and development. Although the bHLH family has been identified in some organisms, a systemic study has not been performed in Brassica napus, which is the third most important oilseed crop worldwide. In the present study, a total of 460 bHLH TFs were identified from the genome of B. napus and clustered into 25 subfamilies. Structural analysis was used to identify the DNA binding type and conserved amino residues of the identified sequence in the bHLH domain. In addition, a comparative genomic analysis of B. napus and its progenitors (Brassica rapa and Brassica oleracea) and two basal angiosperms (Amborella trichopoda and Vitis vinifera) was performed to trace the change during expansion and evolution of the bHLH family. The bHLH TFs in leaves and roots showed various expression patterns. The homologs of the AA-subgenome and CC-subgenome exhibited similar expression patterns, but were more divergent between the homologs caused from other duplicate events.

Melanoidins from Coffee, Cocoa, and Bread Are Able to Scavenge α-Dicarbonyl Compounds under Simulated Physiological Conditions.

Free amino residues react with α-dicarbonyl compounds (DCs) contributing to the formation of advanced glycation end products (AGEs). Phenolic compounds can scavenge DCs, thus controlling the dietary carbonyl load. This study showed that high-molecular weight cocoa melanoidins (HMW-COM), HMW bread melanoidins (HMW-BM), and especially HMW coffee melanoidins (HMW-CM) are effective DC scavengers. HMW-CM (1 mg/mL) scavenged more than 40% DCs within 2 h under simulated physiological conditions, suggesting some physiological relevance. Partial acid hydrolysis of HMW-CM decreased the dicarbonyl trapping capacity, demonstrating that the ability to react with glyoxal, methylglyoxal (MGO), and diacetyl was mainly because of polyphenols bound to macromolecules. Caffeic acid (CA) and 3-caffeoylquinic acid showed a DC-scavenging kinetic profile similar to that of HMW-CM, while mass spectrometry data confirmed that hydroxyalkylation and aromatic substitution reactions led to the formation of a stable adduct between CA and MGO. These findings corroborated the idea that antioxidant-rich indigestible materials could limit carbonyl stress and AGE formation across the gastrointestinal tract.

A metabolic study to decipher amino acid catabolism-directed biofuel synthesis in Acetoanaerobium sticklandii DSM 519

Acetoanaerobium sticklandii DSM 519 is a hyper-ammonia-producing anaerobe. It has the ability to produce organic solvents and acids from protein catabolism through Stickland reactions and specialized pathways. Nevertheless, its protein catabolism-directed biofuel production has not yet been understood. The present study aimed to decipher such growth-associated metabolic potential of this organism at different growth phases using metabolic profiling. A seed culture of this organism was grown separately in metabolic assay media supplemented with gelatin and or a mixture of amino acids. The extracellular metabolites produced by this organism were qualitatively analyzed by gas chromatography-mass spectrometry platform. The residual amino acids after protein degradation and amino acids assimilation were identified and quantitatively measured by high-performance liquid chromatography (HPLC). Organic solvents and acids produced by this organism were detected and the quantity of them determined with HPLC. Metabolic profiling data confirmed the presence of amino acid catabolic products including tyramine, cadaverine, methylamine, and putrescine in fermented broth. It also found products including short-chain fatty acids and organic solvents of the Stickland reactions. It reported that amino acids were more appropriate for its growth yield compared to gelatin. Results of quantitative analysis of amino acids indicated that many amino acids either from gelatin or amino acid mixture were catabolised at a log-growth phase. Glycine and proline were poorly consumed in all growth phases. This study revealed that apart from Stickland reactions, a specialized system was established in A. sticklandii for protein catabolism-directed biofuel production. Acetone-butanol-ethanol (ABE), acetic acid, and butyric acid were the most important biofuel components produced by this organism. The production of these components was achieved much more on gelatin than amino acids. Thus, A. sticklandii is suggested herein as a potential organism to produce butyric acid along with ABE from protein-based wastes (gelatin) in bio-energy sectors.

Modification at the 2'-Position of the 4,5-Series of 2-Deoxystreptamine Aminoglycoside Antibiotics To Resist Aminoglycoside Modifying Enzymes and Increase Ribosomal Target Selectivity.

