Sinisalibacter aestuarii sp. nov., isolated from estuarine sediment of the Arakawa River.

A Gram-stain-negative, rod-shaped, non-motile and strictly aerobic bacterium, which showed biofilm-forming ability on polystyrene, designated as strain B-399T, was isolated from the estuarine sediment of the Arakawa River near Tokyo Bay. It grew at pH 6.0-8.5, at 15-35 °C and in the presence of 0-7.5 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain B-399T was clustered in the genus Sinisalibacter and has 96.94 % sequence similarity to Sinisalibacter lacisalsi X12M-4T, which was the only validly described species in this genus. On the basis of our genome sequencing analyses, the average nucleotide identity and digital DNA-DNA hybridization values between strains B-399T and S. lacisalsi X12M-4T were 79.54 and 22.30 %, respectively, which confirms that strain B-399T represents a novel species of the genus Sinisalibacter. The draft genome size and the DNA G+C content of strain B-399T were 4.12 Mb and 65.2 mol%, respectively. The major fatty acids (>10 %) of strain B-399T were C16 : 0, summed feature 8 (C18 : 1 ω6c and/or C18 : 1 ω7c) and C19 : 0 cyclo ω8c. The polar lipids were phosphatidylcholine, phosphatidylglycerol, an unidentified phospholipid, an unidentified aminolipid and unidentified lipids. The respiratory quinone was Q-10. These chemotaxonomic features were almost coincident with those of the genus Sinisalibacter. Therefore, strain B-399T should be classified as representing a new species of the genus Sinisalibacter, for which the name Sinisalibacter aestuarii sp. nov. is proposed. The type strain is B-399T (=NBRC 115629T=DSM 114148T).

Vallitalea longa sp. nov., an anaerobic bacterium isolated from marine sediment.

A novel bacterium, strain SH18-1T, was isolated from marine sediment collected near Sado Island in the Sea of Japan. This strain was strictly anaerobic, Gram-stain-negative, non-spore-forming, rod-shaped, motile, and mesophilic. It grew at 15-40 °C (optimum, 30-35 °C), at a NaCl concentration of 0.2-5.0 % (w/v; optimum, 1.5-2.5 %), and at pH 5.5-8.5 (optimum, pH 7.0). Results of 16S rRNA gene phylogenetic analysis showed a similarity value of 97.49 % between strain SH18-1T and Vallitalea guaymasensis Ra1766G1T, which was the most closely related species. The genome size of strain SH18-1T was 5.71 Mb and its G+C content was 30.2 mol%. Genome sequence analyses for comparison between strain SH18-1T and V. guaymasensis Ra1766G1T showed values lower than the threshold for species demarcation determined using the Genome-to-Genome Distance Calculator and the Average Nucleotide Identity Calculator. Elemental sulphur, sulphate, thiosulphate, sulphite, fumarate, nitrate, and nitrite were not used as terminal electron acceptors. The major fatty acids in strain SH18-1T were iso-C15 : 0, anteiso-C15 : 0, and C16 : 0, and the detected polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphoglycolipid, glycolipid, three unidentified phospholipids, and one unidentified polar lipid. From these results, strain SH18-1T (=NBRC 115488T=DSM 114058T) is suggested to represent a novel species of the genus Vallitalea and the name Vallitalea longa sp. nov. is proposed.

Imprinting analysis by droplet digital PCR coupled with locked nucleic acid TaqMan probes.

Imprinted genes are differentially expressed in a parent-of-origin-specific manner. Parental origin of the alleles is discriminated by intragenic DNA polymorphisms. Comparisons of parental allelic expression have been analysed by semiquantitative RT-PCR. Here, we developed a novel quantitative method for allelic expression of the imprinted gene Ube3a, which inactivation and mutations cause Angelman syndrome and predominantly expressed by the maternal allele in neuronal tissues. In this method, cDNA was amplified by droplet digital PCR (ddPCR) coupled with allele-specific locked nucleic acid (LNA) TaqMan probes, which labelled by FAM and HEX were designed to detect the SNPs in the target regions. ddPCR assay demonstrated that the sense transcript of Ube3a was equally expressed from both parental alleles in adult tissues except neuronal tissues, where Ube3a expression from the paternal allele was about 10 to 14% of total Ube3a expression in adult brain, and 20% in spinal cord. The antisense transcript of Ube3a was expressed at 60% to 70% of the sense transcript of Ube3a in adult brain. Changes in the Ube3a transcripts during postnatal brain development were also evaluated by ddPCR. The ddPCR method is far more reliable and simpler to use than semiquantitative PCR to analyse skewed or faint allelic expression of imprinted genes.

Transgenic mice with a tandem duplication of the Necdin gene overexpress Necdin.

