Screening and heterologous expression of an antimicrobial peptide SCAK33 with broad-spectrum antimicrobial activity resourced from sea cucumber proteome.

Antimicrobial peptides (AMPs) are a family of short defense proteins that are naturally produced by all organisms and have great potential as effective substitutes for small-molecule antibiotics. The present study aims to excavate AMPs from sea cucumbers and achieve their heterologous expression in prokaryotic Escherichia coli. Using MytC as a probe, a cysteine-stabilized peptide SCAK33 with broad-spectrum antimicrobial activity was discovered from the proteome of Apostichopus japonicas. The SCAK33 showed inhibitory effects on both gram positive and gram negative bacteria with MICs of 3-28 µM, and without significant hemolysis activity in rat blood erythrocyte. Especially, it exhibited good antimicrobial activity against Bacillus megaterium, B. subtilis, and Vibrio parahaemolyticus with the MIC of 3, 7, and 7 µM, respectively. After observation by scanning electronic microscopy (SEM) and confocal laser scanning microscope (CLSM), it was found that the cell membrane of bacteria was severely damaged. Furthermore, the recombinant SCAK33 (reSCAK33) was heterologously expressed by fusion with SUMO tag in E. coli BL21(DE3), and the protein yield reached 70 mg/L. The research will supplement the existing quantity of sea cucumber AMPs and provide data support for rapid mining and biological preparation of sea cucumber AMPs.

Alishewanella maricola sp. nov., isolated from seawater of the Yellow Sea.

A Gram-stain-negative and facultative aerobic bacterium, strain 16-MAT, was isolated from seawater of Yellow Sea in South Korea. Cells were catalase- and oxidase-positive and non-motile rods. Growth occurred at 4-37 °C (optimum, 30 °C) and pH 6.0-11.0 (optimum, 8.0), and in the presence of 0-7.0% NaCl (optimum, 3 %). Strain 16-MAT contained ubiquinone-8 as the sole isoprenoid quinone, C16 : 0 and summed feature three as the major fatty acids (>10 %), and phosphatidylglycerol, phosphatidylethanolamine, an unidentified phospholipid, an unidentified aminophospholipid, and an unidentified polar lipid as the major polar lipids. The genome size and DNA G+C content of strain 16-MAT were 3.69 Mb and 46.0 mol%, respectively. Strain 16-MAT was most closely related to Alishewanella alkalitolerans LNK-7.1T with a 97.9 % 16S rRNA gene sequence similarity. A phylogenomic tree based on whole genome sequences showed that strain 16-MAT formed a phylogenetic lineage within the genus Alishewanella. Based on the phenotypic, chemotaxonomic, and molecular analyses, strain 16-MAT represents a novel species of the genus Alishewanella, for which the name Alishewanella maricola is proposed. The type strain is 16-MAT (=KACC 22238T =JCM 34596T).

Dyadobacter pollutisoli sp. nov., isolated from plastic waste landfill soil.

A Gram-stain-negative, yellow-pigmented and facultatively aerobic bacterium, designated strain U1T, was isolated from plastic dumped soil sampled in the Republic of Korea. Cell of strain U1T were non-motile rods showing catalase-negative and oxidase-positive activities. Strain U1T was shown to grow at 10-37 °C (optimum, 25-30 °C) and pH 6.0-9.0 (optimum, pH 8.0), and in the presence of 0-0.5 % (w/v) NaCl (optimum, 0 %). Strain U1T contained iso-C15 : 0, C16 : 0, C16 : 1 ω5c and summed feature 3 (comprising C16 : 1 ω6c and/or C16 : 1 ω7c) as the major cellular fatty acids (>5 %) and menaquinone-7 as the sole respiratory quinone. Phosphatidylethanolamine, two unidentified aminolipids and three unidentified lipids were identified as the major polar lipids. The DNA G+C content of strain U1T calculated from the whole-genome sequence was 45.5 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain U1T formed a distinct phylogenetic lineage within the genus Dyadobacter. Strain U1T has the highest 16S rRNA sequence similarity to Dyadobacter bucti QTA69T (97.9 %). Average nucleotide identity and digital DNA-DNA hybridization values between strain U1T and D. bucti QTA69T were 74.6 % and 18.9 %, respectively. Based on phenotypic, chemotaxonomic and molecular features, strain U1T represents a novel species of the genus Dyadobacter, for which the name Dyadobacter pollutisoli sp. nov. is proposed. The type strain is U1T (= KACC 22210T=JCM 34491T).

