<Emphasis Type="Italic">Saccharothrix ghardaiensis</Emphasis> sp. nov., an actinobacterium isolated from Saharan soil

The taxonomic position of a new Saccharothrix strain, designated MB46^T, isolated from a Saharan soil sample collected in Mzab region (Ghardaïa province, South Algeria) was established following a polyphasic approach. The novel microorganism has morphological and chemical characteristics typical of the members of the genus Saccharothrix and formed a phyletic line at the periphery of the Saccharothrix espanaensis subcluster in the 16S rRNA gene dendrograms. Results of the 16S rRNA gene sequence comparisons revealed that strain MB46^T shares high degrees of similarity with S. espanaensis DSM 44229^T (99.2%), Saccharothrix variisporea DSM 43911^T (98.7%) and Saccharothrix texasensis NRRL B-16134^T (98.6%). However, the new strain exhibited only 12.5–17.5% DNA relatedness to the neighbouring Saccharothrix spp. On the basis of phenotypic characteristics, 16S rRNA gene sequence comparisons and DNA-DNA hybridizations, strain MB46^T is concluded to represent a novel species of the genus Saccharothrix, for which the name Saccharothrix ghardaiensis sp. nov. (type strain MB46^T = DSM 46886^T = CECT 9046^T) is proposed.     

A rapid and inexpensive labeling method for microarray gene expression analysis

Background

Polyethyleneimine (PEI), a cationic polymer, is one of the successful and widely used vectors for non-viral gene transfection in vitro. However, its in vivo application was greatly limited due to its high cytotoxicity and short duration of gene expression. To improve its biocompatibility and transfection efficiency, PEI has been modified with PEG, folic acid, and chloroquine in order to improve biocompatibility and enhance targeting.

Results

Poly(ε-caprolactone)-Pluronic-Poly(ε-caprolactone) (PCFC) was synthesized by ring-opening polymerization, and PCFC-g-PEI was obtained by Michael addition reaction with GMA-PCFC-GMA and polyethyleneimine (PEI, 25 kD). The prepared PCFC-g-PEI was characterized by ¹H-NMR, SEC-MALLS. Meanwhile, DNA condensation, DNase I protection, the particle size and zeta potential of PCFC-g-PEI/DNA complexes were also determined. According to the results of flow cytometry and MTT assay, the synthesized PCFC-g-PEI, with considerable transfection efficiency, had obviously lower cytotoxicity against 293 T and A549 cell lines compared with that of PEI 25 kD.

Conclusion

The cytotoxicity and in vitro transfection study indicated that PCFC-g-PEI copolymer prepared in this paper was a novel gene delivery system with lower cytotoxicity and considerable transfection efficiency compared with commercial PEI (25 kD).     

<Emphasis Type="Italic">Novosphingobium profundi</Emphasis> sp. nov. isolated from a deep-sea seamount

A marine bacterial strain, F72^T, was isolated from a solitary scleractinian coral, collected in Yap seamounts in the Pacific Ocean. Strain F72^T is a Gram-negative, light-yellow-pigmented, motile, rod-shaped bacterium. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain F72^T is related to the genus Novosphingobium and has high 16S rRNA gene sequence similarities with the type strains of Novosphingobium pentaromativorans US6-1^T (97.7 %), Novosphingobium panipatense SM16^T (97.6 %), Novosphingobium mathurense SM117^T (97.2 %) and Novosphingobium barchaimii LL02^T (97.1 %). Ubiquinone Q-10 was detected as the dominant quinone. The predominant cellular fatty acids were C_18:1ω7c and C_17:1ω6c. The genomic DNA G+C content of strain F72^T was 63.4 mol %. The polar lipids profile contained phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylcholine, sphingoglycolipid and one uncharacterized lipid. Strain F72^T shared DNA relatedness of 25 % with N. pentaromativorans JCM 12182^T, 31 % with N. panipatense DSM 22890^T, 21 % with N. mathurense DSM 23374^T and 26 % with N. barchaimii DSM 25411^T. Combined data from phenotypic, phylogenetic and DNA–DNA relatedness studies demonstrated that the strain F72^T is a representative of a novel species of the genus Novosphingobium, for which we propose the name Novosphingobium profundi sp. nov. (type strain F72^T = KACC 18566^T = CGMCC 1.15390^T).     

