The two-step strategy for enhancing the specific activity and thermostability of alginate lyase AlyG2 with mechanism for improved thermostability.

To overcome the trade-off challenge encountered in the engineering of alginate lyase AlyG2 from Seonamhaeicola algicola Gy8T and to expand its potential industrial applications, we devised a two-step strategy encompassing activity enhancement followed by thermal stability engineering. To enhance the specific activity of efficient AlyG2, we strategically substituted residues with bulky steric hindrance proximal to the active pocket with glycine or alanine. This led to the generation of three promising positive mutants, with particular emphasis on the T91S mutant, exhibiting a 1.91-fold specific activity compared to the wild type. To mitigate the poor thermal stability of T91S, mutants with negative ΔΔG values in the thermal flexibility region were screened out. Notably, the S72Ya mutant not only displayed 17.96 % further increase in specific activity but also exhibited improved stability compared to T91S, manifesting as a remarkable 30.97 % increase in relative activity following a 1-hour incubation at 42 °C. Furthermore, enhanced kinetic stability was observed. To gain deeper insights into the mechanism underlying the enhanced thermostability of the S72Ya mutant, we conducted molecular dynamics simulations, principal component analysis (PCA), dynamic cross-correlation map (DCCM), and free energy landscape (FEL) analysis. The results unveiled a reduction in the flexibility of the surface loop, a stronger correlation dynamic and a narrower motion subspace in S72Ya system, along with the formation of more stable hydrogen bonds. Collectively, our findings suggest amino acids substitutions resulting in smaller side chains proximate to the active site can positively impact enzyme activity, while reducing the flexibility of surface loops emerges as a pivotal factor in conferring thermal stability. These insights offer valuable guidance and a framework for the engineering of other enzyme types.

Silver Nanoparticles Encapped by Dihydromyricetin: Optimization of Green Synthesis, Characterization, Toxicity, and Anti-MRSA Infection Activities for Zebrafish (Danio rerio).

To achieve the environmentally friendly and rapid green synthesis of efficient and stable AgNPs for drug-resistant bacterial infection, this study optimized the green synthesis process of silver nanoparticles (AgNPs) using Dihydromyricetin (DMY). Then, we assessed the impact of AgNPs on zebrafish embryo development, as well as their therapeutic efficacy on zebrafish infected with Methicillin-resistant Staphylococcus aureus (MRSA). Transmission electron microscopy (TEM) and dynamic light-scattering (DLS) analyses revealed that AgNPs possessed an average size of 23.6 nm, a polymer dispersity index (PDI) of 0.197 ± 0.0196, and a zeta potential of -18.1 ± 1.18 mV. Compared to other published green synthesis products, the optimized DMY-AgNPs exhibited smaller sizes, narrower size distributions, and enhanced stability. Furthermore, the minimum concentration of DMY-AgNPs required to affect zebrafish hatching and survival was determined to be 25.0 µg/mL, indicating the low toxicity of DMY-AgNPs. Following a 5-day feeding regimen with DMY-AgNP-containing food, significant improvements were observed in the recovery of the gills, intestines, and livers in MRSA-infected zebrafish. These results suggested that optimized DMY-AgNPs hold promise for application in aquacultures and offer potential for further clinical use against drug-resistant bacteria.

Insights into the Influence of Signal Peptide on the Enzymatic Properties of Alginate Lyase AlyI1 with Removal Effect on Pseudomonas aeruginosa Biofilm.

Most reports on signal peptides focus on their ability to affect the normal folding of proteins, thereby affecting their secreted expression, while few studies on its effects on enzymatic properties were published. Therefore, biochemical characterization and comparison of alginate lyase rALYI1/rALYI1-1 (rALYI1: without signal peptides; rALYI1-1:with signal peptides) were conducted in our study, and the results showed that the signal peptide affected the biochemical properties, especially in temperature and pH. rALYI1 (32.15 kDa) belonging to polysaccharide lyase family 7 was cloned from sea-cucumber-gut bacterium Tamlana sp. I1. The optimum temperature of both rALYI1 and rALYI1-1 was 40 °C, but the former had a wider optimum temperature range and better thermal stability. The optimum pH of rALYI1 and rALYI1-1 were 7.6 and 8.6, respectively. The former was more stable and acid resistant. Noticeably, rALYI1 was a salt-activated enzyme and displayed remarkable salt tolerance. Alginate, an essential polysaccharide in algae and Pseudomonas aeruginosa biofilms, is composed of α-L-guluronate and ß-D-mannuronate. It is also found in our study that rALYI1 is also effective in removing mature biofilms compared with controls. In conclusion, the signal peptide affects several biochemical properties of the enzyme, and alginate lyase rALYI1 may be an effective method for inhibiting biofilm formation of Pseudomonas aeruginosa.

