"Stainomics": identification of mitotracker labeled proteins in mammalian cells.

Organelle-specific cell-permeable fluorescent dyes are invaluable tools in cell biology as they reveal intracellular dynamics in living cells. Mitrotracker is a family of dyes that strongly label the mitochondrion, a key organelle associated with many crucial cellular functions. Despite the popularity of these dyes, little is known about the molecular mechanism behind their staining specificity. Here, we aimed to identify the protein targets of one member of this dye family, mitotracker red (MTR), by 2DE and MS. MTR bound to cellular proteins covalently, and its fluorescence persisted even after cell lysis, protein solubilization, denaturation, and electrophoresis. This enabled us to display MTR-labeled proteins by 2DE. The MTR-specific fluorescent signals on the gel revealed the spots that contained MTR-conjugated proteins. These spots were analyzed by MS, resulting into the identification of ten proteins. We discovered that one major target is the mitochondrial protein HSP60 and that MTR staining could induce production of HSP60, predisposing cells to heat shock-like responses. The identification of the molecular targets of biological dyes, or "stainomics," can help correlate their intracellular staining properties with biochemical affinities. We believe this approach can be applied to a wide range of fluorescent probes.

Complete genome sequence of the pyrene-degrading bacterium Cycloclasticus sp. strain P1.

Cycloclasticus sp. strain P1 was isolated from deep-sea sediments of the Pacific Ocean and characterized as a unique bacterium in the degradation of pyrene, a four-ring polycyclic aromatic hydrocarbon (PAH). Here we report the complete genome of P1 and genes associated with PAH degradation.

A luminescent cyclometalated iridium(III) complex accumulates in mitochondria and induces mitochondrial shortening by conjugation to specific protein targets.

We report the cellular properties of a luminescent cyclometalated iridium(III) complex, [Ir(pq)(2)(phen-ITC)](PF(6)) (Ir-ITC; Hpq=2-phenylquinoline, phen-ITC=5-isothiocyanate-1,10-phenanthroline), that efficiently and specifically labels mitochondria in living mammalian cells. Ir-ITC can be covalently conjugated to its protein targets, and its luminescence survived cell lysis, protein extraction, and gel electrophoresis under denaturing conditions. The conjugation of Ir-ITC with live-cell proteins is rapid and highly selective; the process requires active cellular metabolism, as the conjugation is abolished at nonphysiological temperature or in the presence of sodium azide. Based on measurements of the luminescence intensity, we have devised a biochemical fractionation procedure that allows the enrichment of the conjugated proteins, and their subsequent separation by two-dimensional gel electrophoresis (2DGE). Luminescent protein spots were picked from the gel and analyzed by mass spectrometry; this resulted in the identification of 46 proteins. Many of the strongly luminescently labeled proteins are mitochondrial proteins. One of the targets is VDAC1 (voltage-dependent anion channel 1). Consistent with known phenotypes of VDAC1 deregulation, prolonged exposure of cells to Ir-ITC led to significant mitochondrial shortening and fragmentation. As far as we know, this is the first report on the molecular characterization of the interactions of a luminescent dye with its biological targets. As many biological dyes exhibit specific intracellular staining patterns, the identification of their molecular targets can help elucidate the mechanisms behind their staining specificities and cytotoxicity. We believe our biochemical approach can be applied to identify the targets of a wide range of fluorescent and luminescent probes.

Chinese herbal formula (GCNY)-medicated serum alleviates peroxidation induced by H2O2 in human microglial cells.