Paenibacillus brassicae sp. nov., isolated from cabbage rhizosphere in Beijing, China

A novel Gram-positive, rod-shaped, motile, spore-forming, nitrogen-fixing bacterium, designated strain 112^T, was isolated from cabbage rhizosphere in Beijing, China. The strain was found to grow at 10–40 °C and pH 4–11, with an optimum of 30 °C and pH 7.0, respectively. Phylogenetic analysis based on a fragment of the full-length 16S rRNA gene sequence revealed that strain 112^T is a member of the genus Paenibacillus. High levels of 16S rRNA gene similarities were found between strain 112^T, Paenibacillus sabinae DSM 17841^T (97.82 %) and Paenibacillus forsythiae DSM 17842^T (97.22 %). However, the DNA–DNA hybridization values between strain 112^T and the type strains of these two species were 10.36 and 6.28 %, respectively. The predominant menaquinone was found to be menaquinone 7 (MK-7). The major fatty acids were determined to be anteiso-C_15:0 and C_16:0. The major polar lipids were found to be diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and unknown aminophospholipids. The cell wall peptidoglycan was found to contain meso-diaminopimelic acid. The DNA G+C content was determined to be 55.4 mol%. On the basis of its phenotypic characteristics, 16S rRNA gene sequence analysis and the value of DNA–DNA hybridization, strain 112^T is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus brassicae sp. nov. is proposed. The type strain is 112^T (= ACCC 01125^T = DSM 24983^T).     

Cryptococcus lacticolor sp. nov. and Rhodotorula oligophaga sp. nov., novel yeasts isolated from the nasal smear microbiota of Queensland koalas kept in Japanese zoological parks

A total of 515 yeast strains were isolated from the nasal smears of Queensland koalas and their breeding environments in Japanese zoological parks between 2005 and 2012. The most frequent species in the basidiomycetous yeast biota isolated from koala nasal passages was Cryptococcus neoformans, followed by Rhodotorula minuta. R. minuta was the most frequent species in the breeding environments, while C. neoformans was rare. Seven strains representing two novel yeast species were identified. Analyses of the 26S rDNA (LSU) D1/D2 domain and nuclear ribosomal DNA internal transcribed spacer region sequences indicated that these strains represent new species with close phylogenetic relationships to Cryptococcus and Rhodotorula. A sexual state was not found for either of these two novel yeasts. Key phenotypic characters confirmed that these strains could be placed in Cryptococcus and Rhodotorula. The names Cryptococcus lacticolor sp. nov. (type strain TIMM 10013^T = JCM 15449^T = CBS 10915^T = DSM 21093^T, DDBJ/EMBL/Genbank Accession No.; AB375774 (ITS) and AB375775 (26S rDNA D1/D2 region), MycoBank ID; MB 802688, Fungal Barcoding Database ID; 3174), and Rhodotorula oligophaga sp. nov. (type strain TIMM 10017^T = JCM 18398^T = CBS 12623^T = DSM 25814^T, DDBJ/EMBL/Genbank Accession No.; AB702967 (ITS) and AB702967 (26S rDNA D1/D2 region), MycoBank ID; MB 802689, Fungal Barcoding Database ID; 3175) are proposed for these new species.     

Paenibacillus beijingensis sp. nov., a nitrogen-fixing species isolated from wheat rhizosphere soil

