Regulated N-Terminal Modification of Proteins Synthesized Using a Reconstituted Cell-Free Protein Synthesis System.

The N-terminal modification of nascent proteins, such as acetylation and myristoylation, is one of the most abundant post-translational modifications. To analyze the function of the modification, it is important to compare the modified and unmodified proteins under defined conditions. However, it is technically difficult to prepare unmodified proteins because cell-based systems contain endogenous modification systems. In this study, we developed a cell-free method to conduct N-terminal acetylation and myristoylation of nascent proteins in vitro using a reconstituted cell-free protein synthesis system (PURE system). Proteins synthesized using the PURE system were successfully acetylated or myristoylated in a single-cell-free mixture in the presence of modifying enzymes. Furthermore, we performed protein myristoylation in giant vesicles, which resulted in their partial localization to the membrane. Our PURE-system-based strategy is useful for the controlled synthesis of post-translationally modified proteins.

Phospholipid synthesis inside phospholipid membrane vesicles.

Construction of living artificial cells from genes and molecules can expand our understanding of life system and establish a new aspect of bioengineering. However, growth and division of cell membrane that are basis of cell proliferation are still difficult to reconstruct because a high-yielding phospholipid synthesis system has not been established. Here, we developed a cell-free phospholipid synthesis system that combines fatty acid synthesis and cell-free gene expression system synthesizing acyltransferases. The synthesized fatty acids were sequentially converted into phosphatidic acids by the cell-free synthesized acyltransferases. Because the system can avoid the accumulation of intermediates inhibiting lipid synthesis, sub-millimolar phospholipids could be synthesized within a single reaction mixture. We also performed phospholipid synthesis inside phospholipid membrane vesicles, which encapsulated all the components, and showed the phospholipids localized onto the mother membrane. Our approach would be a platform for the construction of self-reproducing artificial cells since the membrane can grow sustainably.

Proteomic Time-Course Analysis of the Filamentous Anoxygenic Phototrophic Bacterium, Chloroflexus aurantiacus, during the Transition from Respiration to Phototrophy.

Chloroflexus aurantiacus is a filamentous anoxygenic phototrophic bacterium that grows chemotrophically under oxic conditions and phototrophically under anoxic conditions. Because photosynthesis-related genes are scattered without any gene clusters in the genome, it is still unclear how this bacterium regulates protein expression in response to environmental changes. In this study, we performed a proteomic time-course analysis of how C. aurantiacus expresses proteins to acclimate to environmental changes, namely the transition from chemoheterotrophic respiratory to photoheterotrophic growth mode. Proteomic analysis detected a total of 2520 proteins out of 3934 coding sequences in the C. aurantiacus genome from samples collected at 13 time points. Almost all proteins for reaction centers, light-harvesting chlorosomes, and carbon fixation pathways were successfully detected during the growing phases in which optical densities and relative bacteriochlorophyll c contents increased simultaneously. Combination of proteomics and pigment analysis suggests that the self-aggregation of bacteriochlorophyllide c could precede the esterification of the hydrophobic farnesyl tail in cells. Cytoplasmic subunits of alternative complex III were interchanged between oxic and anoxic conditions, although membrane-bound subunits were used for both conditions. These data highlight the protein expression dynamics of phototrophy-related genes during the transition from respiration to phototrophy.

Rapid and Facile Preparation of Giant Vesicles by the Droplet Transfer Method for Artificial Cell Construction.

Giant vesicles have been widely used for the bottom-up construction of artificial (or synthetic) cells and the physicochemical analysis of lipid membranes. Although methods for the formation of giant vesicles and the encapsulation of molecules within them have been established, a standardized protocol has not been shared among researchers including non-experts. Here we proposed a rapid and facile protocol that allows the formation of giant vesicles within 30 min. The quality of the giant vesicles encapsulating a cell-free protein expression system was comparable to that of the ones formed using a conventional method, in terms of the synthesis of both soluble and membrane proteins. We also performed protein synthesis in artificial cells using a lyophilized cell-free mixture and showed an equivalent level of protein synthesis. Our method could become a standard method for giant vesicle formation suited for artificial cell research.

Phylogenomics of Haloarchaea: The Controversy of the Genera Natrinema-Haloterrigena.