Traditional Chinese herbal medicine aiming at nourishing yin formed a distinctive school of thought in history to achieve anti-aging and longevity. In the formula Gancao nourishing yin (GCNY) decoction, all of the ingredients show antioxidant properties. However, in real clinical practice, extractions of herbs are rarely applied alone but are prescribed as the integrated formula. To investigate whether GCNY possesses anti-oxidation potential, we applied GCNY to treat rats to acquire medicated serum, which was then added on H2O2 (200 µM)-modeled human microglial cell line HMC-3 in comparison with its control serum. The results revealed that GCNY-medicated serum decreased reactive oxygen species (ROS) levels. Inflammatory cytokines such as pNF-κB p65 (ser536) and IL-6 were also decreased. Nrf2 and its pathway-related molecules, such as HO1, ABCC2, GLCM, ME1, NQO1, and TKT, were activated by H2O2 modeling while declined by treating with GCNY-medicated serum, which indicated attenuated oxidative stress of GCNY. Furthermore, mRNA-seq analysis showed 58 differential expressed genes (DEGs), which were enriched in pathways including antigen processing and presentation, longevity regulation, oxidative phosphorylation, and Parkinson's disease progression. DEGs that were downregulated by H2O2 modeling but upregulated by GCNY treatment include CENPF, MKI67, PRR11, and TOP2A. Those targets were reported to be associated with the cell cycle and cell proliferation and belong to the category of growth factor genes. In conclusion, this study verified anti-oxidation effects of GCNY and indicated its promising application for cognitive degeneration and aging-related disorders.

mTOR Signaling Pathway Regulates the Release of Proinflammatory Molecule CCL5 Implicated in the Pathogenesis of Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a complex pervasive neurodevelopmental disorder and neuroinflammation may contribute to the pathogenesis of ASD. However, the exact mechanisms of abnormal release of proinflammatory mediators in ASD remain poorly understood. This study reports elevated plasma levels of the proinflammatory chemokine (C-C motif) ligand 5 (CCL5) in children with ASD, suggesting an aberrant inflammatory response appearing in the development of ASD. Mining of the expression data of brain or blood tissue from individuals with ASD reveals that mTOR signaling is aberrantly activated in ASD patients. Our in vitro study shows that suppression of mTOR reduces the gene expression and release of CCL5 from human microglia, supporting that CCL5 expression is regulated by mTOR activity. Furthermore, bacterial lipopolysaccharide (LPS)-induced CCL5 expression can be counteracted by siRNA against NF-κB, suggests a determining role of NF-κB in upregulating CCL5 expression. However, a direct regulatory relationship between the NF-κB element and the mTOR signaling pathway was not observed in rapamycin-treated cells. Our results show that the phosphorylated CREB can be induced to suppress CCL5 expression by outcompeting NF-κB in binding to CREB-binding protein (CREBBP) once the mTOR signaling pathway is inhibited. We propose that the activation of mTOR signaling in ASD may induce the suppression of phosphorylation of CREB, which in turn results in the increased binding of CREBBP to NF-κB, a competitor of phosphorylated CREB to drive expression of CCL5. Our study sheds new light on the inflammatory mechanisms of ASD and paves the way for the development of therapeutic strategy for ASD.

HAPLN1 Affects Cell Viability and Promotes the Pro-Inflammatory Phenotype of Fibroblast-Like Synoviocytes.

