Tear film instability is associated with weakened colocalization between occludin and MUC5AC in scopolamine-induced dry eye disease (DED) rats.

PURPOSE: Dry eye disease (DED) is a disease with tear film instability because of multiple factors. This study was conducted to explore roles of occludin and MUC5AC in tear film instability in DED rat model. METHODS: A total of 20 SD rats were divided into DED group (n = 10) and normal control (NC) group (n = 10). DED rat model was established by subcutaneously injecting with scopolamine hydrobromide. Clinical examinations, including tear breakup time (tBUT), Schirmer's test and corneal fluorescein staining, were conducted to determine corneal functions. Transmission electron microscopy was used to measure the ultrastructures of corneal epithelial cells. Western blotting assay was used to identify occludin expression in corneal tissues of DED rats. Real-time PCR (RT-PCR) was performed to verify gene transcription of occludin and MUC5AC. Colocalization between occludin and MUC5AC was identified with confocal fluorescence microscopy. RESULTS: Tear breakup time was significantly shorter, and corneal fluorescein staining score was predominantly higher in DED rats compared to those in normal rats (P < 0.05). Normal rats showed a steady tear secretion throughout the whole experiments, while DED rats showed a dramatic reduction on day 14. DED rats demonstrated ultrastructural damage of Golgi apparatus and endoplasmic reticulum in corneal epithelial cells. Occludin and MUC5AC expressions were significantly downregulated in corneal tissue of DED rats compared with those of normal rats (P < 0.05). Percentage of occludin-MUC5AC-colocalized corneal epithelial cells in DED rats was significantly less compared with those in normal rats (P < 0.01). CONCLUSIONS: Tear film stability was damaged in scopolamine-induced DED rats because of the weakened colocalization between occludin and MUC5AC molecule. This study would provide a potential clue for the pathogenesis and a promising theoretical basis for clinical work of DED.

Corallococcus silvisoli sp. nov., a novel myxobacterium isolated from subtropical forest soil.

An orange-pigmented myxobacterium, designated strain c25j21T, was isolated from subtropical forest soil collected from the Chebaling National Nature Reserve in Guangdong Province, China. Phylogenetic analysis based on the 16S rRNA gene and core genes clearly showed that the novel strain was affiliated within the genus Corallococcus and most closely related to Corallococcus aberystwythensis DSM 108846T (99.3% 16S rRNA gene sequence similarity), while C. exercitus DSM 108849T (99.2%) and C. carmarthensis DSM 108842T (99.0%) were the next most closely related type strains. The draft genome sequence of strain c25j21T was 9.23 Mb in length with a G + C content of 70.7 mol%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain c25j21T and its closely related type strains were 88.1-89.1 and 34.1-36.3%, respectively. The major fatty acids contained iso-C15:0, iso-C17:0, iso-C17:1ω5c and iso-C17:0 2-OH. The predominant respiratory quinone was menaquinone 7. Based on phylogenetic, phenotypic and chemotaxonomic analysis, strain c25j21T represents a novel species of the genus Corallococcus, for which the name Corallococcus silvisoli sp. nov. is proposed. The type strain is c25j21T (= GDMCC 1.1387T = KCTC 62437T).

Chitinophaga rhizophila sp. nov., isolated from rhizosphere soil of banana.

A Gram-stain-negative, aerobic and rod-shaped bacterium, designated as strain B61T, was isolated from rhizosphere soil of banana collected from Dongguan, Guangdong Province, PR China. Growth occurred at 15-40 °C, within a pH range of pH 6.0-9.0. Results of 16S rRNA gene sequence similarity and phylogenetic analyses showed that strain B61T was most closely related to 'Chitinophaga agri' KACC 21303 (98.9 %) and Chitinophaga pinensis DSM 2588T (98.8 %). The genome size was 7.6 Mb with a G+C content of 45.2 mol%. The genome-inferred average nucleotide identity values between strain B61T and two closely related strains were 79.2 and 79.3 %, respectively, with corresponding digital DNA-DNA hybridization values of 22.3 and 22.6 %. The major fatty acids of the novel strain were iso-C15:0, C16:1 ω5c and iso-C17:0 3-OH and the sole respiratory quinone was menaquinone 7 (MK-7). The polar lipids consisted of phosphatidylethanolamine, five unidentified aminolipids, four unidentified glycolipids and six unidentified lipids. The phenotypic and phylogenetic results clearly supported that strain B61T represents a novel species of the genus Chitinophaga, for which the name Chitinophaga rhizophila, sp. nov. is proposed, with the type strain B61T (=GDMCC 1.2608T=KCTC 82856T).