A series of derivatives of the 4,5-disubstituted class of 2-deoxystreptamine aminoglycoside antibiotics neomycin, paromomycin, and ribostamycin was prepared and assayed for (i) their ability to inhibit protein synthesis by bacterial ribosomes and by engineered bacterial ribosomes carrying eukaryotic decoding A sites, (ii) antibacterial activity against wild type Gram negative and positive pathogens, and (iii) overcoming resistance due to the presence of aminoacyl transferases acting at the 2′-position. The presence of five suitably positioned residual basic amino groups was found to be necessary for activity to be retained upon removal or alkylation of the 2′-position amine. As alkylation of the 2′-amino group overcomes the action of resistance determinants acting at that position and in addition results in increased selectivity for the prokaryotic over eukaryotic ribosomes, it constitutes an attractive modification for introduction into next generation aminoglycosides. In the neomycin series, the installation of small (formamide) or basic (glycinamide) amido groups on the 2′-amino group is tolerated.

Design, synthesis and biological evaluation of some 2-(6-nitrobenzo[d]thiazol-2-ylthio)-N-benzyl-N-(6-nitrobenzo[d]thiazol-2-yl)acetamide derivatives as selective DprE1 inhibitors

Tuberculosis (TB) is an infectious disease and caused by various strains of mycobacteria. In the present study, pharmacophore model was developed using single ligand by ligand-based drug discovery approach. The key features responsible for DprE1 inhibitory activity were taken into consideration for developing pharmacophore. After the virtual screening, top 1000 hits were further subjected to docking study using GLIDE module, Schrödinger. Docking studies have shown promising interaction with amino residues with better glide score. Ligand-based drug design approach yielded a series of 15, 2-(6-nitrobenzo[d]thiazol-2-ylthio)-N-benzyl-N-(6-nitrobenzo[d]thiazol-2-yl)acetamide derivatives. All synthesized derivatives were characterized using NMR, mass, CHN analysis. The synthesized compounds were screened for In vitro antitubercular activity against Mycobacterium tuberculosis (H37Rv). Four compounds, 5g (MIC-1.01 μM); 5i (MIC-0.91 μM); 5k (MIC-0.82 μM); and 5o (MIC-1.04 μM) has shown promising activity compared to MIC of standard isoniazid (INH) and DprE1 enzyme inhibition was compared to BTZ043. Two halogen-substituted compounds have exhibited drastic enzyme inhibition.

Characterization and In Silico Analysis of The Structural Features of G-CSF Derived from Lysates of Escherichia coli.

Objective Granulocyte colony-stimulating factor (G-CSF) has a wide variety of functions including stimulation of hematopoiesis and proliferation of granulocyte progenitor cells. Recombinant human G-CSF (rh-G-CSF) is used for treatment of neutropenia in patients receiving chemotherapy. The mature bloodstream neutrophils express G-CSF receptor (G-CSFR), presenting a significant and specific mechanism for circulating G-CSF clearance. Computational studies are essential bioinformatics methods used for characterization of proteins with regard to their physicochemical properties and 3D configuration, as well as protein–ligand interactions for recombinant drugs. We formerly produced rh-G-CSF in E. coli and showed that the isolated protein had unacceptable biological activity in mice. In the present paper, we aimed to characterize the purified rh-G-CSF by analytical tests and developed an in vivo model by computational modelling of G-CSF. Materials and Methods In this experimental study, we analyzed the purified G-CSF using the analytical experiments. Then, the crystalline structure was extracted from Protein Data Bank (PDB) and molecular dynamics (MD) simulation was performed using Gromacs 5.1 package under an Amber force field. The importance of amino acid contents of G-CSF, to bind the respective receptor was also detected; moreover, the effect of dithiothreitol (DTT) used in G-CSF purification was studied. Results The results revealed that characteristics of the produced recombinant G-CSF were comparable with those of the standard G-CSF and the recombinant G-CSF with the residual amino acid was stable. Also, purification conditions (DTT and existence of extra cysteine) had a significant effect on the stability and functionality of the produced G-CSF. ConclusionExperimental and in silico analyses provided good information regarding the function and characteristics of our recombinant G-CSF which could be useful for industrial researches.

Antityrosinase mechanism of ellagic acid in vitro and its effect on mouse melanoma cells.