HAPLN1 maintains aggregation and the binding activity of extracellular matrix (ECM) molecules (such as hyaluronic acid and proteoglycan) to stabilize the macromolecular structure of the ECM. An increase in HAPLN1 expression is observed in a few types of musculoskeletal diseases including rheumatoid arthritis (RA); however, its functions are obscure. This study examined the role of HAPLN1 in determining the viability, proliferation, mobility, and pro-inflammatory phenotype of RA- fibroblast-like synoviocytes (RA-FLSs) by using small interfering RNA (siHAPLN1), over-expression vector (HAPLN1OE), and a recombinant HAPLN1 (rHAPLN1) protein. HAPLN1 was found to promote proliferation but inhibit RA-FLS migration. Metformin, an AMPK activator, was previously found by us to be able to inhibit FLS activation but promote HAPLN1 secretion. In this study, we confirmed the up-regulation of HAPLN1 in RA patients, and found the positive relationship between HAPLN1 expression and the AMPK level. Treatment with either si-HAPLN1 or HAPLN1OE down-regulated the expression of AMPK-ɑ gene, although up-regulation of the level of p-AMPK-ɑ was observed in RA-FLSs. si-HAPLN1 down-regulated the expression of proinflammatory factors like TNF-ɑ, MMPs, and IL-6, while HAPLN1OE up-regulated their levels. qPCR assay indicated that the levels of TGF-ß, ACAN, fibronectin, collagen II, and Ki-67 were down-regulated upon si-HAPLN1 treatment, while HAPLN1OE treatment led to up-regulation of ACAN and Ki-67 and down-regulation of cyclin-D1. Proteomics of si-HAPLN1, rHAPLN1, and mRNA-Seq analysis of rHAPLN1 confirmed the functions of HAPLN1 in the activation of inflammation, proliferation, cell adhesion, and strengthening of ECM functions. Our results for the first time demonstrate the function of HAPLN1 in promoting the proliferation and pro-inflammatory phenotype of RA-FLSs, thereby contributing to RA pathogenesis. Future in-depth studies are required for better understanding the role of HAPLN1 in RA.

Alcanivorax pacificus sp. nov., isolated from a deep-sea pyrene-degrading consortium.

A taxonomic study was carried out on a novel bacterial strain, designated W11-5(T), which was isolated from a pyrene-degrading consortium enriched from deep-sea sediment of the Pacific Ocean. The isolate was Gram-reaction-negative and oxidase- and catalase-positive. Growth was observed in 0.5-12 % (w/v) NaCl and at 10-42 °C. On the basis of 16S rRNA gene sequence analysis, strain W11-5(T) was shown to belong to the genus Alcanivorax with a close relation to A. dieselolei B-5(T) (93.9 % 16S rRNA sequence similarity), A. balearicus MACL04(T) (93.1 %), A. hongdengensis A-11-3(T) (93.1 %), A. borkumensis SK2(T) (93.0 %), A. venustensis ISO4(T) (93.0 %) and A. jadensis T9(T) (92.9 %). Similarities between the gyrB gene sequences of W11-5(T) and other species of the genus Alcanivorax were between 76.8 and 80.8 %. The principal fatty acids were C(12 : 0) 3-OH (8.0 %), C(16 : 0) (29.1 %) and C(18 : 1)ω7c (27.4 %). The G+C content of the chromosomal DNA was 60.8 mol%. Based on its morphology, physiology and fatty acid composition as well as the results of 16S rRNA and gyrB gene sequence analyses, strain W11-5(T) ( = MCCC 1A00474(T)  = CCTCC AB 208236(T)  = LMG 25514(T)) represents a novel species of the genus Alcanivorax, for which the name Alcanivorax pacificus sp. nov. is proposed.

Nitratireductor pacificus sp. nov., isolated from a pyrene-degrading consortium.

Strain pht-3B(T) was isolated from a pyrene-degrading consortium of an enriched sediment from the Pacific Ocean, collected during the screening of polycyclic aromatic hydrocarbon-degrading bacteria. Cells were Gram-negative, short rods that were motile by means of flagella. Growth was observed at 0-7 % NaCl and 10-41 °C. The isolate was able to reduce nitrate to nitrite, but not to nitrogen. 16S rRNA gene sequence comparisons showed that strain pht-3B(T) was most closely related to Nitratireductor aquibiodomus NL21(T) (97.3 % 16S rRNA gene sequence similarity), N. indicus C115(T) (97.1 %), N. basaltis J3(T) (96.8 %) and N. kimnyeongensis KY 101(T) (96.7 %). DNA-DNA hybridization between strain pht-3B(T) and these reference strains revealed 55, 54, 28 and 42 % DNA-DNA relatedness, respectively. The dominant fatty acids were C(19 : 0)ω8c cyclo (22.6 %) and summed feature 8 (consisting of C(18 : 1)ω7c and/or C(18 : 1)ω6c; 60.4 %). The G+C content of the chromosomal DNA was 63 mol%. These characteristics were in good agreement with those of members of the genus Nitratireductor. According to cell morphology, physiology, fatty acid composition, 16S rRNA gene sequence analysis and DNA-DNA relatedness, the isolate belonged to the genus Nitratireductor but could be readily distinguished from recognized species of the genus. Therefore a novel species is proposed to accommodate strain pht-3B(T), for which the name Nitratireductor pacificus sp. nov. is proposed. The type strain is pht-3B(T) ( = CCTCC AB 209302(T) = LMG 25541(T) = MCCC 1A01024(T)).

