Natrinema soli sp. nov., a novel halophilic archaeon isolated from a hypersaline wetland.

An extremely halophilic archaeon, designated strain 5-3T, was isolated from a soil sample of Meighan wetland in Iran. Strain 5-3T was strictly aerobic, catalase-positive and oxidase-negative. Cells were Gram-stain-negative, non-motile and ovoid. Colonies of strain 5-3T were cream-coloured. The isolate showed optimum growth at 4.0 M NaCl, 40 °C and pH 7.0. The major polar lipids of the strain were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, two unknown phospholipids and three glycolipids (including one that was chromatographically identical to S2-DGD). The major respiratory quinone was menaquinone MK-8. The G+C content of the genomic DNA was 61.5 mol%. The closest relative was Natrinema salaciae JCM 17869T with 97.3 % similarity in the orthologous 16S rRNA gene sequence. Analysis of 16S rRNA and rpoB' gene sequences indicated that strain 5-3T is a member of the genus Natrinema in the family Natrialbaceae and forms a distinct cluster. On the basis of phylogenetic analysis, and phenotypic and chemotaxonomic characteristics, a novel species of the family Natrialbaceae, Natrinema soli sp. nov., is proposed. The type strain is 5-3T (=IBRC-M 11063T=LMG 29247T).

Inter-microcarrier transfer and phenotypic stability of stem cell-derived Schwann cells in stirred suspension bioreactor culture.

Emerging bioreactor technologies offer an effective way for scaled-up production of large numbers of cells for cell therapy applications. One of the clinical paradigms where cell therapy can be an asset is restorative neurosciences. Nerve repair can benefit from the injections of stem cells and/or Schwann cells, acting as a source for axon myelination, myelin debris clearance, and trophic support. We have adapted microcarrier-based suspension bioreactor culture for Schwann cells (SCs) differentiated from a new stem cell source - skin-derived precursors (SKPs). SKP-derived SCs attach and grow on different types of microcarriers in both static and stirred culture, with Cytodex 3 and CultiSpher-S found most effective. Inter-microcarrier migration of SKP-SCs represents a key mechanism for rapid expansion and colonization in stirred suspension culture. We have shown that microcarrier-expanded SKP-SCs cells express Schwann cell markers p75-NTR, GFAP and S100 and retain their key ability to myelinate axons both in vitro and in vivo. Scaled-up microcarrier-based production of SKP-SCs in suspension bioreactors appears feasible for timely generation of sufficient cell numbers for nerve repair strategies.

Feasibility of [18F]-RGD for ex vivo imaging of atherosclerosis in detection of αvß3 integrin expression.

BACKGROUND: Inflammation and angiogenesis play an important role in atherosclerotic plaque rupture. Therefore, molecular imaging of these processes could be used for determination of rupture-prone atherosclerotic plaques. αvß3 integrin is involved in the process of angiogenesis. Targeted imaging of αvß3 integrin has been shown to be possible in previous studies on tumor models, using radiolabeled arginine-glycine-aspartate (RGD). Our aim was to investigate feasibility of ex vivo detection of αvß3 integrin in carotid endarterectomy (CEA) specimens. METHODS AND RESULTS: Nineteen CEA specimens were incubated in 5 MBq [18F]-RGD-K5 for 1 hour followed by 1 hour emission microPET scan. The results were quantified in 4 mm wide segments as percent incubation dose per gram (%Inc/g). Segmental-to-total ratio was calculated and presence of αvß3 integrin and endothelial cells in each segment was confirmed by immunohistochemical staining for CD31 and αvß3 integrin, respectively. [18F]-RGD-K5 uptake was heterogeneously distributed across CEA specimens and was localized within the vessel wall. Significant correlations were observed between segmental-to-total ratio with αvß3 integrin staining score (r = 0.58, P = .038) and CD31 staining score (ρ = 0.67, P < .002). CONCLUSION: This study showed the feasibility of integrin imaging by determination of αvß3 integrin expression in human atherosclerotic plaques.