A novel nitrogen-fixing bacterium, BJ-18^T, was isolated from wheat rhizosphere soil. Strain BJ-18^T was observed to be Gram-positive, facultatively anaerobic, motile and rod-shaped (0.4–0.9 μm × 2.0–2.9 μm). Phylogenetic analysis based on a partial nifH gene sequence and an assay for nitrogenase activity showed its nitrogen-fixing capacity. Phylogenetic analysis based on full 16S rRNA gene sequences suggested that strain BJ-18^T is a member of the genus Paenibacillus. High similarity of 16S rRNA gene sequence was found between BJ-18^T and Paenibacillus peoriae DSM 8320^T (99.05 %), Paenibacillus jamilae DSM 13815^T (98.86 %), Paenibacillus brasiliensis DSM 13188^T (98.55 %), Paenibacillus polymyxa DSM 36^T (98.74 %), Paenibacillus terrae DSM 15891^T (97.99 %) and Paenibacillus kribbensis JCM 11465^T (97.92 %), whereas the similarity was below 96.0 % between BJ-18^T and the other Paenibacillus species. DNA–DNA relatedness between strain BJ-18^T and P. peoriae DSM 8320^T, P. jamilae DSM 13815^T, P. brasiliensis DSM 13188^T, P. polymyxa DSM 36^T, P. kribbensis JCM 11465^T and P. terrae DSM 15891^T was determined to be 43.6 ± 2.7, 34.2 ± 5.3, 47.9 ± 6.6, 36.8 ± 3.5, 27.4 ± 4.3 and 23.6 ± 4.1 % respectively. The DNA G+C content of BJ-18^T was determined to be 45.8 mol %. The major fatty acid was identified as anteiso-C_15:0 (67.1 %). The polar lipids present in strain BJ-18^T were identified as diphosphatidylglycerol, phosphatidyl methylethanolamine, phosphatidylethanolamine and phosphatidylglycerol. The phenotypic and genotypic characteristics, and DNA–DNA relatedness data, suggest that BJ-18^T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus beijingensis sp. nov. (Type strain BJ-18^T=DSM25425^T=CGMCC 1.12045^T) is proposed.     

Paenibacillus taohuashanense sp. nov., a nitrogen-fixing species isolated from rhizosphere soil of the root of Caragana kansuensis Pojark

A nitrogen-fixing bacterium, designated strain gs65^T, was isolated from a rhizosphere soil sample of Caragana kansuensis Pojark. Phylogenetic analysis based on a fragment of the nifH gene and the full-length 16S rRNA gene sequence revealed that strain gs65^T is a member of the genus Paenibacillus. High levels of 16S rRNA gene similarity were found between strain gs65^T and Paenibacillus borealis DSM 13188^T (97.5 %), Paenibacillus odorifer ATCC BAA-93^T (97.3 %), Paenibacillus durus DSM 1735^T (97.0 %) and Paenibacillus sophorae DSM23020^T (96.9 %). Levels of 16S rRNA gene sequence similarity between strain gs65^T and the type strains of other recognized members of the genus Paenibacillus were below 97.0 %. Levels of DNA–DNA relatedness between strain gs65^T and P. borealis DSM 13188^T, P. odorifer ATCC BAA-93^T (97.3 %), P. durus DSM 1735^T and P. sophorae DSM23020^T were 35.9, 38.0, 34.2 and 35.5 % respectively. The DNA G+C content of strain gs65^T was determined to be 51.6 mol%. The major fatty acids were found to be iso-C_14:0, anteiso-C_15:0 and iso-C_16:0. On the basis of its phenotypic characteristics and levels of DNA–DNA hybridization, strain gs65^T is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus taohuashanense sp. nov. is proposed. The type strain is gs65^T (=CGMCC 1.12175^T = DSM 25809^T).     

A targeted high-efficiency angiogenesis strategy as therapy for myocardial infarction

AimsThe aim of this study was to prove that an intramyocardial injection of a mixture of low-dose human growth factor (HGF) plasmid and microbubbles (MB) in combination with insonation was an effective therapy for myocardial infarction.Main methodsTwenty dogs with myocardial infarction were divided into 4 groups: (1) HGF, MB and ultrasound (HGF-US/MB), (2) HGF and US (HGF-US), (3) HGF alone and (4) surgery alone (control). In the HGF-US/MB group, HGF plasmid DNA (500 μg) mixed with 0.5 ml of MB solution was injected 5 min after coronary occlusion followed by insonation. With the exception of the control group, the other dogs were divided into two groups, one treated with the HGF gene and insonation and the other with the HGF gene only.Key findingsCompared to the HGF group, infarct size decreased from 32% ± 7% (control) to 23% ± 5% in the HGF-US/MB group 28 d later (P < 0.05). Capillary density increased from 21.7 ± 4.2/mm² (control) to 114.3 ± 28.9/mm² in the HGF-US/MB group (P < 0.01). Compared to the HGF group, there was a 14% decrease in the ratio of left ventricle weight/body weight and a 25% decrease in hydroxyproline content. We also observed a 29% and 20% decrease in collagen volume fraction of type I and type III collagen, respectively in the HGF-US/MB group.SignificanceIntramyocardial injection of HGF and MB in combination with insonation enhances neovascularization and reduces ventricular remodeling and infarct size.     