Exopolysaccharide-producing bacteria enhanced Pb immobilization and influenced the microbiome composition in rhizosphere soil of pakchoi (Brassica chinensis L.).

Lead (Pb) contamination of planting soils is increasingly serious, leading to harmful effects on soil microflora and food safety. Exopolysaccharides (EPSs) are carbohydrate polymers produced and secreted by microorganisms, which are efficient biosorbent materials and has been widely used in wastewater treatment to remove heavy metals. However, the effects and underlying mechanism of EPS-producing marine bacteria on soil metal immobilization, plant growth and health remain unclear. The potential of Pseudoalteromonas agarivorans Hao 2018, a high EPS-producing marine bacterium, to produce EPS in soil filtrate, immobilize Pb, and inhibit its uptake by pakchoi (Brassica chinensis L.) was studied in this work. The effects of strain Hao 2018 on the biomass, quality, and rhizospheric soil bacterial community of pakchoi in Pb-contaminated soil were further investigated. The results showed that Hao 2018 reduced the Pb concentration in soil filtrate (16%-75%), and its EPS production increased in the presence of Pb2+. When compared to the control, Hao 2018 remarkably enhanced pakchoi biomass (10.3%-14.3%), decreased Pb content in edible tissues (14.5%-39.2%) and roots (41.3%-41.9%), and reduced the available Pb content (34.8%-38.1%) in the Pb-contaminated soil. Inoculation with Hao 2018 raised the pH of the soil, the activity of several enzymes (alkaline phosphatase, urease, and dehydrogenase), the nitrogen content (NH4 +-N and NO3 --N), and the pakchoi quality (Vc and soluble protein content), while also raising the relative abundance of bacteria that promote plant growth and immobilize metals, such as Streptomyces and Sphingomonas. In conclusion, Hao 2018 reduced the available Pb in soil and pakchoi Pb absorption by increasing the pH and activity of multiple enzymes and regulating microbiome composition in rhizospheric soil.

Neobacillus terrae sp. nov., isolated from mountain soil.

A Gram-stain-positive, spore-forming and facultative aerobic bacterium, designated C11T, was isolated from mountain soil collected in the Republic of Korea. The cells were motile rods with peritrichous flagella, and positive for catalase and oxidase activities. Strain C11T grew at 15-45 °C (optimum, 30-37 °C) and pH 6.0-8.0 (optimum, pH 6.0) and in the presence of 0-1 % (w/v) NaCl (optimum, 0.5 %). Strain C11T contained menaquinone-7 as the sole isoprenoid quinone and iso-C15 : 0, iso-C16 : 0 and anteiso-C15 : 0 as the major fatty acids. Diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine were the major polar lipids. The G+C content of the genomic DNA was 38.8 mol%. Strain C11T was most closely related to Neobacillus drentensis IDA1967T and Mesobacillus foraminis CV53T, with 98.0 and 97.7 %, 71.7 and 69.9 %, and 20.1 and 20.3 % 16S rRNA gene sequence similarity, average nucleotide identity, and digital DNA-DNA hybridization values, respectively. Phylogenetic analyses based on 16S rRNA gene and genome sequences showed that strain C11T was included in a phyletic lineage with members of the genus Neobacillus but was distinct from members of the genus Mesobacillus. Phenotypic, chemotaxonomic and molecular properties suggested that strain C11T represents a novel species of the genus Neobacillus, for which the name Neobacillus terrae sp. nov. is proposed. The type strain is C11T (=KACC 21661T=JCM 33943T).