Chitinophaga tropicalis sp. nov., isolated from forest soil.

A novel bacterial strain, designated ysch24T, was isolated from a forest soil sample collected from the Cat Tien National Park, southern Vietnam. Cells were Gram-stain-negative, aerobic, gliding, filamentous or rod-shaped. The results of 16S rRNA gene analyses revealed that strain ysch24T belongs to the genus Chitinophaga, and was most closely related to Chitinophaga silvisoli GDMCC 1.1411T (97.4 %), followed by Chitinophaga oryziterrae JCM 16595T (97.3 %) and Chitinophaga sancti NBRC 15057T (96.9 %). The average nucleotide identity and digital DNA-DNA hybridization values between strain ysch24T and closely related type strains were 72.0-74.0 % and 19.1-19.4 %, respectively. Major fatty acids were iso-C15 : 0, C16 : 1 ω5c and iso-C17 : 0 3-OH and the predominant respiratory quinone was menaquinone 7. Polar lipids consisted of phosphatidylethanolamine, four unidentified aminophospholipids, two unidentified phospholipids and four unidentified lipids. The genomic DNA G+C content was 45.6 mol%. The study clearly showed that strain ysch24T should represent a novel species of the genus Chitinophaga, for which the name Chitinophaga tropicalis sp. nov. is proposed. The type strain is ysch24T (=GDMCC 1.1355T=KACC 21527T).

Methylobacterium nonmethylotrophicum sp. nov., isolated from tungsten mine tailing.

A novel pink-pigmented strain, designated 6HR-1T, was isolated from tungsten mine tailings in Jiangxi Province, PR China. Cells were Gram-stain-negative, aerobic, non-spore-forming, rod-shaped and motile with a polar flagellum (monotrichous). It could not utilize methanol, methylamine, formaldehyde or formate as a sole carbon source. The methanol dehydrogenase mxaF gene was absent but the xoxF gene was present. Phylogenomic and 16S rRNA gene phylogenetic analyses clearly showed that strain 6HR-1T was affiliated to the genus Methylobacterium and closely related to 'Methylobacterium terrae' 17Sr1-28T (98.6 %), Methylobacterium platani JCM 14648T (97.7 %), Methylobacterium variabile DSM 16961T (97.7 %) and Methylobacterium currus KACC 19662T (97.4 %). The average nucleotide identity and digital DNA-DNA hybridization values between strain 6HR-1T and its closely related type species were 87.4-88.7 and 33.2-36.3 %, respectively. It had summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) as the major fatty acid and ubiquinone 10 as the predominant respiratory quinone. Polyphasic characterization supported that strain 6HR-1T represents a novel species of the genus Methylobacterium, for which the name Methylobacterium nonmethylotrophicum sp. nov. is proposed with the type strain 6HR-1T (=GDMCC 1.662T=KCTC 42760T).

Transfer of Sphingorhabdus marina, Sphingorhabdus litoris, Sphingorhabdus flavimaris and Sphingorhabdus pacifica corrig. into the novel genus Parasphingorhabdus gen. nov. and Sphingopyxis baekryungensis into the novel genus Novosphingopyxis gen. nov. within the family Sphingomonadaceae.

During a phylogenetic analysis of Sphingorhabdus and its closely related genera in the family Sphingomonadaceae, we found that the genus Sphingorhabdus and the species Sphingopyxis baekryungensis might not be properly assigned in the taxonomy. Phylogenetic, phenotypic and chemotaxonomic characterizations clearly showed that the genus Sphingorhabdus should be reclassified into two genera (Clade I and Clade II), for which the original genus name, Sphingorhabdus, is proposed to be retained only for Clade I, and a new genus named as Parasphingorhabdus gen. nov. is proposed for Clade II with four new combinations: Parasphingorhabdus marina comb. nov., Parasphingorhabdus litoris comb. nov., Parasphingorhabdus flavimaris comb. nov. and Parasphingorhabdus pacifica comb. nov. Moreover, Sphingopyxis baekryungensis should represent a novel genus in the family Sphingomonadaceae, for which the name Novosphingopyxis gen. nov. is proposed, with a combination of Novosphingopyxis baekryungensis comb. nov. The study provides a new insight into the taxonomy of closely related genera in the family Sphingomonadaceae.

Sphingobium paulinellae and Sphingobium algicola Lee and Jeon 2017 are two later heterotypic synonyms of Sphingobium limneticum Chen et al. 2013 and emended description of the species.