Novel association between a transient receptor potential cation channel subfamily M member 5 expression quantitative trait locus rs35197079 and decreased susceptibility of gestational diabetes mellitus in a Chinese population.

AIMS/INTRODUCTION: Emerging evidence suggests that expression quantitative trait loci (eQTLs) are more likely to associate with complex diseases. Transient receptor potential cation channel subfamily M member 5 (TRPM5) is a ubiquitously expressed voltage-gated cation channel that acts indispensably to trigger insulin secretion in pancreatic ß-cells. The present study evaluated the association between TRPM5 eQTL single-nucleotide polymorphisms and the risk of gestational diabetes mellitus (GDM) in a Chinese population. MATERIALS AND METHODS: A total of 380 unrelated Chinese pregnant women including 241 GDM patients and 139 controls were included in this study. The eQTL single-nucleotide polymorphisms of TRPM5 were obtained from the GTEx eQTL Browser, and were subsequently genotyped using the Agena MassARRAY iPLEX platform. RESULTS: Logistic regression analysis and linear regression analysis showed that rs35197079 and rs74848824 were significantly associated with reduced GDM risk and lower fasting plasma glucose levels after adjusting confounder factors in dominant genetic models. Stratification analysis based on pre-pregnancy body mass index validated a strong association between rs35197079 and GDM susceptibility in underweight and normal weight individuals. Luciferase and electrophoretic mobility shift assays carried out in rat pancreatic ß-cells showed that rs35197079 was functional. CONCLUSIONS: The TRPM5 eQTL single-nucleotide polymorphism rs35197079 was associated with decreased GDM susceptibility in a Chinese population, especially in underweight and normal weight pregnant women, and it was functional in modulating gene transcription.

Albumin/Globulin Ratio as Yin-Yang in Rheumatoid Arthritis and Its Correlation to Inflamm-Aging Cytokines.

PURPOSE: Inflamm-aging is a novel-concept in rheumatoid arthritis (RA) with accelerating aging process. We try to find a correlation between serum albumin/globulin (A/G) ratio and clinical biochemical parameters, incidence of aging-related diseases (ARDs) as well as inflammaging-related molecules. PATIENTS AND METHODS: Healthy controls (HC) and RA patients were compared with their clinical biochemical parameters including albumin and globulin levels, A/G ratio, and levels of serum lipids. Incidence of ARDs in RA was compared with A/G ratio, having a cut off value of 1.2. Expression levels of leptin and Trf2 genes in PBMCs, and inflammatory factors like IL-1ß, IL-6, IL-8 and TNF-ɑ between HC and RA patients were compared, and correlated with the A/G ratio. RESULTS: Compared to HC, RA patients had decreased levels of albumin, while globulin levels were found to be increased, which led to a significantly lower A/G ratio in RA patients. A/G ratio rather than ESR and CRP had significant correlation with dyslipidemia in RA patients. Patients with A/G <1.2 had a higher risk of ARDs than patients with A/G >1.2. The RR was 2.48 (95% CI: 1.79 to 3.64, p <0.0001). In addition, A/G ratio has positively correlated to leptin and Trf2 expression, while an inverse correlation was observed with the levels of inflamm-aging related cytokines like IL-6, IL-8 and TNF-ɑ. CONCLUSION: A decreased A/G ratio in RA patients has significantly correlated with dyslipidemia and ARDs, as well as inflammaging- related adipokine and pro-inflammatory cytokines. Thus, A/G ratio could be a reliable marker for evaluating the inflammaging process during clinical management in ARDs.