The activities of ellagic acid in inhibiting mushroom tyrosinase and cell proliferation were evaluated in this research. The results of enzyme kinetics indicated that ellagic acid could effectively inhibit tyrosinase activity. The value of the semi-inhibitory rate (IC50 ) was 0.2±0.05mM. Ellagic acid inhibited tyrosinase activity in a reversible manner and was a mixed tyrosinase inhibitor. Furthermore, ellagic acid had a good inhibitory effect on the proliferation of mouse melanoma B16 cells and could induce apoptosis. The results acquired from fluorescence spectroscopy revealed that the interaction of ellagic acid with tyrosinase depended on hydrogen bond and electrostatic force. In addition, computational docking showed that ellagic acid interacted with amino acid residues of tyrosinase (Asn19 and Lys372) by hydrogen bond and produced electrostatic interaction with amino residue Lys18. PRACTICAL APPLICATIONS: In the present research, the antityrosinase mechanism of ellagic acid and its effect on mouse melanoma cells were investigated. This study suggested that ellagic acid had a strong inhibitory activity against tyrosinase and cell proliferation,which laid an experimental foundation for the development of new drugs and whitening products. The combined multispectral methods used in this research can be applied to the screening of other antityrosinase inhibitors, further promoting the development and utilization of tyrosinase inhibitors.

Dual functional AgNPs-M13 phage composite serves as antibacterial film and sensing probe for monitoring the corrosion of chromium-containing dental alloys

The preparation of silver nanoparticles (AgNPs) with microbe or plant tissues as bio-template offers green approach, while it suffers from low harvest and purification is needed. Herein, we propose a facile protocol for one-pot preparation of AgNPs using M13 phage as bio-template by simply mixing AgNO3 solution with alkali M13 phage. In the obtained AgNPs-M13 phage composite, Cr(III) selectively coordinates with the amino residues on phage surface and leads to the aggregation of AgNPs through the bridging of M13 phages. This makes it feasible for colorimetric sensing of Cr(III) by measuring the absorbance ratio of AgNPs at 600 and 405 nm, which provides a LOD of 14 nmol/L. The composite also showed favorable bactericidal activity for both Gram-positive and Gram-negative bacteria, making it a promising candidate as antibacterial film in chromium-containing dental alloys and meanwhile serve as a sensing probe for monitoring the corrosion of the dental alloys.

High-throughput virtual screening, ADME analysis, and estimation of MM/GBSA binding-free energies of azoles as potential inhibitors of Mycobacterium tuberculosis H37Rv

Isoniazid, also known as isonicotinyl hydrazide (INH), was used as a frontline drug to treat Mycobacterium tuberculosis. The in silico virtual screening is used to identify the effective inhibitor for CYP 121 of M. tuberculosis. Nearly, 10,000 triazole compounds from various databases have been virtually screened against CYP121 by using the module Glide. A total of 15 molecules with a better docking score compared to marketed drugs and cocrystal ligand were chosen for the MM/GBSA study to recheck the binding affinity. A total of five hit molecules which show significant binding-free energies were chosen for QikProp studies. The triazole molecules which contain 3-benzaimidine moiety interact with a heme cofactor and active site residues of CYP121. These protein–ligand complexes were taken to calculate the potential binding region of the ligand in the receptor using the solvent accessible surface area (SASA). The amino residues Phe168, Val228, Gln385, Trp182, Asp185, Val83, and heme 401 cofactor of the target protein are the important binding residues with the ligands. The in silico ADME studies for the ligand dataset are calculated to determine the druggability of the molecules.

Isolation, characterization and expression of transcription factor ScDREB2 from wild, commercial and interspecific hybrid sugarcane in salinity condition

Dehydration Responsive Element Binding (DREB) gene is one of the essential transcription factors plants use for responding to stress conditions including salinity, drought, and cold stress. The purpose of this study was to isolate the full length and characterize the DREB gene from three different genotypes of sugarcane, wild, commercial cultivar, and interspecific hybrid sugarcane. The length of the gene, designated ScDREB was 789 bp, and coding for a putative polypeptide of 262 amino acid residues. Sequences of the gene were submitted to the GenBank database with accession numbers of KX280722.1, KX280721.1, and KX280719.1 for wild sugarcane, commercial cultivar (KPS94-13), and interspecific hybrid (Biotec2), respectively. In silico characterization indicated that the deduced polypeptide contains a putative nuclear localization signal (NLS) sequence, and a conserved AP2/ERF domain of the DREB family, at 82-140 amino residues. Based on multiple sequence alignment, sequences of the gene from the three sugarcane genotypes were classified in the DREB2 group. Gene expression analysis indicated, that ScDREB2 expression pattern in tested sugarcane was up-regulated by salt stress. When the plants were under 100 mM NaCl stress, relative expressions of the gene in leaves was higher than those in roots. In contrast, under 200 mM NaCl stress, relative expressions of the gene in roots was higher than those in leaves. This is the first report on cloning the full length and characterization, of ScDREB2 gene of sugarcane. Results indicate that ScDREB2 is highly responsive to salt stress.