Phylogenetic analysis of the genus Sphingobium had shown that the type strains of Sphingobium paulinellae, Sphingobium algicola and Sphingobium limneticum shared a very close relationship between each other. The 16S rRNA gene sequences similarity values between each other ranged from 99.65 to 99.93 %. Whole genome sequencing was performed and genomic relatedness values between each pair of the species were 97.49-100 % (ANI) and 79.3-100 % (dDDH), respectively, all higher than the threshold values of 95-96 % ANI and 70 % dDDH suggested for species discrimination, and implicated that the type strains should belong to the same species of the genus Sphingobium. The phenotypic and chemotaxonomic characterizations performed in the original descriptions of S. paulinellae and S. algicola also supported the same conclusion. Due to priority of publication Sphingobium paulinellae and Sphingobium algicola Lee and Jeon 2017, should be taken as two later heterotypic synonyms of Sphingobium limneticum Chen et al. 2013. Correspondingly, the species description of Sphingobium limneticum was emended based on this study.

Ramlibacter humi sp. nov., isolated from tropical forest soil.

A Gram-stain-negative, aerobic and non-motile strain, designated 18x22-1T, was isolated from a forest soil sample collected from Limushan Nature Reserve in Hainan Province, PR China. Growth occurred at 15-37 °C and pH 6.0-8.0 without NaCl. The 16S rRNA gene sequence analyses showed that strain 18x22-1T was closely related to Ramlibacter tataouinensis DSM 14655T (98.5 %), followed by Ramlibacter henchirensis DSM 14656T (97.9 %) and other Ramlibacter species and formed a stable cluster with R. tataouinensis DSM 14655T, R. henchirensis DSM 14656T, Ramlibacter solisilvae JCM 19319T and Ramlibacter rhizophilus CCTCC AB 2015357T. Results of chemotaxonomic analyses showed that ubiquinone-8 (Q-8) was the major respiratory quinone, and the major fatty acids (>10 % of the total amounts) were C16 : 0 and C17 : 0cyclo. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, two unidentified aminopholipids and four unidentified phospholipids. The draft genome sequence was 4.47 Mb long with a G+C content of 68.9 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain 18x22-1T and four closely related type strains were in the range of 79.3-82.3 % and 21.9-25.1 %, respectively. The results of phenotypic, phylogenetic and chemotaxonomic analyses supported that strain 18x22-1T represents a novel species of the genus Ramlibacter, for which the name Ramlibacterhumi sp. nov. is proposed. The type strain is 18x22-1T (=GDMCC 1.1584T=KCTC 52922T).

Whole genome sequences reveal the presence of 11 heterotypic synonyms in the genus Sphingobium and emended descriptions of Sphingobium indicum, Sphingobium fuliginis, Sphingobium xenophagum and Sphingobium cupriresistens.

Phylogenetic analysis based on 16S rRNA gene sequences of the genus Sphingobium showed the presence of four distinguishable clusters, in each of which the species shared almost the same evolutionary distance. They were Sphingobium indicum, Sphinogbium lucknowense, Sphinogbium chinhatense, Sphinogbium francense and Sphinogbium japonicum in cluster I, Sphinogbium barthaii and Sphinogbium fuliginis in cluster II, Sphinogbium hydrophobicum and Sphinogbium xenophagum in cluster III and Sphinogbium czechense and Sphinogbium cupriresistens in cluster IV. The 16S rRNA gene sequence similarities between the species in each cluster were all higher than 98 %. Genome-based average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) relatedness values between the species of each cluster were all higher than the threshold values of 95-96 % ANI and 70 % dDDH for species discrimination, respectively, suggesting that each cluster represents only one species of the genus Sphingobium. Due to priority of publication, S. lucknowense, S. chinhatense, S. francense and S. japonicum should be taken as later heterotypic synonyms of S. indicum, S. barthaii as a later heterotypic synonym of S. fuliginis, S. hydrophobicum as a later heterotypic synonym of S. xenophagum and S. czechense as a later heterotypic synonym of S. cupriresistens. Correspondingly, the descriptions of S. indicum, S. fuliginis, S. xenophagum and S. cupriresistens are also emended based on this study.

Hymenobacter metallilatus sp. nov., isolated from abandoned lead-zinc ore.