Assessment of the association between the polymorphism rs1256031 of the estrogen receptor ß gene and GDM susceptibility.

Estrogen has an important role in regulating glucose homeostasis, and existing evidence indicates that it might be involved in the development of hyperglycemia in pregnancy. It mediates its effect through estrogen receptors including the nuclear receptor ERß encoded by ESR2. The association between the ESR2 polymorphism rs1256031 and GDM susceptibility has not been investigated yet. This study aimed to evaluate the relationship between rs1256031 and GDM risk in Chinese population. A total of 241 GDM patients and 139 healthy pregnant women were recruited for this study. The rs1256031 genotype was examined by time-of-flight mass spectrometry and the association between rs1256031 and GDM susceptibility was assessed by binary logistic regression in three different genetic models. The polymorphism rs1256031 was not associated with GDM susceptibility in additive [OR (95% CI) = 0.871 (0.453,1.675); P = 0.680], dominant [OR (95% CI) = 0.908 (0.495,1.665); P = 0.755] or recessive [OR (95% CI) = 0.912 (0.591,1.408); P = 0.677] models after adjusting for confounding factors. We observed no association between the polymorphism rs1256031 in the ESR2 gene and GDM susceptibility in Chinese pregnant women.

The association between the rs4987105 of 5-lipoxygenase (ALOX5) gene and gestational glucose metabolism in Chinese population.

OBJECTIVE: The arachidonate 5-lipoxygenase (ALOX5) pathway has been investigated in diverse chronic inflammatory diseases including metabolic disorders. Recently, the ALOX5 polymorphism rs4987105 was identified to confer susceptibility to type 2 diabetes mellitus (T2DM), implicating its role in regulating glucose homeostasis. Gestational diabetes mellitus (GDM) shares similar pathogenic mechanism with T2DM. Thus, we aimed to evaluate the association between rs4987105 and gestational glucose metabolism in Chinese pregnant women. RESULTS: A total of 380 unrelated Chinese pregnant women including 241 GDM patients and 139 controls were included in this study. The genotypes of rs4987105 were examined by the Agena MassARRAY iPLEX platform, the association between rs4987105 and fasting plasma glucose (FPG) levels at 24-28 gestational weeks was evaluated using different statistical methods. We found that carriers of rs4987105 CT/TT genotypes exhibited significantly lower FPG levels (P = 0.011). In addition, we observed a significant association between rs4987105 and FPG levels after adjusting confounding variables in the linear regression analysis using dominant genetic model (b = - 0.218; P = 0.01). The present study for the first time reported that the rs4987105 of 5-lipoxygenase (ALOX5) gene was associated with gestational glucose metabolism in Chinese pregnant women.

Leber's Hereditary Optic Neuropathy-Specific Heteroplasmic Mutation m.14495A>G Found in a Chinese Family.

PURPOSE: Leber's hereditary optic neuropathy (LHON) is a mitochondrial DNA (mtDNA)-associated, maternally inherited eye disease. Mutation heteroplasmy level is one of the leading causes to trigger LHON manifestation. In this study, we aimed to identify the causative mutation in a large Han Chinese family with LHON and explore the underlying pathogenic mechanism in this LHON family. METHODS: The whole-mtDNA sequence was amplified by long-range PCR. Mutations were subsequently identified by next-generation sequencing (NGS) and validated by Sanger sequencing. The heteroplasmy rates of those family members were determined by digital PCR (dPCR). Mitochondrial haplogroups were assigned based on mtDNA tree build 17. RESULTS: The m.14495A>G mutation was identified as causative due to its higher heteroplasmy level (>50%) in patients than in their unaffected relatives. All mutation carriers belong to M7b1a1 and are assigned to Asian mtDNA lineage. Interestingly, our result revealed that high mtDNA copy number in carrier might prevent LHON manifestation. CONCLUSIONS: This is the first report of m.14495A>G mutation in Asian individuals with LHON. Our study shows that dPCR technology can provide more reliable results in mutation heteroplasmy assay and determination of the cellular mtDNA content, making it a potentially promising tool for clinical precise diagnosis of LHON. Furthermore, our results also add evidence to the opinion that higher mtDNA content may protect mutation carriers from LHON. TRANSLATIONAL RELEVANCE: dPCR can be used for the assessment of LHON disease, and a new genetic-based diagnostic strategy has been proposed for LHON patients with the m.14495A>G mutation.