The haloarchaeal genera Natrinema and Haloterrigena were described almost simultaneously by two different research groups and some strains studied separately were described as different species of these genera. Furthermore, the description of additional species were assigned to either Natrinema or Haloterrigena, mainly on the basis of the phylogenetic comparative analysis of single genes (16S rRNA gene and more recently rpoB' gene), but these species were not adequately separated or assigned to the corresponding genus. Some studies suggested that the species of these two genera should be unified into a single genus, while other studies indicated that the genera should remain but some of the species should be reassigned. In this study, we have sequenced or collected the genomes of the type strains of species of Natrinema and Haloterrigena and we have carried out a comparative genomic analysis in order to clarify the controversy related to these two genera. The phylogenomic analysis based on the comparison of 525 translated single-copy orthologous genes and the Overall Genome Relatedness Indexes (i.e., AAI, POCP, ANI, and dDDH) clearly indicate that the species Haloterrigena hispanica, Haloterrigena limicola, Haloterrigena longa, Haloterrigena mahii, Haloterrigena saccharevitans, Haloterrigena thermotolerans, and Halopiger salifodinae should be transferred to the genus Natrinema, as Natrinema hispanicum, Natrinema limicola, Natrinema longum, Natrinema mahii, Natrinema saccharevitans, Natrinema thermotolerans, and Natrinema salifodinae, respectively. On the contrary, the species Haloterrigena turkmenica, Haloterrigena salifodinae, and Haloterrigena salina will remain as the only representative species of the genus Haloterrigena. Besides, the species Haloterrigena daqingensis should be reclassified as a member of the genus Natronorubrum, as Natronorubrum daqingense. At the species level, Haloterrigena jeotgali and Natrinema ejinorense should be considered as a later heterotypic synonyms of the species Haloterrigena (Natrinema) thermotolerans and Haloterrigena (Natrinema) longa, respectively. Synteny analysis and phenotypic features also supported those proposals.

Haloarcula mannanilytica sp. nov., a galactomannan-degrading haloarchaeon isolated from commercial salt.

A mannan-degrading halophilic archaeal strain, MD130-1T, was isolated from a commercial salt sample. Cells were motile, rod-shaped, and stained Gram-negative. Colonies were pink pigmented. Strain MD130-1T was able to grow at 1.5-4.6 M NaCl (optimum, 3.6 M) at pH 6.0-8.0 (optimum, pH 7.0) and at 25-50 °C (optimum, 40 °C). The DNA G+C content was 62.1 mol% (genome). The orthologous 16S rRNA gene sequence showed the highest similarity (99.4 %) to those of Haloarcula japonica JCM 7785T and Haloarcula hispanica JCM 8911T. The values of genome relatedness between strain MD130-1T and Haloarcula species were 84.33-85.96 % in ANIb and 30.4-32.9 % using GGDC formula 2. The polar lipids of strain MD130-1T were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and triglycosyl diether-2. Based on the results of phenotypic and phylogenetic analyses, the strain represents a new species of the genus Haloarcula, for which the name Haloarcula mannanilytica sp. nov. is proposed. The type strain is MD130-1T (=JCM 33835T=KCTC 4287T) isolated from commercial salt made in Ishikawa prefecture, Japan.

Structural comparisons of phosphoenolpyruvate carboxykinases reveal the evolutionary trajectories of these phosphodiester energy conversion enzymes.

Inorganic pyrophosphate (PPi) consists of two phosphate molecules and can act as an energy and phosphate donor in cellular reactions, similar to ATP. Several kinases use PPi as a substrate, and these kinases have recently been suggested to have evolved from ATP-dependent functional homologs, which have significant amino acid sequence similarity to PPi-utilizing enzymes. In contrast, phosphoenolpyruvate carboxykinase (PEPCK) can be divided into three types according to the phosphate donor (ATP, GTP, or PPi), and the amino acid sequence similarity of these PEPCKs is too low to confirm that they share a common ancestor. Here we solved the crystal structure of a PPi-PEPCK homolog from the bacterium Actinomyces israelii at 2.6 Å resolution and compared it with previously reported structures from ATP- and GTP-specific PEPCKs to assess the degrees of similarities and divergences among these PEPCKs. These comparisons revealed that they share a tertiary structure with significant value and that amino acid residues directly contributing to substrate recognition, except for those that recognize purine moieties, are conserved. Furthermore, the order of secondary structural elements between PPi-, ATP-, and GTP-specific PEPCKs was strictly conserved. The structure-based comparisons of the three PEPCK types provide key insights into the structural basis of PPi specificity and suggest that all of these PEPCKs are derived from a common ancestor.