An aerobic, non-motile, Gram-stain-negative, red-to-pinkish and rod-shaped bacterium, designated 9PBR-2T, was isolated from an abandoned lead-zinc ore sample collected from Meizhou, Guangdong Province, PR China. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain 9PBR-2T belongs to the genus Hymenobacter and was most closely related to Hymenobacter rigui KCTC 12533T (98.0 %), Hymenobacter swuensis KCTC 32018T (97.8 %) and Hymenobacter perfusus LMG 26000T (97.6 %). The calculated average nucleotide identity values based on whole genome sequences between strain 9PBR-2T and closely related type strains ranged from 81.3 to 84.1 %. Correspondingly, the digital DNA-DNA hybridization values ranged from 25.5 to 28.1 %. The major fatty acids of strain 9PBR-2T were iso-C15:0, anteiso-C15:0, C16:1ω5c, summed feature 3 (C16:1ω6c and/or C16:1ω7c) and summed feature 4 (iso-C17:1 I and/or anteiso-C17:1 B). It contained menaquinone 7 (MK-7) as the major isoprenoid quinone and phosphatidylethanolamine as the major polar lipid. The genomic DNA G+C content based on whole genome sequence was 59.8 mol%. Characterization based on phylogenetic, chemotaxonomic and phenotypic analyses clearly indicated that strain 9PBR-2T represents a novel species of the genus Hymenobacter, for which the name Hymenobactermetallilatus sp. nov. is proposed. The type strain is 9PBR-2T (=GDMCC 1.1492T=JCM 32699T).

Sphingomonas lenta sp. nov., a slowly growing bacterium isolated from an abandoned lead-zinc mine.

A novel slowly growing member of the genus Sphingomonas, designated 1PNM-20T, was isolated from an abandoned lead-zinc mine in Meizhou, Guangdong Province, PR China. A polyphasic taxonomic study was performed to characterize the novel strain. Growth occurred on Reasoner's 2A (R2A) agar and peptone-yeast extract (PYE) agar, but not in liquid R2A or PYE media. Cells were Gram-stain-negative, aerobic, non-spore-forming, rod-shaped and motile with a polar flagellum (monotrichous). 16S rRNA gene sequence comparison showed that it shared the highest similarity with Sphingomonas carriPR0302T (97.2 %), followed by Sphingomonas spermidinifaciens 9NM-10T (97.0 %), Sphingomonas floccifaciens FQM01T (97.0 %) and other species of Sphingomonas (<97 %). Phylogenetic analyses clearly showed that strain 1PNM-20T fell into the cluster of Sphingomonas, and was most closely related to S. carri. The draft genome sequence was 3.76 Mb in length with a DNA G+C content of 69.8 mol%. Major fatty acids were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C16 : 0 and 11-methyl C18 : 1ω7c, with C14 : 0 2-OH as the main hydroxy fatty acid. Ubiquinone 10 (Q-10) was the predominant respiratory quinone, and sym-homospermidine was displayed as the major polyamine. The polar lipids were composed of sphingoglycolipid, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, an unidentified phospholipid and an unidentified glycolipid. The phenotypic, phylogenetic and chemotaxonomic results supported the hypothesis that strain 1PNM-20T represents a novel species of the genus Sphingomonas, for which the name Sphingomonas lenta sp. nov. is proposed. The type strain is 1PNM-20T (=GDMCC 1.660T=DSM 27572T).

Microvirga flavescens sp. nov., a novel bacterium isolated from forest soil and emended description of the genus Microvirga.

An aerobic, Gram-stain-negative, motile and rod-shaped bacterium, designated c27j1T, was isolated from a forest soil sample from the Chebaling National Nature Reserve in Guangdong Province, China. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain c27j1T belongs to the genus Microvirga and was most closely related to Microvirga aerophila DSM 21344T (97.7 %) and Microvirga subterranea DSM 14364T (96.7 %). The average nucleotide identity and digital DNA-DNA hybridization values based on whole genome sequences of strain c27j1 T and M. aerophila DSM 21344T were 77.2 and 22.4 %, respectively. It contained ubiquinone 10 as the predominant quinone, and C19 : 0 cycloω8c and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) as the major fatty acids. The polar lipids consisted of phosphatidylglycerol, phosphatidylcholine, diphosphatidylglycerol, phosphatidylethanolamine and two unidentified glycolipids. The genomic DNA G+C content based on the whole genome sequence was 62.2 mol%. Phenotypic, chemotaxonomic, phylogenetic and genomic analyses suggested that strain c27j1T should represent a novel species of the genus Microvirga, for which the name Microvirgaflavescens sp. nov. is proposed. The type strain is c27j1T (=GDMCC 1.1356T=KCTC 62433T). The description of the genus Microvirga is also emended, including the major fatty acids, genome size and DNA G+C content.

Lysobacter silvisoli sp. nov., isolated from forest soil.