Development of Fullerene Thin-Film Assemblies and Fullerene-Diamine Adducts towards Practical Nanocarbon-Based Electronic Materials

Abstract Fullerenes are attractive spherical aromatic molecules with good electron acceptor capabilities and good utility as an n-type organic semiconductor. By using a fullerene-amine addition reaction, it was possible to fabricate ultrathin-film assemblies of fullerene on the surface of substrates, which were confirmed by photoelectric conversion applications. Addition reactions between fullerenes and primary aliphatic diamines can also occur to form insoluble adduct particles consisting of fullerenes and diamines. In one example, C60-ethylenediamine adduct particles can be solubilized by addition of alkylacid chloride to residual amino groups of the adducts. Spin-coated or dip-coated thin-films of C60-ethylenediamine adducts from their solutions are useful as n-type organic semiconductors which was confirmed with solar cell application. In this account, the history of the fabrication and application of fullerene thin-film assemblies and fullerene-diamine adducts using the fullerene-amine addition reaction is introduced and summarized from the early studies to more recent developments.

Biogenic synthesis of silver nanoparticles using Jatropha curcas seed cake extract and characterization: evaluation of its antibacterial activity

ABSTRACT Nanoparticles synthesis using biological methods is gaining importance over the physicochemical methods because of its nontoxic nature and less environmental burden. In this paper, we report the biosynthesis of silver nanoparticles (AgNPs) from its precursor salt AgNO3, using Jatropha curcas seed cake extract, which will provide value addition to the byproduct of the biodiesel industry. Jatropha curcas seed oil is usually used as a source for biodiesel production as fatty acyl methyl esters (FAME). Jatropha curcas seed cake extract was screened for phytochemicals present in it which may help in reducing the precursor metal salt to nanoparticles as well as coat around the nanoparticles, thereby increasing their stability. Phytochemical screening revealed the presence of carbohydrates, proteins, alkaloids, flavonoids, saponins, tannins, phenols and terpenoids in the extract. Biosynthesized nanoparticles were characterized by UV-visible spectroscopy, SEM and FTIR. The Uv-visible spectroscopic analysis confirmed the production of silver nanoparticles at 400–460 nm, where the colour change in the solution from pale yellow to brown indicated the formation of silver nanoparticles. SEM image showed the size of the AgNPs ranged from 80 nm to 95 nm. FTIR analysis confirmed the capping of nanoparticles with organic residues like, proteins, amino acids, and polyphenols present in the extract, which led to increased stabilization of nanoparticles. Negative zeta values indicated the stability of nanoparticles. The synthesized AgNPs were tested for their antibacterial activity against both Gram positive (Bacillus subtilis) and Gram negative (E. coli, Pseudomonas aeruginosa) bacteria with known antibiotic as a control. Biosynthesized AgNPs showed significantly high bacteriostatic effect.

Identification of promising anti-DNA gyrase antibacterial compounds using de novo design, molecular docking and molecular dynamics studies

The rapidly increasing rate of antibiotic resistance is of great concern. Approximately two million deaths result annually from bacterial infections worldwide. Therefore, there is a paramount requirement to develop innovative and novel antibacterial agents with new mechanisms of action and activity against resistant bacterial strains. For this purpose, a set of benzothiazole and N-phenylpyrrolamides derivatives reported as DNA Gyrase B (GyrB) inhibitors were collected from the literature and docked inside the receptor cavity of DNA Gyrase B (PDB ID: 5L3J). The best 10 docked complexes were used to identify novel antibacterial chemical agents through a de novo design approach. Out of initial 300 chemical analogues, the best six analogues were identified using screening with a set of criteria followed by pharmacokinetic analysis. The binding interactions of the best six analogues revealed that all molecules formed a number of critical interactions with catalytic amino residues of DNA Gyrase B with high binding energy. The predicted inhibitory constant biological activity based on binding energy supported the potential of the molecules as DNA Gyrase B ligands. The RMSD, RMSF, and radius of gyration parameters obtained from the 100 ns molecular dynamics simulation study clearly demonstrated that all six analogues were efficient enough to form stable complexes with DNA Gyrase B. High negative binding energy of all ligands obtained from MM-GBSA approach undoubtedly explained the strong affinity toward the DNA Gyrase B. Therefore, the proposed de novo designed molecules can be considered as promising antibacterial chemical agents subject to experimental validation, in vitro.Communicated by Ramaswamy H. Sarma