Accurate high-throughput screening based on digital protein synthesis in a massively parallel femtoliter droplet array.

We report a general strategy based on digital counting principle that enables an efficient acquisition of enzyme mutants with desired activities from just a few clones within a day. We prepared a high-density femtoliter droplet array, consisting of 1 million uniform droplets per 1 cm2 to carry out high-throughput protein synthesis and screening. Single DNA molecules were randomly distributed into each droplet following a Poisson process to initiate the protein synthesis with coupled cell-free transcription and translation reactions and then recovered by a microcapillary. The protein yield in each droplet was proportional to the number of DNA molecules, meaning that droplets with apparent intensities higher than the Poisson distribution-predicted maximum can be readily identified as the exact hits exhibiting the desired increased activity. We improved the activity of an alkaline phosphatase up to near 20-fold by using less than 10 nl of reagents.

Computational Analysis of the Interaction Energies between Amino Acid Residues of the Measles Virus Hemagglutinin and Its Receptors.

Measles virus (MV) causes an acute and highly devastating contagious disease in humans. Employing the crystal structures of three human receptors, signaling lymphocyte-activation molecule (SLAM), CD46, and Nectin-4, in complex with the measles virus hemagglutinin (MVH), we elucidated computationally the details of binding energies between the amino acid residues of MVH and those of the receptors with an ab initio fragment molecular orbital (FMO) method. The calculated inter-fragment interaction energies (IFIEs) revealed a number of significantly interacting amino acid residues of MVH that played essential roles in binding to the receptors. As predicted from previously reported experiments, some important amino-acid residues of MVH were shown to be common but others were specific to interactions with the three receptors. Particularly, some of the (non-polar) hydrophobic residues of MVH were found to be attractively interacting with multiple receptors, thus indicating the importance of the hydrophobic pocket for intermolecular interactions (especially in the case of Nectin-4). In contrast, the electrostatic interactions tended to be used for specific molecular recognition. Furthermore, we carried out FMO calculations for in silico experiments of amino acid mutations, finding reasonable agreements with virological experiments concerning the substitution effect of residues. Thus, the present study demonstrates that the electron-correlated FMO method is a powerful tool to search exhaustively for amino acid residues that contribute to interactions with receptor molecules. It is also applicable for designing inhibitors of MVH and engineered MVs for cancer therapy.

Complete Genome Sequence of Sphingobium sp. Strain YG1, a Lignin Model Dimer-Metabolizing Bacterium Isolated from Sediment in Kagoshima Bay, Japan.

Sphingobium sp. strain YG1 is a lignin model dimer-metabolizing bacterium newly isolated from sediment in Kagoshima, Japan, at a depth of 102 m. Here, we report the complete genome nucleotide sequence of strain YG1.

Complete Genome Sequence of Altererythrobacter sp. Strain B11, an Aromatic Monomer-Degrading Bacterium, Isolated from Deep-Sea Sediment under the Seabed off Kashima, Japan.

Altererythrobacter sp. strain B11 is an aromatic monomer-degrading bacterium newly isolated from sediment under the seabed off Kashima, Japan, at a depth of 2,100 m. Here, we report the complete nucleotide sequence of the genome of strain B11.

Salinarchaeum chitinilyticum sp. nov., a chitin-degrading haloarchaeon isolated from commercial salt.

Two chitin-degrading halophilic archaeal strains, MC-74T and MC-23, were isolated from commercial salt samples. Cells were motile, rod-shaped and stained Gram-negative. Colonies were vermillion-pigmented. Strains MC-74T and MC-23 were able to grow with 1.5-5.1 M NaCl (optimum, 2.6-3.1 M) at pH 6.0-10.0 (optimum, pH 7.0) and at 20-50 °C (optimum, 40 °C). The orthologous 16S rRNA gene sequence similarity between the two strains was 99.8 %, and the closest phylogenetic relative was Salinarchaeum laminariae JCM 17267T with 99.3-99.5 % similarity. The level of DNA-DNA relatedness between the two strains was 93 and 94 % (reciprocally), and those between the two strains and Salinarchaeumlaminariae JCM 17267T were 35-36 % and 38-39 % (reciprocally). The polar lipids of both strains were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and phosphatidylglycerol sulfate. Glycolipids were not detected. Based on the phenotypic and phylogenetic analyses, the strains represent a novel species of the genus Salinarchaeum, for which the name Salinarchaeum chitinilyticum sp. nov. is proposed. The type strain is MC-74T (=JCM 19597T=KCTC 4262T), isolated from solar salt produced in France. Strain MC-23, isolated from a commercial solar salt sample produced in China, is an additional strain of the species.