Haloplanus litoreus sp. nov. and Haloplanus ruber sp. nov., from a marine solar saltern and an aquaculture farm,respectively

Two halophilic archaea, strains GX21^T and R35^T, were isolated from a marine solar saltern and an aquaculture farm in China, respectively. Cells of the two strains were observed to be pleomorphic, flat, to contain gas vesicles, stain Gram-negative and produce red-pigmented colonies. Strain GX21^T was found to be able to grow at 25–50 °C (optimum 37 °C), at 2.6–4.8 M NaCl (optimum 3.4 M NaCl), at 0.05–1.0 M MgCl₂ (optimum 0.1 M MgCl₂) and at pH 6.0–8.5 (optimum pH 6.5) while strain R35^T was found to be able to grow at 25–45 °C (optimum 37 °C), at 2.1–4.8 M NaCl (optimum 3.1 M NaCl), at 0–0.7 M MgCl₂ (optimum 0.03 M MgCl₂) and at pH 5.5–9.5 (optimum pH 6.5–7.0). The cells of both isolates were observed to lyse in distilled water. The minimum NaCl concentrations that prevented cell lysis were determined to be 15 % (w/v) for strain GX21^T and 12 % (w/v) for strain R35^T. The major polar lipids of the two strains were identified as phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, one major glycolipid and a minor lipid chromatographically identical to sulfated mannosyl glucosyl diether and mannosyl glucosyl diether, respectively. 16S rRNA gene sequence analysis revealed that strains GX21^T and R35^T show 97.1 % sequence similarity to each other and are closely related to Haloplanus aerogenes TBN37^T (96.8 and 95.8 %), Haloplanus vescus RO5-8^T (96.7 and 96.1 %), Haloplanus salinus YGH66^T (96.4 and 95.8 %) and Haloplanus natans JCM 14081^T (96.3 and 95.4 %). The rpoB′ gene similarity between strains GX21^T and R35^T is 90.5 % and show 88.5–90.8 % similarity to the Haloplanus species with validly published names. The DNA G+C content of strain GX21^T and R35^T were determined to be 65.8 and 66.0 mol%, respectively. The DNA–DNA hybridization values between strain GX21^T and strain R35^T, and the two strains with the Haloplanus species with validly published names, showed less than 50 % DNA–DNA relatedness. It was concluded that strain GX21^T (=CGMCC 1.10456^T = JCM 17092^T) and strain R35^T (=CGMCC 1.10594 ^T = JCM 17271^T) represent two new species of Haloplanus, for which the names Haloplanus litoreus sp. nov. and Haloplanus ruber sp. nov. are proposed.     

Haloarchaeobius litoreus sp. nov., isolated from a marine solar saltern

Two extremely halophilic archaeal strains GX1^T and GX60 were isolated from the Gangxi marine solar saltern, China. Cells from the two strains were observed to be rod-shaped and stained Gram-negative, with red-pigmented colonies. Strains GX1^T and GX60 were found to be able to grow at 25–50 °C (optimum 37 °C), at 1.4–4.8 M NaCl (optimum 2.6 M), at pH 5.5–9.5 (optimum pH 7.0) and neither strain required Mg²⁺ for growth. The cells lysed in distilled water and the minimal NaCl concentration to prevent cell-lysis was found to be 8 % (w/v). The major polar lipids of the two strains were identified as phosphatidic acid, phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and three glycolipids chromatographically identical to those of Haloarchaeobius iranensis IBRC-M 10013^T. 16S rRNA gene analysis revealed that each strain had two dissimilar 16S rRNA genes and both strains were phylogenetically related to Hab. iranensis IBRC-M 10013^T (94.9–98.9 % nucleotide identity). The rpoB′ gene similarity between strains GX1^T and GX60, and between these strains and Hab. iranensis IBRC-M 10013^T were found to be 99.6, 96.0 and 95.8 %, respectively. The DNA G + C content of strain GX1^T and GX60 were determined to be 67.7 and 67.8 mol %, respectively. The DNA–DNA hybridization value of strains GX1^T and GX60 was 86 % and the two strains showed low DNA–DNA relatedness with Hab. iranensis IBRC-M 10013^T (38 and 32 %). It was concluded that strain GX1^T (= CGMCC 1.10390^T = JCM 17114^T) and strain GX60 (= CGMCC 1.10389 = JCM 17120) represent a new species of Haloarchaeobius, for which the name Haloarchaeobius litoreus sp. nov. is proposed.     