A pyrene-degrading consortium from deep-sea sediment of the West Pacific and its key member Cycloclasticus sp. P1.

A pyrene-degrading bacterial consortium was obtained from deep-sea sediments of the Pacific Ocean. The consortium degraded many kinds of polycyclic aromatic hydrocarbons (PAHs), including naphthalene, phenanthrene, pyrene, acenaphthene, fluorene, anthracene, fluoranthene, 2-methylnaphthalene and 2,6-dimethylnaphthalene, but it did not grow with chrysene and benzo[alpha]pyrene. With methods of plate cultivation and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), 72 bacteria belonging to 22 genera were detected from this consortium. Among the detected bacteria, the following genera frequently occurred: Flavobacterium, Cycloclasticus, Novosphingobium, Halomonas, Achromobacter, Roseovarius and Alcanivorax. The first two genera showed the strongest bands in denaturing gradient gel electrophoresis (DGGE) profiles and appeared in all PAH treatments. By now, only one isolate designated P1 was confirmed to be a pyrene degrader. It was identified to be Cycloclasticus spirillensus (100%). Although P1 can degrade pyrene independently, other bacteria, such as Novosphingobium sp. (Band 14), Halomonas sp. (Band 16) and an unidentified bacterium (Band 35), were involved in pyrene degradation in some way; they persist in the consortium in the test of dilution to extinction if only the consortium was motivated with pyrene. However, the secondary most important member Flavobacterium sp. evaded from the community at high dilutions. As a key member of the consortium, P1 distinguished itself by both cell morphology and carbon source range among the isolates of this genus. Based on intermediate analyses of pyrene degradation, P1 was supposed to take an upper pathway different from that previously reported. Together with the results of obtained genes from P1 homology with those responsible for naphthalene degradation, its degradation to pyrene is supposed to adopt another set of genes unique to presently detected. Summarily, an efficient pyrene-degrading consortium was obtained from the Pacific Ocean sediment, in which Cycloclasticus bacterium played a key role. This is the first report to exploit the diversity of pyrene-degrading bacteria in oceanic environments.

[Diversity of bacteria isolated from the South China Sea sediments].

OBJECTIVE: To isolate bacteria from Nansha area of South China sea, METHODS: Sediment samples of 22 sites were used. Bacterial isolation was conducted on plates of marine medium, followed by 16S rRNA identification and phylogenetic analysis. RESULTS: In total 349 bacteria were obtained, belonging to 87 species. Analyses of 16S rRNA sequence showed that Bacillus and other spore-forming bacteria occupied the majority of isolates in 10 sites. Bacillus was the most abundant bacterium and of high diversity; with 34 species and 8 possible novel species. Halobacillus also occurred frequently while other spore-forming bacteria including Brevibacillus, Paenibacillus, Pontibacillus and Thalassobacillus were also found, but less occurred in this area. In addition to these low-G+C-content bacteria, gamma-Proteobacteria were the second subgroup of high occurrence, among which Pseudomonas, Marinobacter and Alcanivorax were relatively abundant. Generally, isolates of 750-2000 m deep mainly consist of low-G+C-content bacteria, while mainly composed of gamma-Proteobacteria when the depth is over 2000 m. CONCLUSION: Marine sediments of South China Sea are rich in spore-forming bacteria, which deserve further study and exploitation.