A yellow-pigmented, Gram-stain-negative, gliding and rod-shaped bacterial strain, designated zong2l5T, was isolated from a forest soil sample at Dinghu Mountain, Guangdong Province, PR China. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain zong2l5T belongs to the genus Lysobacter, and was most closely related to Lysobacter enzymogenes KCTC 12131T (97.7 %) and Lysobacter soli KCTC 22011T (97.6 %). The novel strain showed an average nucleotide identity (ANI) value of 81.5 % and a digital DNA-DNA hybridization (dDDH) value of 25.3 % with L. enzymogenes KCTC 12131T based on draft genome sequences, followed by L. soli KCTC 22011T with ANI and dDDH values of 79.4 % and 22.7 %, respectively. The DNA G+C content of strain zong2l5T based on the whole genome sequence was 69.2 mol%. The major fatty acids were iso-C15 : 0, iso-C17 : 0 and summed feature 9 (iso-C17 : 1ω9c and/or 10-methyl C16 : 0). Strain zong2l5T contained Q-8 as the major isoprenoid quinone and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidyl-N-methylethanolamine, phosphatidylethanolamine, three unidentified phospholipids and an unidentified aminolipid. The phenotypic, genotypic and chemotaxonomic anlyses clearly showed that strain zong2l5T represents a novel species of the genus Lysobacter, for which the name Lysobactersilvisoli sp. nov. is proposed. The type strain is zong2l5T (=GDMCC 1.1489T=KCTC 52923T).

Chitinophaga varians sp. nov., isolated from forest soil.

A Gram-stain-negative, aerobic, non-motile bacterium, designated strain 10-7W-9003T, was isolated from the forest soil of Limushan National Forest Park, south-east China (19° 10' 42″ N, 109° 44' 45″ E). Strain 10-7W-9003T showed a shape change during the course of culture from long filamentous cells (5-10×0.4-0.5 µm) at 5-36 h, to rod shaped (1.0-1.5×0.5-0.7 µm) with inoculation after 2 days. It grew optimally at 28-30 °C and pH 6.5-7.5. On the basis of 16S rRNA gene sequence analysis, it belongs to the genus Chitinophaga and is most closely related to Chitinophaga eiseniae KACC 13774T and Chitinophaga qingshengii JCM 30026T, with 16S rRNA gene sequences similarities of 98.8 and 98.3 %, respectively. However, the DNA-DNA hybridization study showed that strain 10-7W-9003T shared relatively low relatedness values with KACC 13774T (21.8 %) and JCM 30026T (20.4 %), respectively. The major fatty acids (>10 %) were iso-C15 : 0, C16 : 1ω5c and iso-C17 : 0 3-OH. The genomic DNA G+C content was 50.7 mol%. It contained MK-7 as the major quinone. The phenotypic, chemotaxonomic and phylogenetic data clearly showed that strain 10-7W-9003T represents a novel species of the genus Chitinophaga, for which the name Chitinophaga varians sp. nov. is proposed. The type strain is 10-7W-9003T (=GDMCC 1.1252T=KACC 19415T=KCTC 52926T).

A high ratio of dietary n-3/n-6 polyunsaturated fatty acids improves obesity-linked inflammation and insulin resistance through suppressing activation of TLR4 in SD rats.

Dietary ratios of n-3/n-6 polyunsaturated fatty acids (PUFAs) have been implicated in controlling markers of metabolic disorders, including obesity, insulin resistance (IR), inflammation, and lipid profiles, which are also presumed to be partly related to type 2 diabetes mellitus (T2DM). However, molecular mechanisms of the different PUFAs related to metabolic disorders have not been systematically addressed. The present study aimed to investigate the impact of dietary n-3/n-6 PUFA ratios on obesity and IR and, further, to determine the underlying mechanisms. For 16 weeks, 32 SD male rats, randomly divided into four groups (n = 8 per group), received one of the following diets: normal chow, high saturated fatty acid (SFA), high n-3/n-6 PUFA ratio (1∶1, PUFA¹:¹), or low n-3/n-6 PUFA ratio (1∶4, PUFA¹:4). Following the experimental diet period, metabolic parameters related to obesity and IR were measured. Compared to SFA diet-fed rats, PUFA¹:¹ diet-fed rats exhibited decreased body and visceral fat weight, lowered blood lipids, and improved glucose tolerance and insulin sensitivity. Interestingly, these changes were accompanied with decreased expression levels of circulating pro-inflammatory cytokines, including tumor necrosis factor α, interleukin-6, and C-reactive protein. Moreover, the TLR4 protein and mRNA levels were markedly down-regulated by PUFA¹:¹ compared with SFA; however, PUFA¹:4 diet-fed rats failed to exhibit these changes. Cumulatively, our data highlight a role for a PUFA¹:¹ diet in the prevention of obesity and related metabolic disorders by suppressing the activation of TLR4, a critical modulator of pro-inflammatory cytokines.