Nanoscale characterization coupled to multi-parametric optimization of Hi5 cell transient gene expression

Polyethylenimine (PEI)-based transient gene expression (TGE) is nowadays a well-established methodology for rapid protein production in mammalian cells, but it has been used to a much lower extent in insect cell lines. A fast and robust TGE methodology for suspension Hi5 (Trichoplusia ni) cells is presented. Significant differences in size and morphology of DNA:PEI polyplexes were observed in the different incubation solutions tested. Moreover, minimal complexing time (&lt; 1 min) between DNA and PEI in 150 mM NaCl solution provided the highest transfection efficiency. Nanoscopic characterization by means of cryo-EM revealed that DNA:PEI polyplexes up to 300–400 nm were the most efficient for transfection. TGE optimization was performed using eGFP as model protein by means of the combination of advanced statistical designs. A global optimal condition of 1.5 × 10⁶ cell/mL, 2.1 μg/mL of DNA, and 9.3 μg/mL PEI was achieved through weighted-based optimization of transfection, production, and viability responses. Under these conditions, a 60% transfection and 0.8 μg/10⁶ transfected cell·day specific productivity were achieved. The TGE protocol developed for Hi5 cells provides a promising baculovirus-free and worthwhile approach to produce a wide variety of recombinant proteins in a short period of time.

     

Halobellus rarus sp. nov., a halophilic archaeon from an inland salt lake of China

Two halophilic archaeal strains, YC21^T and YC77, were isolated from an inland salt lake of China. Both have pleomorphic rod-shaped cells that lyse in distilled water, stain Gram-negative and form red-pigmented colonies. They are neutrophilic, require at least 2.1 M NaCl for growth under the optimum growth temperature of 37 °C. The major polar lipids of the two strains were phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me), phosphatidylglycerol sulfate (PGS), two major glycolipids (GL1 and GL2) chromatographically identical to sulfated mannosyl glucosyl diether (S-DGD-1) and mannosyl glucosyl diether (DGD-1), respectively. Trace amounts of two unidentified lipids (GL0-1 and GL0-2) were also detected. The 16S rRNA gene sequences of the two strains are 99.9 % identical, show 94.0–98.9 % similarity to the closest relative members of Halobellus of the family Halobacteriaceae. The rpoB′ gene similarity between strains YC21^T and YC77 is 99.8 % and show 90.3–95.3 % similarity to the closest relative members of Halobellus. The DNA G+C content of strains YC21^T and YC77 were 66.1 and 66.2 mol%, respectively. The DNA–DNA hybridization value between strain YC20^T and strain YC77 was 89 %, and the two strains showed low DNA–DNA relatedness with Halobellus limi TBN53^T, the most related member of Halobellus. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strains YC21^T and YC77 represent a novel species of the genus Halobellus, for which the name Halobellus rarus sp. nov. is proposed. The type strain is YC21^T (=CGMCC 1.12121^T = JCM 18362^T).     

Pseudomonas oryzae sp. nov. isolated from a paddy soil in South China

A Gram-staining-negative, rod-shaped and motile with several polar flagellums bacterium, designated WM-3^T, was isolated from a rice paddy soil in South China. Growth occurred with 0–3.0 % (w/v) NaCl (optimum 2.0 %), at pH 5.5–9.0 (optimum pH 7.0) and at 25–42 °C (optimum 30–37 °C) in liquid Reasoner’s 2A medium. Analysis of the 16S rRNA gene and gyrB gene sequences revealed that strain WM-3^T was most closely related to the type strains of the species Pseudomonas linyingensis and Pseudomonas sagittaria. Its sequence similarities with P. linyingensis CGMCC 1.10701^T and P. sagittaria JCM 18195^T were 97.4 and 97.3 %, respectively, for 16S rRNA gene, and were 94.1 and 94.2 %, respectively, for gyrB gene. DNA–DNA hybridization between strain WM-3^T and these two type strains showed relatedness of 35.6 and 30.9 %, respectively. G+C content of genomic DNA was 69.4 mol%. The whole-cell fatty acids mainly consisted of C_16:0 (30.0 %), C_16:1 ω6c and/or C_16:1 ω7c (19.3 %) and C_18:1 ω6c and/or C_18:1 ω7c (16.3 %). The results of phenotypic, chemotaxonomic and genotypic analyses clearly indicated that strain WM-3^T belongs to genus Pseudomonas but represents a novel species, for which the name Pseudomonas oryzae sp. nov. is proposed. The type strain is WM-3^T (=KCTC 32247^T =CGMCC 1.12417^T).     