Taxogenomics of the Genus Cyclobacterium: Cyclobacterium xiamenense and Cyclobacterium halophilum as Synonyms and Description of Cyclobacterium plantarum sp. nov.

The genus Cyclobacterium belongs to the phylum Bacteroidetes and includes eight species. Our study, based on the genomic parameters in silico DNA-DNA hybridization (GGDC), average nucleotide identity (OrthoANI), and average amino acid identity (AAI), confirmed that all current species of Cyclobacterium belong to this genus and constitute a coherent phylogenomic group, but with species forming two separate branches. In addition, the genome-based analyses revealed that Cyclobacterium xiamenense and Cyclobacterium halophilum are members of the same species. Besides, we carried out a taxonomic characterization of the new strain GBPx2T, isolated from the halophytic plant Salicornia sp. Analysis of its 16S rRNA gene sequence showed the highest sequence similarity (97.5%) to Cyclobacterium lianum HY9T. Percentages of GGDC and OrthoANI between strain GBPx2T and species of the genus Cyclobacterium were lower than the threshold value for species delineation. The DNA G+C content was 43.0 mol%. The polar lipids included phosphatidylethanolamine as well as one unidentified phospholipid and four unidentified lipids, and its major cellular fatty acids were iso-C15:0 and summed feature 3 (C16:1ω7c and/or iso-C15:0 2-OH). The only quinone present was menaquinone 7. Based on a combination of phenotypic, chemotaxonomic, and phylogenomic features, the GBPx2T strain represents a novel species of the genus Cyclobacterium, for which the name Cyclobacterium plantarum sp. nov. is proposed. The type strain of Cyclobacterium plantarum is GBPx2T (= IBRC-M 10634T = LMG 28551T).

Phosphoramidite accelerated copper(i)-catalyzed [3 + 2] cycloadditions of azides and alkynes.

Monodentate phosphoramidite ligands are used to accelerate the copper(i)-catalyzed 1,3-dipolar cycloaddition of azides and alkynes (CuAAC) rapidly yielding a wide variety of functionalized 1,4-disubstituted-1,2,3-triazoles; Cu(i) and Cu(ii) salts both function as the copper source in aqueous solution to provide excellent yields.

Copper-free 'click': 1,3-dipolar cycloaddition of azides and arynes.

Arynes formed through fluoride-promoted ortho-elimination of o-(trimethylsilyl)aryl triflates can undergo [3 + 2] cycloaddition with various azides to form substituted benzotriazoles. The rapid reaction times and mild conditions make this an attractive variation of the classical 'click' reaction of azides and alkynes.

Serum-free bioprocessing of adult human and rodent skin-derived Schwann cells: implications for cell therapy in nervous system injury.

Peripheral nerve injury affects 2.8% of trauma patients with severe cases often resulting in long-lived permanent disability, despite nerve repair surgery. Autologous Schwann cell (SC) therapy currently provides an exciting avenue for improved outcomes for these patients, particularly with the possibility to derive SCs from easily-accessible adult skin. However, due to current challenges regarding the efficient expansion of these cells, further optimization is required before they can be seriously considered for clinical application. Here, a microcarrier-based bioreactor system is proposed as a means to scale-up large numbers of adult skin-derived SCs for transplantation into the injured nerve. Bioprocessing parameters that allow for the expansion of adult rodent SCs have been identified, whilst maintaining similar rates of proliferation (as compared to static-grown SCs), expression of SC markers, and, importantly, their capacity to myelinate axons following transplant into the injured sciatic nerve. The same bioprocessing parameters can be applied to SCs derived from adult human skin, and like rodent cells, they sustain their proliferative potential and expression of SC markers. Taken together, this dataset demonstrates the basis for a scalable bioprocess for the production of SCs, an important step towards clinical use of these cells as an adjunct therapy for nerve repair. Copyright © 2017 John Wiley & Sons, Ltd.