Jeotgalibacillus aurantiacus sp. nov., a novel orange-pigmented species with a carotenoid biosynthetic gene cluster, isolated from wetland soil.

A Gram-stain-positive, orange-pigmented, rod-shaped and flagellated bacterial strain T12T was isolated from wetland soil in Kunyu Mountain Wetland in Yantai, China. The strain was able to grow at 15-40 °C (optimum 37 °C), at 0.0-9.0% NaCl (optimum 2%, w/v) and at pH 5.5-9.0 (optimum 8.5). A phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain T12T is a member of the family Planococcaceae, sharing 97.6% and 97.1% sequence similarity with the type strains of Jeotgalibacillus salarius and Jeotgalibacillus marinus, respectively. Genome-based analyses revealed a genome size of 3,506,682 bp and a DNA G + C content of 43.7%. Besides, the genome sequence led to 55.0-74.6% average amino acid identity values and 67.8-74.7% average nucleotide identity values between strain T12T and the current closest relatives. Digital DNA-DNA hybridization of strain T12T with the type strains of Jeotgalibacillus proteolyticus and J. marinus demonstrated 19.0% and 20.3% relatedness, respectively. The chemotaxonomic analysis showed that the sole quinone was MK-7. The predominant cellular fatty acids were iso-C15:0, anteiso-C15:0, C16:1ω7c alcohol and iso-C14:0. The polar lipids consisted of an unidentified aminolipid, phosphatidylglycerol, diphosphatidylglycerol and two unidentified phospholipids. Based on the polyphasic characterization, strain T12T is considered to represent a novel species, for which the name Jeotgalibacillus aurantiacus sp. nov. is proposed. The type strain is T12T (= KCTC 43296 T = MCCC 1K07171T).

Kangiella shandongensis sp. nov., a novel species isolated from saltern in Yantai, China.

A Gram-stain-negative, wheat, rod-shaped, non-motile, non-spore forming, and facultatively anaerobic bacterium strain, designated as PIT, was isolated from saline silt samples collected in saltern in Yantai, Shandong, China. Growth was observed within the ranges 4-45 °C (optimally at 33 °C), pH 6.0-9.0 (optimally at pH 7.0) and 1.0-11.0% NaCl (optimally at 3.0%, w/v). Strain PIT showed highest 16S rRNA gene sequence similarity to Kangiella sediminilitoris BB-Mw22T (98.3%) and Kangiella taiwanensis KT1T (98.3%). The major cellular fatty acids (> 10% of the total fatty acids) were iso-C15:0 (52.7%) and summed featured 9 (iso-C17:1ω9c/C16:0 10-methyl, 11.8%). The major polar lipids identified were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine and phosphatidylglycerol. The major respiratory isoprenoid quinone was Q-8. The G + C content of the genomic DNA was 45.8%. Average Nucleotide Identity values between whole genome sequences of strain PIT and next related type strains supported the novel species status. Based on physiological, biochemical, chemotaxonomic characteristics and genomic analysis, strain PIT is considered to represent a novel species within the genus Kangiella, for which the name Kangiella shandongensis sp. nov. is proposed. The type strain is PIT (= KCTC 82509 T = MCCC 1K04352T).

Planococcus maritimu ML1206 Strain Enhances Stress Resistance and Extends the Lifespan in Caenorhabditis elegans via FOXO/DAF-16.