Synthesis of new 1-benzyl tetrahydropyridin-4-ylidene piperidinium salts and their antiplasmodial and antitrypanosomal activities

Our last study revealed the distinct antiplasmodial and antitrypanosomal activities of 1-benzyl tetrahydropyridin-4-ylidene pyrrolidinium salts. Therefore, we prepared a series of new 1-benzyl tetrahydropyridin-4-ylidene ammonium salts with an alternative amino residue and varying substitution patterns at the benzyl group to investigate the influence of these modifications on the biological activities. All new compounds were characterized by spectroscopic methods like ultraviolet, infrared, nuclear magnetic resonance, as well as high- resolution mass spectra. They were tested for their activities against Plasmodium falciparum and Trypanosoma brucei rhodesiense as well as for their cytotoxicity against L6 cells using microplate assays. The results show that the structure of the amino residue as well as the substitution pattern of the benzyl group influences the biological activities distinctly. Physicochemical parameters are calculated and structure–activity relationships are discussed.

Epigenetic Process Monitoring in Live Cultures with Peptide Biosensors.

Acetyltransferase is a member of the transferase group responsible for transferring an acetyl group from acetyl-CoA to amino group of a histone lysine residue. Past efforts on histone acetylation monitoring involved biochemical analysis that do not provide spatiotemporal information in a dynamic format. We propose a novel approach to monitor acetyltransferase acetylation in live single cells using time correlated single photon counting fluorescence lifetime imaging (TCSPC-FLIM) with peptide biosensors. Utilizing 2D and 3D cultures we show that the peptide sensor has a specific response to acetyltransferase enzyme activity in a fluorescence lifetime dependent manner ( P < 0.001). Our FLIM biosensor concept enables real-time longitudinal measurement of acetylation activity with high spatial and temporal resolution in live single cells to monitor cell function or evaluate drug effects to treat cancer or neurological diseases.

Abstract B163: Regulation of translation by the interferon-induced antiviral protein viperin

Abstract The innate immune response involves the induction of hundreds of interferon-stimulated genes (ISGs), many of which play a role in cancer immunosurveillance and antiviral immunity. Therefore, understanding how ISGs function and coordinate with the cellular network is of critical importance. We focus on one such ISG, viperin (virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible). Viperin has been reported to inhibit a broad spectrum of DNA and RNA viruses, including many flaviviruses, human cytomegalovirus (HCMV), Chikungunya virus, influenza A virus, Sindbis virus, vesicular stomatitis virus, tick-borne encephalitis virus, HIV-1 and more. It is highly conserved in evolution, from protozoans to humans. Viperin is a peripheral membrane protein, associating on the cytosolic face of the ER via its N-terminal amphipathic helix. The cytosolic domain contains a radical SAM domain with a [4Fe-4S] cluster coordinated by three cysteine residues in the active site. Although the enzymatic function and substrate of viperin is still under debate, a recent study reveals that viperin catalyzes the conversion of cytidine triphosphate to 3′-deoxy-3′,4′-didehydro-CTP (ddhCTP) via its radical SAM activity. However, a general mechanism for viperin action and its potential roles within the innate immune response remain to be defined. Here, we demonstrate that viperin inhibits global protein translation via its radical SAM-dependent enzymatic activity. Using immortalized mouse macrophages from wild-type and viperin knockout mice we show that, although other ISGs are induced, type I IFN treatment of WT but not viperin KO mouse microphages leads to a global change in polysome profile with an increase in the ribosome fraction and a decrease in actively translating ribosomes. In parallel, the translational regulation function was evaluated in 293T-cells with transient transfection and doxycycline-inducible system. Despite no IFN stimulation and therefore no other ISG expression, viperin effectively represses translation. Furthermore, we find that viperin reduces global protein translation by ~30% in live cells using [35S]-methionine metabolic pulse-labeling or O-propargyl-puromycin (OPP) labelling. The radical SAM enzymatic activity is required for translational inhibition: site-directed mutagenesis of amino residues involved in iron-sulfur cluster and substrate binding abolishes the translational inhibition activity. We also demonstrate that ddhCTP, a recently identified vipern product, inhibits global protein translation in live cells. Viral replication requires the host translation machinery to make viral proteins, and our results suggest that much of its antiviral activity may be attributable to translational regulation. We show that viperin inhibits viral protein synthesis and viral replication of the Kunjin virus, a West Nile Virus variant, in tissue culture cells. Our study suggests a partially unifying mechanism for the broad antiviral function of viperin based on translational regulation. Citation Format: Chun-Chieh Hsu, Maudry Laurent-Rolle, Peter Cresswell. Regulation of translation by the interferon-induced antiviral protein viperin [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B163.