Microvirga terrae sp. nov., Isolated from Soil.

A Gram-stain-negative and strictly aerobic bacterium, strain R24T, was isolated from soil in South Korea. Cells were non-motile short rods showing catalase- and oxidase-positive activities. Growth was observed at 15-40 °C (optimum, 25-30 °C) and pH 6.0-10.0 (optimum, 8.0-9.0), and in the presence of 0-3.0% NaCl (optimum, 0%). Strain R24T contained ubiquinone-10 as the sole respiratory quinone, C16:0, C18:0, and summed feature 8 (comprising C18:1 ω7c and/or C18:1 ω6c) as the major fatty acids, and phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylcholine as the major polar lipids. The DNA G+C content calculated from the whole genome sequence was 64.4%. Strain R24T was most closely related to Microvirga aerilata 5420S-16T with a 98.6% 16S rRNA gene sequence similarity. Average nucleotide identity and digital DNA-DNA hybridization values between strain R24T and all Microvirga species were less than 82.5 and 23.8%, respectively. Phylogenetic analyses based on the 16S rRNA gene and whole genome sequences revealed that strain R24T formed a phyletic lineage within the genus Microvirga. Based on its phenotypic, chemotaxonomic, and molecular characteristics, strain R24T represents a novel species of the genus Microvirga, for which the name Microvirga terrae sp. nov. is proposed. The type strain is R24T (= KACC 21784T = JCM 34259T).

Mettl14-driven senescence-associated secretory phenotype facilitates somatic cell reprogramming.

The METTL3-METTL14 complex, the "writer" of N6-methyladenosine (m6A), plays an important role in many biological processes. Previous studies have shown that Mettl3 overexpression can increase the level of m6A and promote somatic cell reprogramming. Here, we demonstrate that Mettl14, another component of the methyltransferase complex, can significantly enhance the generation of induced pluripotent stem cells (iPSCs) in an m6A-independent manner. In cooperation with Oct4, Sox2, Klf4, and c-Myc, overexpressed Mettl14 transiently promoted senescence-associated secretory phenotype (SASP) gene expression in non-reprogrammed cells in the late stage of reprogramming. Subsequently, we demonstrated that interleukin-6 (IL-6), a component of the SASP, significantly enhanced somatic cell reprogramming. In contrast, blocking the SASP using a senolytic agent or a nuclear factor κB (NF-κB) inhibitor impaired the effect of Mettl14 on reprogramming. Our results highlight the m6A-independent function of Mettl14 in reprogramming and provide new insight into the interplay between senescence and reprogramming in vitro.

[Intrinsically disordered proteins (IDPs) and the impact on cell stress resistance].

Intrinsically disordered proteins (IDPs) are proteins or protein regions that fail to get folded into definite three-dimensional structures but participate in various biological processes and perform specific functions. Defying the traditional protein "sequence-structure-function" paradigm, they enrich the protein "structure-function" diversity. Ubiquitous in organisms, they show extreme hydrophilicity, charged amino acids, and highly repetitive amino acid sequences, with simple arrangement. As a result, they feature highly variable binding affinities and high coordination, which facilitate their functions. IDPs play an important role in cell stress response, which can improve the tolerance to a variety of stresses, such as freezing, high salt, heat shock, and desiccation. In this study, we briefed the characteristics, classifications, and identification of IDPs, summarized the molecular mechanism in improving cell stress resistance, and described the potential applications.

Pan-Genomic and Transcriptomic Analyses of Marine Pseudoalteromonas agarivorans Hao 2018 Revealed Its Genomic and Metabolic Features.