The antioxidant effect of probiotics has been widely recognized across the world, which is of great significance in food, medicine, and aquaculture. There are abundant marine microbial resources in the ocean, which provide a new space for humans to explore new probiotics. Previously, we reported on the anti-infective effects of Planococcus maritimu ML1206, a potential marine probiotic. The antioxidant activity of ML1206 in C. elegans was studied in this paper. The study showed that ML1206 could improve the ability of nematodes to resist oxidative stress and effectively prolong their lifespan. The results confirmed that ML1206 could significantly increase the activities of CAT and GSH-PX, and reduce the accumulation of reactive oxygen species (ROS) in nematodes under oxidative stress conditions. In addition, ML1206 promoted DAF-16 transfer to the nucleus and upregulated the expression of sod-3, hsp-16.2, and ctl-2, which are downstream antioxidant-related genes of DAF-16. Furthermore, the expression of the SOD-3::GFP and HSP-16.2::GFP was significantly higher in the transgenic strains fed with ML1206 than that in the control group fed with OP50, with or without stress. In summary, these findings suggest that ML1206 is a novel marine probiotic with an antioxidant function that stimulates nematodes to improve their defense abilities against oxidative stress and prolong the lifespan by regulating the translocation of FOXO/DAF-16. Therefore, ML1206 may be explored as a potential dietary supplement in aquaculture and for anti-aging and antioxidant purposes.

Pseudaestuariivita rosea sp. nov., isolated from Acmaea sp., a marine mollusk.

A Gram-stain-negative, pink-pigmented, aerobic, non-motile and rod-shaped bacterium, designated as strain H15T, was isolated from Acmaea sp., collected from Weihai, Shandong Province, China. The novel isolate was able to grow at 4-37 °C (optimum 33 °C), pH 5.5-9.0 (optimum 7.0) and with 0.0-7.0% NaCl (optimum 4%, w/v). Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that the strain belonged to the family Rhodobacteraceae and was associated to the type strain of Pseudaestuariivita atlantica (96.7%). Genome analysis showed that the genome size was 3,893,398 bp and the DNA G + C content obtained from the draft genome sequence was 56.7%. The secondary metabolites predicated that the strain H15T contained one cluster of lasso peptide, one cluster of bacteriocin, two clusters of terpene production, two clusters of homoserine lactone and one cluster of beta lactone. The average amino acid identity, average nucleotide identity and digital DNA-DNA hybridization values between genome sequences of strain H15T and all the related strains compared were lower than 63.1, 72.0 and 19.7%, respectively. Based on the analysis of chemical components, the predominant cellular fatty acids were summed featured 8 (C18:1ω7c/ω6c, 46.1%), C20:1 ω7c (17.1%), the major polar lipids contained phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and an unidentified lipid and the predominant menaquinone was Q10. Therefore, the combined chemotaxonomic, phenotypic and phylogenetic data indicated that the strain was considered to represent a novel species of the genus Pseudaestuariivita and the name Pseudaestuariivita rosea sp. nov. was proposed for strain H15T (MCCC 1K04420T = KCTC 82505T).

Williamsia soli sp. nov., an actinobacterium isolated from soil at a thermal power plant in Yantai, China.

Strain C17T, a novel strain belonging to the phylum Actinobacteria, was isolated from a thermal power plant in Yantai, Shandong Province, China. Cells of strain C17T were Gram stain positive, aerobic, pink, non-motile and round with neat edges, showing optimum growth at 28 °C. Phylogenetically, strain C17T was a member of the class Actinobacteria, order Mycobacteriales, family Gordoniaceae. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that the related strains were Williamsia faeni JCM 17784 T and Williamsia limnetica KCTC 19981 T with pairwise sequence similarity of 98.5% for both strains. According to the draft genome sequence, the DNA G + C content was 64.7%. The average amino acid identity (AAI), average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between genome sequences of strain C17T and the closest type strain W. faeni JCM 17784 T were 77.5, 77.9, and 20.7%, respectively. Predominant fatty acids were C16:0 (31.7%) and C18:1ω9c (26.8%). The major menaquinone was MK-9. The diagnostic phospholipids were phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), and phosphatidylinositol (PI). Therefore, the combined phenotypic, chemotaxonomic and phylogenetic data indicated that strain C17T was considered to represent a novel species of the genus Williamsia. Williamsia soli sp. nov. was proposed for strain C17T (= KCTC 49567 T = MCCC 1K04355T).