[(18)F]-(fluoromethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)propan-2-ol ([(18)F FPTC) a novel PET-ligand for cerebral beta-adrenoceptors.

UNLABELLED: Cerebral ß-adrenergic receptors (ß-ARs) play important roles in normal brain and changes of ß-AR expression are associated with several neuropsychiatric illnesses. Given the high density of ß-AR in several brain regions, quantification of ß-AR levels using PET is feasible. However, there is a lack of radiotracers with suitable biological properties and meeting safety requirements for use in humans. We developed a PET tracer for ß-AR by (18)F-fluorination of 1-((9H-carbazol-4-yl)oxy)-3-4(4-((2-(2-(fluoromethoxy)-ethoxy)methyl)-1H-1,2,3-triazol-1-yl)propan-2-ol ((18)F-FPTC). METHODS: [(18)F] FPTC was synthesized by Cu(I)-catalyzed alkyne-azide cycloaddition. First, (18)F-PEGylated alkyne was prepared by (18)F-fluorination of the corresponding tosylate. Next (18)F-PEGylated alkyne was reacted with an azidoalcohol derivative of 4-hydroxycarbazol in the presence of the phosphoramidite Monophos as a ligand and Cu(I) as a catalyst. After purification with radio-HPLC, the binding properties of [(18)F FPTC were tested in ß-AR-expressing C6-glioma cells in vitro and in Wistar rats in vivo using microPET. RESULTS: The radiochemical yield of (18)F-PEGylated alkyne was 74%-89%. The click reaction to prepare [(18)F]FPTC proceeded in 10min with a conversion efficiency of 96%. The total synthesis time was 55min from the end of bombardment. Specific activities were >120GBq/µmol. Propranolol strongly and dose-dependently inhibited the binding of both [(125)I]-ICYP and [(18)F]FPTC to C6 glioma cells, with IC50 values in the 50-60 nM range. However, although both FPTC and propranolol inhibited cellular [(125)I]ICYP binding, FPTC decreased [(125)I]ICYP uptake by only 25%, whereas propranolol reduced it by 83%. [(18)F]FPTC has the appropriate lipophilicity to penetrate the blood brain barrier (logP +2.48). The brain uptake reached a maximum within 2min after injection of 20-25MBq [(18)F]FPTC. SUV values ranged from 0.4 to 0.6 and were not reduced by propranolol. Cerebral distribution volume of the tracer (calculated from a Logan plot) was increased rather than decreased after propranolol treatment. CONCLUSION: 'Click chemistry' was successfully applied to the synthesis of [(18)F]FPTC resulting in high radiochemical yields. [(18)F]FPTC showed specific binding in vitro, but not in vivo. Based on the logP value and its ability to block [(125)I]ICYP binding to C6 cells, FPTC may be a lead to suitable cerebral ß-AR ligands.

Synthesis of [18F]RGD-K5 by catalyzed [3 + 2] cycloaddition for imaging integrin αvß3 expression in vivo.

In the last few years click chemistry reactions, and in particular copper-catalyzed cycloadditions have been used extensively for the preparation of new bioconjugated molecules such as (18)F-radiolabeled radiopharmaceuticals for positron emission tomography (PET). This study is focused on the synthesis of the Siemens imaging biomarker [(18)F]RGD-K5. This cyclic peptide contains an amino acid sequence which is a well known binding motif for integrin αvß3 involved in cellular adhesion to the extracellular matrix. We developed an improved "click" chemistry method using Cu(I)-Monophos as catalyst to conjugate [(18)F]fluoropentyne to the RGD-azide precursor yielding [(18)F]RGD-K5. A comparison is made with the registered Siemens method with respect to synthesis, purification and quality control. [(18)F]RGD-K5 was obtained after 75 min overall synthesis time with an overall radiochemical yield of 35% (EOB). The radiochemical purity was >98% and the specific radioactivity was 100-200 GBq/µmol at the EOS.