New strains of Japanese encephalitis virus circulating in Shanghai, China after a ten-year hiatus in local mosquito surveillance

BackgroundContinuous vector pathogen surveillance is essential for preventing outbreaks of mosquito-borne diseases. Several mosquito species acting as vectors of Japanese encephalitis virus (JEV), dengue virus, Zika virus, malaria parasites and other pathogens are primary mosquito species in Shanghai, China. However, few surveys of human pathogenic arboviruses in mosquitoes in Shanghai have been reported in the last ten years. Therefore, in this study, we evaluated mosquito activity in Shanghai, China during 2016 and tested for the presence of alphaviruses, flaviviruses, orthobunyaviruses and several parasitic pathogens.ResultsFive pooled samples were JEV-positive [4/255 pools of Culex tritaeniorhynchus and 1/256 pools of Cx. pipiens (s.l.)] based on analysis of the NS5 gene. Alphaviruses, orthobunyaviruses, Plasmodium and filariasis were not found in this study. Phylogenetic and molecular analyses revealed that the JEV strains belonged to genotype I. Moreover, newly detected Shanghai JEV strains were genetically close to previously isolated Shandong strains responsible for transmission during the 2013 Japanese encephalitis (JE) outbreak in Shandong Province, China but were more distantly related to other Shanghai strains detected in the early 2000s. The E proteins of the newly detected Shanghai JEV strains differed from that in the live attenuated vaccine SA14-14-2-derived strain at six amino residues: E130 (Ile→Val), E222 (Ala→Ser), E327 (Ser→Thr), E366 (Arg→Ser/Pro), E393 (Asn→Ser) and E433 (Val→Ile). However, no differences were observed in key amino acid sites related to antigenicity. Minimum JEV infection rates were 1.01 and 0.65 per 1000 Cx. tritaeniorhynchus and Cx. pipiens (s.l.), respectively.ConclusionsFive new Shanghai JEV genotype I strains, detected after a ten-year hiatus in local mosquito surveillance, were genetically close to strains involved in the 2013 Shandong JE outbreak. Because JEV is still circulating, vaccination in children should be extensively and continuously promoted. Moreover, JEV mosquito surveillance programmes should document the genotype variation, intensity and distribution of circulating viruses for use in the development and implementation of disease prevention and control strategies.

Superoxide dismutase transcellular shuttle constructed from dendritic MOF and charge reversible protein derivatives.

The development of molecular biology has led to the identification of protein-based therapeutics as an intriguing approach for the treatment of a wide range of diseases. To manufacture transcellular protein delivery shuttles, we attempted charge reversal chemistry on native proteins [e.g., superoxide dismutase (SOD): an enzyme capable of scavenging detrimental reactive oxygen species] by the selective conversion of the positively charged amino residues of native SOD to conjugated negatively charged citraconic moieties, eliciting overall negatively charged polyelectrolytes for the subsequent electrostatic self-assembly with cationic metal-organic framework (MOF) derivatives into protein delivery systems. Please note that the charge conversion was reversible, restoring the original amino groups in intracellular acidic endosome compartments (pH 5), which afforded facile charge reversible functions to reclaim the active SOD in the cell interior. In particular, the strategic manufacture of dendritic MOF supramolecular architectures as transcellular shuttles for the aforementioned charge-reversible SOD derivatives is noteworthy. The MOF was surface-functionalized with several polycationic segments, thus contributing to the hyper-charged architecture for the easy accommodation of the negatively charged SOD derivatives. Consequently, the SOD derivatives managed to internalize into cells by hitchhiking via endocytosis of the positively charged MOF. Once they resided in the acidic endosomes, the charge reversal of the SOD derivatives could occur smoothly and result in reduced interactions between the charged-reversed SOD and MOF, leading to the release of active SOD. Simultaneously, the dendritic MOF due to protein release presented a highly positive-charged architecture to provoke endosome membrane disruption, consequently spurring the translocation of SOD to the cytosol for the execution of its enzymatic activities. Herein, the intracellular ROS level of the activated macrophages was validated to be markedly suppressed by our proposed transcellular SOD shuttles, thereby indicating their wide availability to diverse functional proteins for biomedical applications.