Structure Characteristics, Biochemical Properties, and Pharmaceutical Applications of Alginate Lyases.

Alginate, the most abundant polysaccharides of brown algae, consists of various proportions of uronic acid epimers α-L-guluronic acid (G) and ß-D-mannuronic acid (M). Alginate oligosaccharides (AOs), the degradation products of alginates, exhibit excellent bioactivities and a great potential for broad applications in pharmaceutical fields. Alginate lyases can degrade alginate to functional AOs with unsaturated bonds or monosaccharides, which can facilitate the biorefinery of brown algae. On account of the increasing applications of AOs and biorefinery of brown algae, there is a scientific need to explore the important aspects of alginate lyase, such as catalytic mechanism, structure, and property. This review covers fundamental aspects and recent developments in basic information, structural characteristics, the structure-substrate specificity or catalytic efficiency relationship, property, molecular modification, and applications. To meet the needs of biorefinery systems of a broad array of biochemical products, alginate lyases with special properties, such as salt-activated, wide pH adaptation range, and cold adaptation are outlined. Withal, various challenges in alginate lyase research are traced out, and future directions, specifically on the molecular biology part of alginate lyases, are delineated to further widen the horizon of these exceptional alginate lyases.

Echinicola salinicaeni sp. nov., a novel bacterium isolated from saltern mud.

A novel gram-negative, aerobic, pink, motile, gliding, rod-shaped bacterium, designated P51T, was isolated from saline silt samples in Yantai, China. It was able to grow at 4-42 °C (optimum 33 °C), pH 4.0-9.0 (optimum 7.0), and in 0-11.0% NaCl (optimum 4.0%, w/v). It grew at 4 °C, which was lower than the minimum temperature for related strains. The genome consisted of 4111 genes with a total length of 5 139 782 bp. The 16S rRNA gene sequence analysis indicated that strain P51T was a member of the genus Echinicola and most closely related to 'Echinicola shivajiensis'. A genome analysis identified genes encoding proteins associated with carbon source utilisation, and the carotenoid biosynthesis and ß-lactam resistance pathways. Strain P51T shared an average nucleotide identity value below 84.7%, an average amino acid identity value between 70.8 and 89.3%, and a digital DNA-DNA hybridisation identity of between 17.9-28.2% with closely related type strains within the genus Echinicola. The sole menaquinone was MK-7, and the major fatty acids were iso-C15:0, summed feature 3 (C16:1ω7c and/or C16:1ω6c), summed feature 4 (anteiso-C17:1 B and/or iso-C17:1 I), and summed feature 9 (iso-C17:1ω9c and/or 10-methyl C16:0). The polar lipids included one phosphatidylethanolamine, one unidentified aminophospholipid, one unidentified phospholipid, three unidentified aminolipids, and one unknown lipid. The phenotypic, chemotaxonomic, and phylogenetic analyses suggest that strain P51T is a novel species of the genus Echinicola, for which the name Echinicola salinicaeni sp. nov. is proposed. The type strain was P51T (KCTC 82513T = MCCC 1K04413T).

Oceaniglobus trochenteri sp. nov., isolated from the gut microflora of top shell (Trochus maculatus Linnaeus).

A Gram-stain negative, non-flagellated, beige-pigmented, circular, catalase-positive, oxidase-positive bacterium, designated G4T, was isolated from gut microflora of top shell (Trochus maculatus Linnaeus) collected from Diwanggong market, Weihai, People's Republic of China. The novel isolate was able to grow at 4-42 °C (optimum 25-33 °C), pH 7.0-9.0 (optimum 6.5-7.0) and with 0.0-11.0% NaCl (optimum 2.0-3.0%, w/v). Analysis of 16S rRNA gene sequence revealed that strain G4T shared the highest 16S rRNA gene sequence similarities with Oceaniglobus ichthyenteri YLY08T (96.6%), followed by Oceaniglobus indicus 1-19bT (95.3%). The genome of strain G4T, with 32 assembled contigs, was 4.5 Mb long with a G+C content of 65.3 mol%. DNA-DNA hybridization values of the isolate against the closely related type strains were far below the 70% limit for species delineation. The average amino acid identity, average nucleotide identity and digital DNA-DNA genome hybridization relatedness between strain G4T and the closely related members of the genus Oceaniglobus, Oceaniglobus indicus1-19bT and Oceaniglobus ichthyenteri YLY08T were 71.3, 76.4 and 20.0%, and 75.0, 76.3 and 19.4%. The major cellular fatty acid was summed feature 8 (C18:1ω7c and/or C18:1ω6c). The sole respiratory quinone was Q-10. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidyldimethylethanolamine. The results of phenotypical, phylogenetic and biochemical analyses indicated that strain G4T represents a novel species in genus Oceaniglobus within the family Rhodobacteraceae, for which the name Oceaniglobus trochenteri sp. nov. is proposed. The type strain is G4T (= MCCC 1K04356T = KCTC 82506T).