Transfection of bovine fetal fibroblast with polyethylenimine (PEI) nanoparticles: effect of particle size and presence of fetal bovine serum on transgene delivery and cytotoxicity

The development of efficient transfection protocols for livestock cells is crucial for implementation of cell-based transgenic methods to produce genetically modified animals. We synthetized fully deacylated linear 22, 87 and 217 kDa polyethylenimine (PEI) nanoparticles and compared their transfection efficiency and cytotoxicity to commercial branched 25 kDa PEI and linear 58 kDa poly(allylamine) hydrochloride. We studied the effect of PEI size and presence of serum on transfection efficiency on primary cultures of bovine fetal fibroblasts and established cells lines (HEK 293 and Hep G2). We found that transfection efficiency was affected mainly by polymer/pDNA ratio and DNA concentration and in less extent by PEI MW. In bovine fibroblast, preincubation of PEI nanoparticles with fetal bovine serum (FBS) greatly increased percentage of cells expressing the transgene (up to 82%) while significantly decreased the polymer cytotoxic effect. 87 and 217 kDa PEI rendered the highest transfection rates in HEK 293 and Hep G2 cell lines (>50% transfected cells) with minimal cell toxicity. In conclusion, our results indicate that fully deacylated PEI of 87 and 217 kDa are useful DNA vehicles for non-viral transfection of primary cultures of bovine fetal fibroblast and HEK 293 and Hep G2 cell lines.     

Halobellus litoreus sp. nov., a Halophilic Archaeon Isolated from a Chinese Marine Solar Saltern

Halophilic archaeal strain GX31^T was isolated from a marine solar saltern of China. The cells of the strain were rod-shaped and lysed in distilled water, stain Gram-negative and formed red-pigmented colonies. It was neutrophilic, and required at least 0.9 M NaCl and 0–1.0 M MgCl₂ for growth under the optimum growth temperature of 37 °C. The major polar lipids of the strain were phosphatidylglycerol (PG), PG phosphate methyl ester, PG sulphate, and two major glycolipids chromatographically identical to sulphated mannosyl glucosyl diether (S-DGD-1) and mannosyl glucosyl diether (DGD-1), respectively. Trace amounts of two unidentified lipids were also detected. On the basis of 16S rRNA gene sequence analysis, strain GX31^T was closely related to the members of Halobellus of the family Halobacteriaceae with similarities of 94.1–98.7 %. Strain GX31^T showed 89.8–95.4 % of the rpoB′ gene similarity to the members of Halobellus. The DNA G+C content of strain GX31^T was 66.8 mol%. Strain GX31^T showed low DNA–DNA relatedness with two most related members of the genus Halobellus. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strain GX31^T represent a novel species of the genus Halobellus, for which the name Halobellus litoreus sp. nov. is proposed. The type strain is GX31^T (=CGMCC 1.10387^T = JCM 17118^T).     

Ultrasound-Targeted Microbubble Destruction (UTMD) Assisted Delivery of shRNA against PHD2 into H9C2 Cells