A Novel Potentially Causative Variant of NDUFAF7 Revealed by Mutation Screening in a Chinese Family With Pathologic Myopia.

Purpose: Pathologic myopia described as myopia accompanied by severe deformation of the eye besides excessive elongation of eye, is usually a genetic heterogeneous disorder characterized by extreme, familial, early-onset vision loss. However, the exact pathogenesis of pathologic myopia remains unclear. In this study, we screened a Han Chinese family with pathologic myopia to identify the causative mutation and explore the possible pathogenic mechanism based on evaluation of the biological functions of the mutation. Methods: We identified the mutations in a family with pathologic myopia by single nucleotide polymorphism array combined with short tandem repeat microsatellite marker analysis and exome sequencing. Mutations were validated among family members by direct Sanger sequencing. The subcellular localization of the protein variant was investigated by immunofluorescence, and the stability of the mutant protein was determined by immunoblotting. Intracellular levels of adenosine triphosphate and reactive oxygen species and complex I activity were measured by traditional biochemical methods to determine the functional role of the disease-associated mutation. Results: The novel missense mutation: c.798C>G (p.Asp266Glu) in NDUFAF7, cosegregated with the disease and the resulting amino acid substitution affected a highly conserved residue in its protein. The mutation D266E in NDUFAF7 impaired complex I activity, which resulted in decreased ATP levels in cultured cells. Conclusions: We propose that the heterozygous mutation (c.798C>G) in NDUFAF7 may contribute to the pathogenesis of pathologic myopia, possibly by interfering with the phototransduction cascade. Mitochondrial dysfunction during eye development may lead to pathologic myopia.

Graphene oxide: a nonspecific enhancer of cellular growth.

There have been multiple conflicting reports about the biocompatibility and antimicrobial activity of graphene oxide. To address this, we conducted a study to characterize the antimicrobial properties of graphene oxide (GO) and its biocompatibility with mammalian cells. When GO was added to a bacterial culture at 25 µg/mL, the results showed that bacteria grew faster and to a higher optical density than cultures without GO. Scanning electron microscopy indicated that bacteria formed dense biofilms in the presence of GO. This was shown by a large mass of aggregated cells and extracellular polymeric material. Bacterial growth on filters coated with 25 and 75 µg of GO grew 2 and 3 times better than on filters without GO. Closer analysis showed that bacteria were able to attach and proliferate preferentially in areas containing the highest GO levels. Graphene oxide films failed to produce growth inhibition zones around them, indicating a lack of antibacterial properties. Also, bacteria were able to grow on GO films to 9.5 × 10(9) cells from an initial inoculation of 1.0 × 10(6), indicating that it also lacks bacteriostatic activity. Thus, silver-coated GO films were able to produce clearing zones and cell death. Also, graphene oxide was shown to greatly enhance the attachment and proliferation of mammalian cells. This study conclusively demonstrates that graphene oxide does not have intrinsic antibacterial, bacteriostatic, and cytotoxic properties in both bacteria and mammalian cells. Furthermore, graphene oxide acts as a general enhancer of cellular growth by increasing cell attachment and proliferation.

Roseovarius nanhaiticus sp. nov., a member of the Roseobacter clade isolated from marine sediment.