Thirdhand cigarette smoke leads to age-dependent and persistent alterations in the cecal microbiome of mice.

The gut microbiome composition is influenced by many factors including environmental exposures. Here, we investigated the effect of thirdhand cigarette smoke (THS) and exposure age on gut microbiome diversity. C57BL/6 mice were exposed to THS at human exposure relevant levels for three weeks during three different life stages: postnatal (0-3 weeks of age), pubescent (4-7 weeks of age), and adult (9-12 weeks of age), respectively. Cecal microbiome profiles were assessed at 17 weeks of age by 16S rRNA gene sequencing. We found that age at THS exposure strongly influenced the cecal microbiome composition. Although postnatal THS exposure significantly influenced the microbial composition, pubescent and adulthood exposures only had minor effects. The microbiome of postnatally THS-exposed mice significantly increased several degradation pathways that regulate glycolysis and pyruvate decarboxylation, and significantly decreased coenzyme A biosynthesis and pyrimidine deoxyribonucleoside salvage. Our results indicate that mouse postnatal development is particularly susceptible to persistent THS exposure effects on the gut microbiome.

Planococcus maritimus ML1206 Isolated from Wild Oysters Enhances the Survival of Caenorhabditis elegans against Vibrio anguillarum.

With the widespread occurrence of aquaculture diseases and the broad application of antibiotics, drug-resistant pathogens have increasingly affected aquatic animals' health. Marine probiotics, which live under high pressure in a saltwater environment, show high potential as a substitute for antibiotics in the field of aquatic disease control. In this study, twenty strains of non-hemolytic bacteria were isolated from the intestine of wild oysters and perch, and a model of Caenorhabditis elegans infected by Vibrio anguillarum was established. Based on the model, ML1206, which showed a 99% similarity of 16S rRNA sequence to Planococcus maritimus, was selected as a potential marine probiotic, with strong antibacterial capabilities and great acid and bile salt tolerance, to protect Caenorhabditis elegans from being damaged by Vibrio anguillarum. Combined with plate counting and transmission electron microscopy, it was found that strain ML1206 could significantly inhibit Vibrio anguillarum colonization in the intestinal tract of Caenorhabditis elegans. Acute oral toxicity tests in mice showed that ML1206 was safe and non-toxic. The real-time qPCR results showed a higher expression level of genes related to the antibacterial peptide (ilys-3) and detoxification (ugt-22, cyp-35A3, and cyp-14A3) in the group of Caenorhabditis elegans protected by ML1206 compared to the control group. It is speculated that ML1206, as a potential probiotic, may inhibit the infection caused by Vibrio anguillarum through stimulating Caenorhabditis elegans to secrete antibacterial effectors and detoxification proteins. This paper provides a new direction for screening marine probiotics and an experimental basis to support the potential application of ML1206 as a marine probiotic in aquaculture.

Characterization of a New Biofunctional, Exolytic Alginate Lyase from Tamlana sp. s12 with High Catalytic Activity and Cold-Adapted Features.