Gene therapy has great potential for human diseases. Development of efficient delivery systems is critical to its clinical translation. Recent studies have shown that microbubbles in combination with ultrasound (US) can be used to facilitate gene delivery. An aim of this study is to investigate whether the combination of US-targeted microbubble destruction (UTMD) and polyethylenimine (PEI) (UTMD/PEI) can mediate even greater gene transfection efficiency than UTMD alone and to optimize ultrasonic irradiation parameters. Another aim of this study is to investigate the biological effects of PHD2-shRNA after its transfection into H9C2 cells. pEGFP-N1 or eukaryotic shPHD2-EGFP plasmid was mixed with albumin-coated microbubbles and PEI to form complexes for transfection. After these were added into H9C2 cells, the cells were exposed to US with various sets of parameters. The cells were then harvested and analyzed for gene expression. UTMD/PEI was shown to be highly efficient in gene transfection. An US intensity of 1.5 W/cm², a microbubble concentration of 300μl/ml, an exposure time of 45s, and a plasmid concentration of 15μg/ml were found to be optimal for transfection. UTMD/PEI-mediated PHD2-shRNA transfection in H9C2 cells significantly down regulated the expression of PHD2 and increased expression of HIF-1α and downstream angiogenesis factors VEGF, TGF-β and bFGF. UTMD/PEI, combined with albumin-coated microbubbles, warrants further investigation for therapeutic gene delivery.     

Halococcus sediminicola sp. nov., an extremely halophilic archaeon isolated from a marine sediment

A novel, red-pigmented and coccoid haloarchaeon, designated strain CBA1101^T, was isolated from a marine sediment. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain CBA1101^T is most closely related to the genus Halococcus in the family Halobacteriaceae. Strain CBA1101^T had a highest 16S rRNA gene sequence similarity of 98.4 % with Halococcus dombrowskii DSM 14522^T, followed by 93.7–98.3 % with sequences of other type strains in the genus Halococcus. The RNA polymerase subunit B′ gene sequence similarity of strain CBA1101^T with that of Halococcus qingdaonensis JCM 13587^T is 89.5 % and lower with those of other members of the genus Halococcus. Strain CBA1101^T was observed to grow at 25–40 °C, pH 6.0–9.0 and in the presence of 15–30 % (w/v) NaCl, with optimal growth at 35–40 °C, pH 7.0 and with 20 % NaCl. The cells of strain CBA1101^T are Gram-negative and did not lyse in distilled water. The major polar lipids were identified as phosphatidylglyerol, phosphatidylglycerol phosphate methyl ester, sulfated diglycosyl diether, unidentified phospholipids and unidentified glycolipids. The genomic DNA G+C content was determined 66.0 mol%. The DNA–DNA hybridization experiment showed that there was less than 40 % relatedness between strain CBA1101^T and the reference species in the genus Halococcus. Based on this polyphasic taxonomic analysis, strain CBA1101^T is considered to represent a new species in the genus Halococcus, for which the name Halococcus sediminicola sp. nov. is proposed. The type strain is CBA1101^T (=JCM 18965^T = CECT 8275^T).     

Pannonibacter indica sp. nov., a highly arsenate-tolerant bacterium isolated from a hot spring in India

A novel aerobic bacterium, strain HT23^T, able to grow on 500 mM sodium arsenate was isolated from a hot-spring sediment sample collected from Athamallik, Orissa, India. Cells of this isolate were Gram negative. Heterotrophic growth was observed at pH 6.0–11.0 and 20–45 °C. Optimum growth was observed at 37 °C and pH 7.0–10.0. The major polar lipids are diphosphatidyl glycerol, phosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl choline and phosphatidyl monomethyl ethanolamine. The major isoprenoid quinone was Q-10. 16S rRNA gene sequence analysis indicated that the bacterium clustered with the genus Pannonibacter and showed 98.9 % similarity with Pannonibacter phragmitetus C6-19^T (DSM 14782^T) and 98 % with the P. phragmitetus group B and P. phragmitetus group E strains. Levels of DNA–DNA relatedness between the strain HT23^T and P. phragmitetus C6-19^T (DSM 14782^T) and other strains of P. phragmitetus group B and group E strains were below 55 %. On the basis of phenotypic and chemotaxonomic characteristics, 16S rRNA gene sequence analysis and DNA–DNA hybridization data, strain HT23^T is considered to represent a novel species of the genus Pannonibacter, for which the name Pannonibacter indica sp. nov. is proposed. The type strain is HT23^T (=JCM 16851^T = DSM 23407^T = LMG 25769^T).     