Haloparvum alkalitolerans sp. nov., alkali-tolerant haloarchaeon isolated from commercial salt.

A Gram-stain-negative, rod-pleomorphic, aerobic, halophilic archaeon, strain MK62-1T, was isolated from commercial salt made from seawater in the Philippines. Strain MK62-1T was able to grow at 2.1-4.7 M NaCl (with optimum at 2.1-2.6 M NaCl), pH 6.5-9.5 (optimum, pH 7.0-7.5) and 20-55 °C (optimum, 45-50 °C). Based on the orthologous 16S rRNA gene sequence, the closest relative was Haloparvum sedimenti JCM 30891T with 99.2 % similarity. The RNA polymerase subunit B' gene sequence also showed the highest similarity (97.4 %) to that of Haloparvum sedimenti DYS4T. The DNA G+C content of MK62-1T was 70.1 mol%, while that of Haloparvum sedimenti JCM 30891T was 69.5 mol% by the HPLC method. The levels of DNA-DNA relatedness between MK62-1T and Haloparvum sedimenti JCM 30891T were 60.6 and 60.8 % (reciprocally). The major polar lipids of the isolate were C20C20 archaeol derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and phosphatidylglycerol sulfate. Based on the phenotypic and phylogenetic analyses, it is proposed that the isolate represents species within the genus Haloparvum, for which the name Haloparvum alkalitolerans sp. nov. is proposed. The type strain is MK62-1T (=JCM 30442T =KCTC 4214T).

Halopiger thermotolerans sp. nov., a thermo-tolerant haloarchaeon isolated from commercial salt.

Three thermo-tolerant halophilic archaeal strains, SR-441T, SR-412 and SR-188, were isolated from commercial salt samples. Cells were non-motile pleomorphic rod-shaped, and stained Gram-negative. Colonies were pink-pigmented. The three strains were able to grow with 1.7-4.6 M NaCl (optimum, 2.5 M), at pH 6.5-9.0 (optimum, pH 8.0) and at 35-60 °C (optimum, 45 °C). The orthologous 16S rRNA gene sequence similarities amongst the three strains were 98.8-99.3 %, and the level of DNA-DNA relatedness was 71-74 and 72-75 % (reciprocally). The closest relative was Halopiger aswanensis JCM 11628T with 98.6 %-99.1 % similarity in the orthologous 16S rRNA gene sequences, followed by two more Halopiger species, Halopiger xanaduensis JCM 14033T (98.5 %-99.1 %) and Halopiger salifodinae JCM 9578T (95.5 %-95.6 %). DNA-DNA relatednesses between the three strains and H. aswanensis JCM 11628T and H. xanaduensis JCM 14033T were 61 and 54 %, respectively. The polar lipids of the three novel strains were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, and bis-sulfated diglycosyl archaeol-1. The most distinctive feature of the three strains was the ability to grow at 60 °C, while the maximum growth temperature of H. aswanensis is 55 °C. Based on phenotypic and phylogenetic analyses, the isolates are considered to represent a novel species of the genus Halopiger, for which the name Halopiger thermotolerans sp. nov. is proposed. The type strain is SR-441T (=JCM 19583T=KCTC 4248T) isolated from solar salt produced in Australia. SR-412 (=JCM 19582) and SR-188 (=JCM 19581) isolated from commercial salt samples are additional strains of the species.

Halorubrum gandharaense sp. nov., an alkaliphilic haloarchaeon from commercial rock salt.

A Gram-stain-negative, non-motile, pleomorphic rod-shaped, orange-red-pigmented, facultatively aerobic and haloalkaliphilic archaeon, strain MK13-1T, was isolated from commercial rock salt imported from Pakistan. The NaCl, pH and temperature ranges for growth of strain MK13-1T were 3.0-5.2 M NaCl, pH 8.0-11.0 and 15-50 °C, respectively. Optimal growth occurred at 3.2-3.4 M NaCl, pH 9.0-9.5 and 45 °C. Addition of Mg2+ was not required for growth. The major polar lipids of the isolate were C20C20 and C20C25 archaeol derivatives of phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester. Glycolipids were not detected. The DNA G+C content was 64.1 mol%. The 16S rRNA gene sequence of strain MK13-1T was most closely related to those of the species of the genus Halorubrum, Halorubrum luteum CECT 7303T (95.9% similarity), Halorubrum alkaliphilum JCM 12358T (95.3%), Halorubrum kocurii JCM 14978T (95.3%) and Halorubrum lipolyticum JCM 13559T (95.3%). The rpoB' gene sequence of strain MK13-1T had < 90% sequence similarity to those of other members of the genus Halorubrum. Based on the phylogenetic analysis and phenotypic characterization, strain MK13-1T may represent a novel species of the genus Halorubrum, for which the name Halorubrum gandharaense sp. nov. is proposed, with the type strain MK13-1T ( = JCM 17823T = CECT 7963T).