The genomic and carbohydrate metabolic features of Pseudoalteromonas agarivorans Hao 2018 (P. agarivorans Hao 2018) were investigated through pan-genomic and transcriptomic analyses, and key enzyme genes that may encode the process involved in its extracellular polysaccharide synthesis were screened. The pan-genome of the P. agarivorans strains consists of a core-genome containing 2331 genes, an accessory-genome containing 956 genes, and a unique-genome containing 1519 genes. Clusters of Orthologous Groups analyses showed that P. agarivorans harbors strain-specifically diverse metabolisms, probably representing high evolutionary genome changes. The Kyoto Encyclopedia of Genes and Genomes and reconstructed carbohydrate metabolic pathways displayed that P. agarivorans strains can utilize a variety of carbohydrates, such as d-glucose, d-fructose, and d-lactose. Analyses of differentially expressed genes showed that compared with the stationary phase (24 h), strain P. agarivorans Hao 2018 had upregulated expression of genes related to the synthesis of extracellular polysaccharides in the logarithmic growth phase (2 h), and that the expression of these genes affected extracellular polysaccharide transport, nucleotide sugar synthesis, and glycosyltransferase synthesis. This is the first investigation of the genomic and metabolic features of P. agarivorans through pan-genomic and transcriptomic analyses, and these intriguing discoveries provide the possibility to produce novel marine drug lead compounds with high biological activity.

Effect of Different Initial Fermentation pH on Exopolysaccharides Produced by Pseudoalteromonas agarivorans Hao 2018 and Identification of Key Genes Involved in Exopolysaccharide Synthesis via Transcriptome Analysis.

Exopolysaccharides (EPSs) are carbohydrate polymers produced and secreted by microorganisms. In a changing marine environment, EPS secretion can reduce damage from external environmental disturbances to microorganisms. Meanwhile, EPSs have promising application prospects in the fields of food, cosmetics, and pharmaceuticals. Changes in external environmental pH have been shown to affect the synthesis of EPSs in microorganisms. In this study, we analyzed the effects of different initial fermentation pHs on the production, monosaccharide composition, and antioxidant activity of the EPSs of Pseudoalteromonas agarivorans Hao 2018. In addition, the transcriptome sequence of P. agarivorans Hao 2018 under different initial fermentation pH levels was determined. GO and KEGG analyses showed that the differentially expressed genes were concentrated in the two-component regulatory system and bacterial chemotaxis pathways. We further identified the expression of key genes involved in EPS synthesis during pH changes. In particular, the expression of genes encoding the glucose/galactose MFS transporter, phosphomannomutase, and mannose-1-phosphate guanylyltransferase was upregulated when the environmental pH increased, thus promoting EPS synthesis. This study not only contributes to elucidating the environmental adaptation mechanisms of P. agarivorans, but also provides important theoretical guidance for the directed development of new products using biologically active polysaccharides.

Colwellia maritima sp. nov. and Polaribacter marinus sp. nov., isolated from seawater.

Two Gram-stain-negative, catalase- and oxidase-positive, and aerobic bacteria, strains MSW7T and MSW13T, were isolated from seawater. Cells of strains MSW7T and MSW13T are motile and non-motile rods, respectively. Strain MSW7T optimally grew at 25 °C and pH 7.0 and in the presence of 3 % (w/v) NaCl, whereas strain MSW13T optimally grew at 25 °C and pH 6.0-7.0 and in the presence of 2 % NaCl. As the sole respiratory quinone and the major fatty acids and polar lipids, strain MSW7T contained ubiquinone-8, C16 : 0, C15 : 1 ω8c, C17 : 1 ω8c and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), and phosphatidylethanolamine and phosphatidylglycerol, respectively, whereas strain MSW13T contained menaquinone-6, C15 : 1 ω6c, iso-C15 : 0, anteiso-C15 : 0, and iso-C15 : 0 3-OH, and phosphatidylethanolamine, respectively. The DNA G+C contents of strains MSW7T and MSW13T were 37.3 and 29.9 %, respectively. Phylogenetic analyses based on 16S rRNA gene sequences showed that strains MSW7T and MSW13T were most closely related to Colwellia echini A3T and Polaribacter atrinae WP25T with 98.8 and 98.1 % sequence similarities, respectively. The average nucleotide identity and digital DNA-DNA hybridization values between strain MSW7T and C. echini A3T and between strain MSW13T and P. atrinae KACC 17473T were 73.6 and 22.6 % and 80.4 and 23.8 %, respectively. Based on phenotypic, chemotaxonomic and phylogenetic data, strains MSW7T and MSW13T represent novel species of the genera Colwellia and Polaribacter, respectively, for which the names Colwellia maritima sp. nov. and Polaribacter marinus sp. nov. are proposed, respectively. The type strains of C. maritima sp. nov. and P. marinus sp. nov. are MSW7T (=KACC 22339T=JCM 35001T) and MSW13T (=KACC 22341T=JCM 35021T), respectively.