Halorubrum rubrum sp. nov., an extremely halophilic archaeon from a Chinese salt lake

Two halophilic archaeal strains, YC87^T and YCA11, were isolated from Yuncheng salt lake in Shanxi, China. Cells of the two strains were observed to be pleomorphic rod-shaped, stained Gram-negative and produced red-pigmented colonies. Strain YC87^T was able to grow at 20–50 °C (optimum 37 °C), at 1.4–4.8 M NaCl (optimum 2.1 M NaCl), at 0.05–1.0 M MgCl₂ (optimum 0.3 M MgCl₂) and at pH 6.0–9.0 (optimum pH 7.0) while strain YCA11 was able to grow at 20–50 °C (optimum 37 °C), at 2.1–4.8 M NaCl (optimum 3.1 M NaCl), at 0.01–0.7 M MgCl₂ (optimum 0.1 M MgCl₂) and at pH 6.0–9.0 (optimum pH 7.5). The cells of both isolates were observed to lyse in distilled water. The minimum NaCl concentrations that prevented cell lysis were determined to be 8 % (w/v) for strain YC87^T and 12 % (w/v) for strain YCA11. The major polar lipids of the two strains were identified as phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and one major glycolipid chromatographically identical to sulfated mannosyl glucosyl diether; another major glycolipid and trace amounts of several unidentified lipids were also detected. The 16S rRNA gene sequences of the two strains were 99.8 % identical, showing 93.2–98.2 % similarity to members of the genus Halorubrum of the family Halobacteriaceae. The rpoB′ gene similarity between strains YC87^T and YCA11 was 99.3 % and showed 87.5–95.2 % similarity to the closest relative members of the genus Halorubrum. The DNA G+C content of strains YC87^T and YCA11 were determined to be 64.9 and 64.5 mol%, respectively. The DNA–DNA hybridization value between strain YC20^T and strain YC77 was 87 % and the two strains showed low DNA–DNA relatedness with Halorubrum cibi JCM 15757^T and Halorubrum aquaticum CGMCC 1.6377^T, the most related members of the genus Halorubrum. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strains YC87^T and YCA11 represent a novel species of the genus Halorubrum, for which the name Halorubrum rubrum sp. nov. is proposed. The type strain is YC87^T (=CGMCC 1.12124^T = JCM 18365^T).     

Solirubrobacter taibaiensis sp. nov., isolated from a stem of Phytolacca acinosa Roxb.

A white-coloured bacterium, designated strain GTJR-20^T, was isolated from a stem of Phytolacca acinosa Roxb. collected from Taibai Mountain in Shaanxi Province, north-west China, and was subjected to a taxonomic study by using a polyphasic approach. The novel isolate was found to grow optimally at 28–30 °C, at pH 7.5–8.0 and in the absence of NaCl. Cells were observed to be Gram-stain positive, strictly aerobic, rod-shaped and non-motile. The predominant respiratory quinone was identified as MK-7(H₄) and the major cellular fatty acids were identified as iso-C_16:0 (35.8 %), C_18:1 ω9c (17.7 %), C_17:1 ω6c (11.0 %), C_17:1 ω8c (7.8 %) and C_18:3 ω6c (6, 9, 12) (7.2 %). The DNA G+C content was determined to be 71.6 mol %. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain GTJR-20^T is a member of the genus Solirubrobacter and is closely related to Solirubrobacter phytolaccae GTGR-8^T (16S rRNA gene sequence similarity, 98.4 %), Solirubrobacter soli KCTC 12628^T (97.8 %), Solirubrobacter pauli KCTC 9974^T (97.7 %) and Solirubrobacter ginsenosidimutans KCTC 19420^T (97.6 %). No other recognized bacterial species showed more than 94.6 % 16S rRNA gene sequence similarity to the novel isolate. DNA–DNA relatedness values for strain GTJR-20^T with respect to its closely related neighbours S. phytolaccae GTGR-8^T, S. soli KCTC 12628^T, S. pauli KCTC 9974^T and S. ginsenosidimutans KCTC 19420^T were 48.3 ± 8.6, 21.3 ± 5.2, 36.8 ± 6.2 and 36.0 ± 5.5 %, respectively. Based on the phenotypic, phylogenetic and genotypic data, strain GTJR-20^T is considered to represent a novel species of the genus Solirubrobacter, for which the name Solirubrobacter taibaiensis sp. nov. is proposed. The type strain is GTJR-20^T (=CCTCC AB 2013308^T = KCTC 29222^T).