Aneurinibacillus tyrosinisolvens sp. nov., a tyrosine-dissolving bacterium isolated from organics- and methane-rich seafloor sediment.

A novel Gram-positive-staining, strictly aerobic and heterotrophic bacterium, designated strain LL-002T, was isolated from organics- and methane-rich seafloor sediment at a depth of 100 m in Kagoshima Bay, Kagoshima, Japan. Colonies were lustreless and translucent white in colour. The temperature, pH and salt concentration ranges for growth were 10-30 °C, pH 6.0-6.5 and 0-1 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences confirmed that strain LL-002T belongs to the genus Aneurinibacillus of the family Paenibacillaceae. 16S rRNA gene sequence similarities between strain LL-002T and the type strains of species of the genus Aneurinibacillus were 92.8-95.7 %; the highest sequence identity was with the type strain of Aneurinibacillus migulanus. The DNA G+C content of strain LL-002T was 46.2 mol%. MK-7 was the predominant menaquinone. The predominant cellular fatty acids were iso-C15 : 0 and anteiso-C15 : 0, and the cell-wall peptidoglycan contained meso-diaminopimelic acid and glutamic acid, glycine and alanine in addition to muramic acid and glucosamine. The peptidoglycan type was A1γ. In DNA-DNA hybridization assays between strain LL-002T and the type strains of the other species of the genus Aneurinibacillus, the level of hybridization was 6.3-30.1 %. On the basis of its biological features and the 16S rRNA gene sequence comparison presented here, strain LL-002T is considered to represent a novel species of the genus Aneurinibacillus, for which the name Aneurinibacillus tyrosinisolvens sp. nov. is proposed; the type strain is LL-002T ( = NBRC 110097T = CECT 8536T).

Halococcus agarilyticus sp. nov., an agar-degrading haloarchaeon isolated from commercial salt.

Two agar-degrading halophilic archaeal strains, 62 E(T) and 197 A, were isolated from commercial salt samples. Cells were non-motile cocci, approximately 1.2-2.0 µm in diameter and stained Gram-negative. Colonies were pink-pigmented. Strain 62 E(T) was able to grow with 24-30% (w/v) NaCl (optimum, 27%), at pH 6.5-8.5 (optimum, pH 7.5) and at 22-47 °C (optimum, 42 °C). The 16S rRNA gene sequences of strains 62 E(T) and 197 A were identical, and the level of DNA-DNA relatedness between them was 90 and 90% (reciprocally). The closest relative was Halococcus saccharolyticus JCM 8878(T) with 99.7% similarity in 16S rRNA orthologous gene sequences, followed by Halococcus salifodinae JCM 9578(T) (99.6%), while similarities with other species of the genus Halococcus were equal to or lower than 95.1%. The rpoB' gene tree strongly supported that the two strains were members of the genus Halococcus . Mean DNA-DNA relatedness between strain 62 E(T) and H. saccharolyticus JCM 8878(T) and H. salifodinae JCM 9578(T) was 46 and 44%, respectively. The major polar lipids were archaeol derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, derived from both C20C20 and C20C25 archaeol, and sulfated diglycosyl archaeol-1. Several unidentified glycolipids were present. Based on the phenotypic and phylogenetic analyses, the isolates are considered to represent a novel species of the genus Halococcus , for which the name Halococcus agarilyticus sp. nov. is proposed. The type strain is 62 E(T) ( = JCM 19592(T) =KCTC 4143(T)).

Halocalculus aciditolerans gen. nov., sp. nov., an acid-tolerant haloarchaeon isolated from commercial salt.