An aerobic, Gram-staining-negative, rod or ovoid-shaped bacterial isolate, strain NH52J(T), was isolated from a sandy sediment sample from the South China Sea. Strain NH52J(T) exhibited tumbling motility, formed beige or faint pink colonies, gave a positive reaction in tests for catalase and oxidase and required NaCl for growth. Optimal growth was observed at pH 7.8-9.3, at 30 degrees C and in the presence of 2.0-4.0 % (w/v) NaCl. The novel strain did not synthesize bacteriochlorophyll a, and the DNA G+C content was 62 %. The major fatty acids were C(18 : 1)omega7c, C(16 : 0) and C(18 : 1)omega7c 11-methyl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain NH52J(T) was affiliated to the genus Roseovarius of the class Alphaproteobacteria. Roseovarius pacificus and Roseovarius aestuarii were the most closely related recognized species to strain NH52J(T) with 16S rRNA gene sequence similarity values of 95.0 and 95.7 %, respectively. Sequence similarity values between strain NH52J(T) and other phylogenetically related species were all below 95.0 %. Based on the phenotypic, chemotaxonomic and phylogenetic data presented, strain NH52J(T) is considered to represent a novel species of the genus Roseovarius, for which the name Roseovarius nanhaiticus sp. nov. is proposed. The type strain is NH52J(T) (=LMG 24840(T)=CCTCC AB 208317(T)=MCCC 1A03543(T)).

Meridianimaribacter flavus gen. nov., sp. nov., a member of the family Flavobacteriaceae isolated from marine sediment of the South China Sea.

A Gram-staining-negative, rod-shaped marine bacterium, designated strain NH57N(T), isolated from sandy sediment in the Mischief Reef of the South China Sea, was characterized based on its physiological and biochemical features, fatty acid profile and phylogenetic position. 16S rRNA gene sequence analysis revealed a clear affiliation with the family Flavobacteriaceae. Strain NH57N(T) showed the closest phylogenetic relationship with members of the genera Gaetbulibacter, Gelidibacter, Subsaxibacter, Subsaximicrobium and Yeosuana; levels of 16S rRNA gene sequence similarity between strain NH57N(T) and the type strains of related species ranged from 94.9 to 91.2 %. Cells of strain NH57N(T) were motile by gliding and grew on solid media as yellow colonies at 9-37 degrees C, pH 6.5-8.5 and in the presence of 0.5-4.0 % NaCl. The DNA G+C content was 32.7 mol% and the predominant fatty acids were iso-C(15 : 1) (22.7 % of the total), iso-C(15 : 0) (20.7 %), iso-C(17 : 0) 3-OH (9.5 %), iso-C(16 : 0) 3-OH (8.3 %), C(15 : 0) (7.8 %) and iso-C(15 : 0) 3-OH (5.8 %). Based on the physiological and phylogenetic data, and on the fatty acid composition, strain NH57N(T) is considered to represent a novel species of a new genus in the family Flavobacteriaceae, for which the name Meridianimaribacter flavus gen. nov., sp. nov. is proposed. The type strain of Meridianimaribacter flavus is NH57N(T) (=CCTCC AB 208318(T)=LMG 24839(T)=MCCC 1A03544(T)).

Arenibacter nanhaiticus sp. nov., isolated from marine sediment of the South China Sea.

An aerobic, Gram-stain-negative, rod-shaped bacterial isolate, strain NH36A(T), was isolated from a sandy sediment sample from the South China Sea. Colonies of the isolate were dark orange on M2 agar. Optimal growth was observed at pH 7.0-8.5, 30 degrees C and in the presence of 0.5-4.0 % (w/v) NaCl. The major fatty acids were C(15 : 0), iso-C(15 : 0), anteiso-C(15 : 0), iso-C(15 : 1), iso-C(15 : 0) 3-OH, iso-C(17 : 0) 3-OH and summed feature 3 (comprising iso-C(15 : 0) 2-OH and/or C(16 : 1)omega7c). The DNA G+C content was 38.9 mol%. 16S rRNA gene sequence analysis revealed that strain NH36A(T) was most closely related to members of the genus Arenibacter, exhibiting 94.3-96.2 % sequence similarity to the type strains of Arenibacter species. On the basis of phenotypic, chemotaxonomic and phylogenetic data, this organism should be classified as a representative of a novel species in the genus Arenibacter. The name Arenibacter nanhaiticus sp. nov. is proposed and the type strain is NH36A(T) (=LMG 24842(T)=CCTCC AB 208315(T)=MCCC 1A04137(T)).