Alginate, a major acidic polysaccharide in brown algae, has attracted great attention as a promising carbon source for biorefinery systems. Alginate lyases, especially exo-type alginate lyase, play a critical role in the biorefinery process. Although a large number of alginate lyases have been characterized, few can efficiently degrade alginate comprised of mannuronate (M) and guluronate (G) at low temperatures by means of an exolytic mode. In this study, the gene of a new exo-alginate lyase-Alys1-with high activity (1350 U/mg) was cloned from a marine strain, Tamlana sp. s12. When sodium alginate was used as a substrate, the recombinant enzyme showed optimal activity at 35 °C and pH 7.0-8.0. Noticeably, recombinant Alys1 was unstable at temperatures above 30 °C and had a low melting temperature of 56.0 °C. SDS and EDTA significantly inhibit its activity. These data indicate that Alys1 is a cold-adapted enzyme. Moreover, the enzyme can depolymerize alginates polyM and polyG, and produce a monosaccharide as the minimal alginate oligosaccharide. Primary substrate preference tests and identification of the final oligosaccharide products demonstrated that Alys1 is a bifunctional alginate lyase and prefers M to G. These properties make Alys1 a valuable candidate in both basic research and industrial applications.

Correction to: Genetic and metabolic links between the murine microbiome and memory.

An amendment to this paper has been published and can be accessed via the original article.

Genetic and metabolic links between the murine microbiome and memory.

BACKGROUND: Recent evidence has linked the gut microbiome to host behavior via the gut-brain axis [1-3]; however, the underlying mechanisms remain unexplored. Here, we determined the links between host genetics, the gut microbiome and memory using the genetically defined Collaborative Cross (CC) mouse cohort, complemented with microbiome and metabolomic analyses in conventional and germ-free (GF) mice. RESULTS: A genome-wide association analysis (GWAS) identified 715 of 76,080 single-nucleotide polymorphisms (SNPs) that were significantly associated with short-term memory using the passive avoidance model. The identified SNPs were enriched in genes known to be involved in learning and memory functions. By 16S rRNA gene sequencing of the gut microbial community in the same CC cohort, we identified specific microorganisms that were significantly correlated with longer latencies in our retention test, including a positive correlation with Lactobacillus. Inoculation of GF mice with individual species of Lactobacillus (L. reuteri F275, L. plantarum BDGP2 or L. brevis BDGP6) resulted in significantly improved memory compared to uninoculated or E. coli DH10B inoculated controls. Untargeted metabolomics analysis revealed significantly higher levels of several metabolites, including lactate, in the stools of Lactobacillus-colonized mice, when compared to GF control mice. Moreover, we demonstrate that dietary lactate treatment alone boosted memory in conventional mice. Mechanistically, we show that both inoculation with Lactobacillus or lactate treatment significantly increased the levels of the neurotransmitter, gamma-aminobutyric acid (GABA), in the hippocampus of the mice. CONCLUSION: Together, this study provides new evidence for a link between Lactobacillus and memory and our results open possible new avenues for treating memory impairment disorders using specific gut microbial inoculants and/or metabolites. Video Abstract.

Prenylated Phenolic Compounds from the Aerial Parts of Glycyrrhiza uralensis as PTP1B and α-Glucosidase Inhibitors.

Glycyrrhiza uralensis (liquorice) is a well-known medicinal plant. Its roots and rhizomes are used as the popular Chinese herbal medicine Gan-Cao. An ethanol extract of the aerial parts of G. uralensis showed antidiabetic effects on db/db mice. It decreased the blood glucose level by 30.3% and increased the serum insulin level by 41.8% compared to the control group. Eighty-six phenolic compounds (1-86) were obtained from the aerial parts, including the new prenylated isoflavanones (1-5), isoflavans (6-9), and a 2-phenylbenzofuran (10). The structures were identified by NMR and HRESIMS data analyses, and the absolute configurations were established by comparing the calculated and experimental ECD spectroscopic data. Compounds 2, 6, and 10 inhibited PTP1B with IC50 values of 5.9, 6.7, and 5.3 µM, respectively. Compound 2 and the known compounds glycycoumarin (76) and glyurallin A (79) inhibited α-glucosidase with IC50 values of 20.1, 0.1, and 0.3 µM, respectively. Compound 4 at 10 µM increased the glucose uptake rate to 95% in an insulin resistance HepG2 cell model (p < 0.01).

Diverse tumour susceptibility in Collaborative Cross mice: identification of a new mouse model for human gastric tumourigenesis.