Necdin (Ndn) transgenic (Tg) mice were generated with a bacterial artificial chromosome (BAC) clone. Droplet digital PCR (ddPCR) and inverse PCR methods revealed that the transgene consisted of four fragments with a total length of 171 kb. Two of these fragments were tandem tail-to-tail duplicates of 77 kb and 37 kb that both contained a Ndn gene. The transgene was inserted in chromosome 15qD1. Ndn is a paternally expressed imprinted gene; however, the total expression level of Ndn in hemizygous Tg mice was approximately twofold higher than that in wild-type mice. ddPCR assays with locked nucleic acid (LNA) TaqMan probes revealed that transgenic Ndn expression was almost equal to endogenous Ndn expression, despite there being two copies of the Ndn gene in the transgene, indicating an interaction between the transcriptional regulation of endogenous Ndn and the transgene. ddPCR assays with LNA TaqMan probes were also applied for imprinting analysis to confirm exclusive paternal expression in tissues with low Ndn expression. This is the first report of a Tg mouse with a tandem duplication of a Ndn transgene and Ndn overexpression, which will be useful for the in vivo study of Ndn overexpression and for rescue experiments of the neonatal lethality seen in the Ndn knockout mouse.

Application of droplet digital PCR in the analysis of genome integration and organization of the transgene in BAC transgenic mice.

Transgenic (Tg) mice containing bacterial artificial chromosome (BAC) DNA are widely used for gene expression analysis and gene therapy models because BAC transgenes provide gene expression at physiological levels with the same developmental timing as endogenous genes. To ensure correct interpretation of transgene functions, investigation of the genomic organisation and integration of the BAC transgene is required. Here, we describe a reliable method based on droplet digital PCR (ddPCR) and inverse PCR to estimate copy number, genomic organisation and insertion sites of BAC transgenes in the mouse genome. We generated BAC Tg mice containing fragments of BAC clone RP23-59P20. ddPCR and iPCR analysis showed that the transgene consisted of five fragments of the BAC clone containing the Mkrn3 gene region, and that the transgene was inserted into Bckdhb, homozygous deletion of which causes the maple syrup urine disease phenotype. The ddPCR method described here should prove useful for analysis of genomic organisation and integration of BAC transgenes.

The Y54(L)W mutation of anti-leukotriene C4 single-chain antibody increases affinity to leukotriene E4.

The X-ray crystal structure of an anti-leukotriene (LT) C4 monoclonal antibody (mAbLTC) in complex with LTC4 was determined, however, crystallographic studies alone are not enough to fully understand the structures of the antigen-binding site. To elucidate the individual contribution of Tyr-54 and Asn-58 in the light chain of mAbLTC, both of which formed a hydrogen bond with glutamic acid of LTC4, we examined whether substitution of the residues affects the antigen binding affinity and specificity using an anti-LTC4 single chain variable fragment (scFvLTC). Among the Tyr-54(L) mutants, Y54(L)W showed a dramatic increase in the affinity to LTE4 which was comparable to that to LTD4 Essentially the same results were obtained using the Y54(L)W mutant expressed in Escherichia coli and Pichia pastoris. The structural modeling suggested the formation of a novel hydrogen bond between the substituted tryptophan in the antibody and the cysteine residue in LTE4 The affinity of Y54(L)R, Y54(L)E and Y54(L)L to LTC4 was markedly reduced, whereas other tested Tyr-54(L) mutants as well as Asn-58(L) mutants did not show significant change in LT binding. The results may provide an insight into the molecular basis of specific LT recognition by the antibody.

Neutralization of leukotriene C4 and D4 activity by monoclonal and single-chain antibodies.

BACKGROUND: Cysteinyl leukotrienes (LTs) are key mediators in inflammation. To explore the structure of the antigen-recognition site of a monoclonal antibody against LTC4 (mAbLTC), we previously isolated full-length cDNAs for heavy and light chains of the antibody and prepared a single-chain antibody comprising variable regions of these two chains (scFvLTC). METHODS: We examined whether mAbLTC and scFvLTC neutralized the biological activities of LTC4 and LTD4 by competing their binding to their receptors. RESULTS: mAbLTC and scFvLTC inhibited their binding of LTC4 or LTD4 to CysLT1 receptor (CysLT1R) and CysLT2 receptor (CysLT2R) overexpressed in Chinese hamster ovary cells. The induction by LTD4 of monocyte chemoattractant protein-1 and interleukin-8 mRNAs in human monocytic leukemia THP-1 cells expressing CysLT1R was dose-dependently suppressed not only by mAbLTC but also by scFvLTC. LTC4- and LTD4-induced aggregation of mouse platelets expressing CysLT2R was dose-dependently suppressed by either mAbLTC or scFvLTC. Administration of mAbLTC reduced pulmonary eosinophil infiltration and goblet cell hyperplasia observed in a murine model of asthma. Furthermore, mAbLTC bound to CysLT2R antagonists but not to CysLT1R antagonists. CONCLUSIONS: These results indicate that mAbLTC and scFvLTC neutralize the biological activities of LTs by competing their binding to CysLT1R and CysLT2R. Furthermore, the binding of cysteinyl LT receptor antagonists to mAbLTC suggests the structural resemblance of the LT-recognition site of the antibody to that of these receptors. GENERAL SIGNIFICANCE: mAbLTC can be used in the treatment of inflammatory diseases such as asthma.