Three halophilic archaeal strains, MH2-243-1(T), MH2-93-1 and MH2-91-1 were isolated from commercial salt samples from Japan, Australia, and Bolivia. Strain MH2-243-1(T) was able to grow in the presence of 12-30% (w/v) NaCl (optimum, 18% NaCl), at pH 4.5-7.0 (optimum, pH 6.0) and at 20-60 °C (optimum, 40 °C). Strains MH2-91-1 and MH2-93-1 grew in slightly different ranges. The orthologous 16S rRNA gene sequences of the three strains were almost identical (99.8-99.9% similarities), and the closest relative was Salarchaeum japonicum JCM 16327(T) with 94.2-94.3% 16S rRNA gene sequence similarities, followed by strains of members of the closely related genera Halobacterium and Halarchaeum . The RNA polymerase subunit B' gene (rpoB') sequence also showed the highest similarity (86.6%) to that of Salarchaeum japonicum JCM 16327(T). The DNA G+C contents of strains MH2-243-1(T), MH2-93-1 and MH2-91-1 were 68.5, 68.8 and 68.3 mol%, respectively. DNA-DNA relatedness values amongst the three strains were 97-99%. The polar lipids of the three strains were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, and at least seven unidentified glycolipids. The polar lipid composition differed from those of Salarchaeum japonicum and species of the genera Halobacterium and Halarchaeum . Based on the phenotypic and phylogenetic analyses, it is proposed that the isolates represent a novel species of a new genus, for which the name Halocalculus aciditolerans gen. nov., sp. nov. is proposed. The type strain of the type species is MH2-243-1(T) ( = JCM 19596(T) =KCTC 4149(T)) isolated from solar salt produced in Japan. MH2-93-1 ( = JCM 19595) and MH2-91-1 ( = JCM 19594) are additional strains of the type species.

Halarchaeum grantii sp. nov., a moderately acidophilic haloarchaeon isolated from a commercial salt sample.

Three moderately acidophilic, halophilic archaeal strains, MH1-243-3T, MH1-243-5 and MH1-243-6, were isolated from a commercial salt sample made from seawater in Okinawa, Japan. Cells of the three strains were pleomorphic and stained Gram-negative. Colonies of the strains were orange-red-pigmented. Strain MH1-243-3T was able to grow at 15-27 % (w/v) NaCl (optimum 24 °C), at pH 4.5-6.5 (pH 5.5) and at 35-50 °C (45 °C). Strains MH1-243-5 and MH1-243-6 grew within slightly different ranges (shown in text). The 16S rRNA gene sequences of the three strains were identical, and the closest phylogenetic relative was Halarchaeum salinum MH1-34-1T with 97.0 % similarity. The rpoB' gene sequences of the three strains were also identical, and the closest phylogenetic relative was Halarchaeum acidiphilum JCM 16109T with 92.0 % similarity. The DNA G+C content of MH1-243-3T, MH1-243-5 and MH1-243-6 was 65.2 mol%. The levels of DNA-DNA relatedness amongst the three strains were 84.1-99.8 %, while that between MH1-243-3T and H. salinum MH1-34-1T was 30.6 % and 31.6 % (reciprocally), and those between MH1-243-3T and type strains of other species in the genus Halarchaeum were 42.3-29.4 %. Based on the phenotypic, genotypic and phylogenetic analyses, it is proposed that the isolates should represent a novel species of the genus Halarchaeum, for which the name Halarchaeum grantii sp. nov. is proposed. The type strain is MH1-243-3T ( = JCM 19585T = KCTC 4142T), isolated from commercial sea salt produced in Okinawa, Japan. MH1-243-5 ( = JCM 19586) and MH1-243-6 ( = JCM 18422) are additional strains of the species.

Structurally distinct hybrid polymer/lipid nanoconstructs harboring a type-I ribotoxin as cellular imaging and glioblastoma-directed therapeutic vectors.

A nanoformulation composed of a ribosome inactivating protein-curcin and a hybrid solid lipid nanovector has been devised against glioblastoma. The structurally distinct nanoparticles were highly compatible to human endothelial and neuronal cells. A sturdy drug release from the particles, recorded upto 72 h, was reflected in the time-dependent toxicity. Folate-targeted nanoparticles were specifically internalized by glioma, imparting superior toxicity and curbed an aggressively proliferating in vitro 3D cancer mass in addition to suppressing the anti-apoptotic survivin and cell matrix protein vinculin. Combined with the imaging potential of the encapsulated dye, the nanovector emanates as a multifunctional anti-cancer system.