OBJECTIVE: The Collaborative Cross (CC) is a mouse population model with diverse and reproducible genetic backgrounds used to identify novel disease models and genes that contribute to human disease. Since spontaneous tumour susceptibility in CC mice remains unexplored, we assessed tumour incidence and spectrum. DESIGN: We monitored 293 mice from 18 CC strains for tumour development. Genetic association analysis and RNA sequencing were used to identify susceptibility loci and candidate genes. We analysed genomes of patients with gastric cancer to evaluate the relevance of genes identified in the CC mouse model and measured the expression levels of ISG15 by immunohistochemical staining using a gastric adenocarcinoma tissue microarray. Association of gene expression with overall survival (OS) was assessed by Kaplan-Meier analysis. RESULTS: CC mice displayed a wide range in the incidence and types of spontaneous tumours. More than 40% of CC036 mice developed gastric tumours within 1 year. Genetic association analysis identified Nfκb1 as a candidate susceptibility gene, while RNA sequencing analysis of non-tumour gastric tissues from CC036 mice showed significantly higher expression of inflammatory response genes. In human gastric cancers, the majority of human orthologues of the 166 mouse genes were preferentially altered by amplification or deletion and were significantly associated with OS. Higher expression of the CC036 inflammatory response gene signature is associated with poor OS. Finally, ISG15 protein is elevated in gastric adenocarcinomas and correlated with shortened patient OS. CONCLUSIONS: CC strains exhibit tremendous variation in tumour susceptibility, and we present CC036 as a spontaneous laboratory mouse model for studying human gastric tumourigenesis.

No difference in 4-nitroquinoline induced tumorigenesis between germ-free and colonized mice.

Variations in oral bacterial communities have been linked to oral cancer suggesting that the oral microbiome is an etiological factor that can influence oral cancer development. The 4-nitroquinoline 1-oxide (4-NQO)-induced murine oral and esophageal cancer model is frequently used to assess the effects of preventive and/or therapeutic agents. We used this model to assess the impact of the microbiome on tumorigenesis using axenic (germ-free) and conventionally housed mice. Increased toxicity was observed in germ-free mice, however, no difference in tumor incidence, multiplicity, and size was observed. Transcriptional profiling of liver tissue from germ-free and conventionally housed mice identified 254 differentially expressed genes including ten cytochrome p450 enzymes, the largest family of phase-1 drug metabolizing enzymes in the liver. Gene ontology revealed that differentially expressed genes were enriched for liver steatosis, inflammation, and oxidative stress in livers of germ-free mice. Our observations emphasize the importance of the microbiome in mediating chemical toxicity at least in part by altering host gene expression. Studies on the role of the microbiome in chemical-induced cancer using germ-free animal models should consider the potential difference in dose due to the microbiome-mediated changes in host metabolizing capacity, which might influence the ability to draw conclusions especially for tumor induction models that are dose dependent.

Quantitative proteomics reveals mitochondrial respiratory chain as a dominant target for carbon ion radiation: Delayed reactive oxygen species generation caused DNA damage.

Heavy ion radiotherapy has shown great promise for cancer therapy. Understanding the cellular response mechanism to heavy ion radiation is required to explore measures of overcoming devastating side effects. Here, we performed a quantitative proteomic analysis to investigate the mechanism of carbon ion irradiation on human AHH-1 lymphoblastoid cells. We identified 4602 proteins and quantified 4569 proteins showing high coverage in the mitochondria. Data are available via ProteomeXchange with identifier PXD008351. After stringent filtering, 290 proteins were found to be significantly up-regulated and 16 proteins were down-regulated. Functional analysis revealed that these up-regulated proteins were enriched in the process of DNA damage repair, mitochondrial ribosome, and particularly mitochondrial respiratory chain, accounting for approximately 50% of the accumulated proteins. Bioinformatics and functional analysis demonstrated that these up-regulated mitochondrial respiratory chain proteins enhanced ATP production and simultaneously reactive oxygen species release. More importantly, increased reactive oxygen species led to secondary organelle injury and lagged DNA double-strand breaks. Consistently, the expression of antioxidant enzymes was up-regulated for free radical scavenging. The mechanism of lagged secondary injury originated from disturbances in the mitochondrial respiratory chain. Our results provided a novel target for cell self-repair against heavy ion radiation-induced cellular damage.