An acidic-groups detection method and its application to analysis of Chinese humic acid samples.

The method of non-aqueous conductivity titration (NACT) of organic weak acids was applied to quickly and accurately determine the phenolic-hydroxyl and carboxyl-groups contents in humic acid. By varying the pH of the humic-acid sample, the concentration of the titrant, and the nitrogen-gas flow rate, the optimal titration conditions were determined to be a sample pH of 4, titrant concentration of 0.05 mol/L, and nitrogen-gas flow rate of 80 mL/min. Applying the detection method to p-hydroxybenzoic acid showed that its phenolic-hydroxyl content was 758.82±111.76 cmol/kg and carboxyl content was 744.44±51.11 cmol/kg. The theoretical phenolic-hydroxyl and carboxyl-groups contents of the p-hydroxybenzoic acid were 723.96 cmol/kg respectively, indicating that the method can accurately quantify the carboxyl and phenolic-hydroxyl groups in the sample. The NACT was used to measure the phenolic-hydroxyl and carboxyl-groups contents in humic acid quickly and accurately. In addition, 29 humic acid samples from 8 provinces of China covering the main humic-acid producing areas were collected and analyzed for acidic-groups content using the reported method.

Distribution of Zinc in Mycelial Cells and Antioxidant and Anti-Inflammatory Activities of Mycelia Zinc Polysaccharides from Thelephora ganbajun TG-01.

This study demonstrates that Thelephora ganbajun had a strong ability to absorb zinc, and zinc can be compartmentally stored in the small vesicles and mainly accumulated in the form of zinc-enriched polysaccharides (zinc content was 25.0 ± 1.27 mg/g). Mycelia zinc polysaccharides (MZPS) and its fractions were isolated. The main fraction (MZPS-2) with the highest antioxidant activity in vitro was composed of mannose : galacturonic acid : glucose : galactose in a molar ratio of 61.19 : 1 : 39.67 : 48.67, with a weight-averaged molecular weight of 5.118 × 105 Da. MZPS-2 had both α-pyranose and ß-pyranose configuration and had a triple helical conformation. By establishing zebrafish models, we found that MZPS-2 can significantly scavenge free radicals, reduce the generation of reactive oxygen species caused by inflammation, and inhibit the recruitment of neutrophils toward the injury site. Therefore, MZPS-2 exhibited antioxidant and anti-inflammatory effects and can be used as a zinc supplement with specific biological activities to alleviate zinc deficiency complications, such as chronic oxidative stress or inflammation.

Isolation, Optimization of Fermentation Conditions, and Characterization of an Exopolysaccharide from Pseudoalteromonas agarivorans Hao 2018.

In recent years, the wide application of exopolysaccharides (EPSs) in food, cosmetics, medicine, and other fields has drawn tremendous attention. In this study, an EPS produced by Pseudoalteromonas agarivorans Hao 2018 was isolated and purified, and its fermentation conditions were optimized. Its structure and biological functions were also studied. The purity and molecular weight of EPS were determined by high performance liquid chromatography (HPLC), and the EPS exhibited a number average of 2.26 × 105 and a weight average of 2.84 × 105. EPS has good adsorption for Cu2+ and Pb2+. The adsorption rates can reach up to 69.79% and 82.46%, respectively. The hygroscopic property of EPS was higher than that of chitosan, but slightly lower than that of sodium hyaluronate. However, the water-retaining activity of EPS was similar to that of chitosan and sodium hyaluronate. EPS has strong ability to scavenge free radicals, including OH radical and O2- radical. Further, its activity on O2- radicals has similarities with that of vitamin C. EPS has broad application prospects in many fields, such as cosmetics, environmental protection.

Genome sequence and transcriptomic profiles of a marine bacterium, Pseudoalteromonas agarivorans Hao 2018.

Members of the marine genus Pseudoalteromonas have attracted great interest because of their ability to produce a large number of biologically active substances. Here, we report the complete genome sequence of Pseudoalteromonas agarivorans Hao 2018, a strain isolated from an abalone breeding environment, using second-generation Illumina and third-generation PacBio sequencing technologies. Illumina sequencing offers high quality and short reads, while PacBio technology generates long reads. The scaffolds of the two platforms were assembled to yield a complete genome sequence that included two circular chromosomes and one circular plasmid. Transcriptomic data for Pseudoalteromonas were not available. We therefore collected comprehensive RNA-seq data using Illumina sequencing technology from a fermentation culture of P. agarivorans Hao 2018. Researchers studying the evolution, environmental adaptations and biotechnological applications of Pseudoalteromonas may benefit from our genomic and transcriptomic data to analyze the function and expression of genes of interest.

Isolation, Characterization, and Pharmaceutical Applications of an Exopolysaccharide from Aerococcus Uriaeequi.

Many marine bacteria secrete exopolysaccharides (EPSs), which are made up of a substantial component of the macro-molecules surrounding cells. Recently, the wide demand for EPSs for food, cosmetics, pharmaceutical and other applications has led to great interest in them. In this study, an EPS produced by marine bacteria Aerococcus uriaeequi HZ strains (EPS-A) was isolated and purified to examine its structure and biological function. The molecular weight of EPS-A analyzed by high-performance liquid gel filtration chromatography (HPGFC) is found to have a number average of 2.22 × 105 and weight average of 2.84 × 105, respectively. High-performance liquid chromatography (HPLC) and Fourier-transform⁻infrared (FT⁻IR) analysis indicate that EPS-A was a polysaccharide composed of glucose and a little mannose. In addition, the flocculating rate of sewage of EPS-A was 79.90%. The hygroscopicity studies showed that hygroscopicity of EPS-A was higher than chitosan but lower than that of sodium hyaluronate. The moisture retention of EPS-A showed similar retention activity to both chitosan and sodium hyaluronate. EPS-A also can scavenge free radicals including both OH• free radical and O2•- free radical and the activity to O2•- free radical is similar to vitamin C. Safety assessment on mice indicated that the EPS-A is safe for external use and oral administration. EPS-A has great potential for applications in medicine due to its characteristics mentioned above.

New Insight Into the Diversity of SemiSWEET Sugar Transporters and the Homologs in Prokaryotes.

Sugars will eventually be exported transporters (SWEETs) and SemiSWEETs represent a family of sugar transporters in eukaryotes and prokaryotes, respectively. SWEETs contain seven transmembrane helices (TMHs), while SemiSWEETs contain three. The functions of SemiSWEETs are less studied. In this perspective article, we analyzed the diversity and conservation of SemiSWEETs and further proposed the possible functions. 1,922 SemiSWEET homologs were retrieved from the UniProt database, which is not proportional to the sequenced prokaryotic genomes. However, these proteins are very diverse in sequences and can be classified into 19 clusters when >50% sequence identity is required. Moreover, a gene context analysis indicated that several SemiSWEETs are located in the operons that are related to diverse carbohydrate metabolism. Several proteins with seven TMHs can be found in bacteria, and sequence alignment suggested that these proteins in bacteria may be formed by the duplication and fusion. Multiple sequence alignments showed that the amino acids for sugar translocation are still conserved and coevolved, although the sequences show diversity. Among them, the functions of a few amino acids are still not clear. These findings highlight the challenges that exist in SemiSWEETs and provide future researchers the foundation to explore these uncharted areas.

Enhanced anti-proliferative efficacy of epothilone B loaded with Escherichia coli Nissle 1917 bacterial ghosts on the HeLa cells by mitochondrial pathway of apoptosis.

Epothilones constitute a new class of microtubule-stabilizing anti-cancer agents with promising preclinical and clinical activity. However, its systemic application still causes some toxic side effects. To reduce these undesired effects, advanced drug delivery systems based on cell targeting carriers are needed currently. In this study, the high quality bacterial ghosts of the probiotic Escherichia coli Nissle 1917 (EcN) were prepared in a large scale and retained fully intact surface structures for specific attachment to mammalian cells. The EcN ghosts could be efficiently loaded with the low hydrophilic drug Epothilone B (Epo B) and the maximal load efficiency was approximately 2.5% (w/w). Cytotoxicity assays revealed that Epo B-ghosts exhibited enhanced anti-proliferative properties on the HeLa cells. The Epo B associated with EcN ghosts was more cytotoxic at least 10 times than the free Epo B at the same concentrations. Apoptosis assays showed that both Epo B-ghosts and free Epo B induced time course-dependent apoptosis and necrosis in HeLa cells, respectively. While the former induced more apoptosis and necrosis than the latter. Furthermore, the cytochrome C release and the activation of caspase-3 were more remarkable after treatment with the Epo B-ghosts compared to the free Epo B, which implied that Epo B-ghosts might more effectively induce the apoptosis mediated by mitochondrial pathway in HeLa cells. Therefore, the higher anti-proliferative effects of the Epo B-ghosts on the HeLa cells were mediated by mitochondrial pathway of apoptosis. The EcN ghosts may provide a useful drug delivery carrier for drug candidates in cancer therapy.

Molecular characterization of a thermostable aldehyde dehydrogenase (ALDH) from the hyperthermophilic archaeon Sulfolobus tokodaii strain 7.

Aldehyde dehydrogenase (ALDH) is a widely distributed enzyme in nature. Although many ALDHs have been reported until now, the detailed enzymatic properties of ALDH from Archaea remain elusive. Herein, we describe the characterization of an ALDH from the hyperthermophilic archaeon Sulfolobus tokodaii. The enzyme (stALDH) could utilize various aldehydes as substrates, and maximal activity was found with acetaldehyde and the coenzyme NAD. The optimal temperature and pH were 80 °C and 8, respectively, and high thermostability was found with the half-life at 90 °C to be 4 h. The enzyme was considerably resistant to nitroglycerin (GTN) inhibition, which could be restored by reducing agent DTT or (±)-α-lipoic acid. Coenzyme NAD or NADP could regulate the enzymatic thermostability, as well as the esterase activity. Molecular modeling suggested that the enzyme harbored similar structural arrangement with its eukaryotic and bacterial counterparts. Sequence alignment showed the conserved catalytic residues E240 and C274 and cofactor interactive sites N142, K165, I168 and E370, the function of which were verified by site-directed mutagenesis analysis. This is the most thermostable ALDH reported until now and the unique property of this enzyme is potentially beneficial in the fields of biotechnology and biomedicine.

Crystal structure of RIG-I C-terminal domain bound to blunt-ended double-strand RNA without 5' triphosphate.

RIG-I recognizes molecular patterns in viral RNA to regulate the induction of type I interferons. The C-terminal domain (CTD) of RIG-I exhibits high affinity for 5' triphosphate (ppp) dsRNA as well as blunt-ended dsRNA. Structures of RIG-I CTD bound to 5'-ppp dsRNA showed that RIG-I recognizes the termini of dsRNA and interacts with the ppp through electrostatic interactions. However, the structural basis for the recognition of non-phosphorylated dsRNA by RIG-I is not fully understood. Here, we show that RIG-I CTD binds blunt-ended dsRNA in a different orientation compared to 5' ppp dsRNA and interacts with both strands of the dsRNA. Overlapping sets of residues are involved in the recognition of blunt-ended dsRNA and 5' ppp dsRNA. Mutations at the RNA-binding surface affect RNA binding and signaling by RIG-I. These results provide the mechanistic basis for how RIG-I recognizes different RNA ligands.