Albumin Fusion at the N-Terminus or C-Terminus of HM-3 Leads to Improved Pharmacokinetics and Bioactivities.

HM-3, an integrin antagonist, exhibits anti-tumor biological responses and therefore has potential as a therapeutic polypeptide. However, the clinical applications of HM-3 are limited by its short half-life. In this study, we genetically fused human serum albumin (HSA) to the N or C-terminus of HM-3 to improve HM-3 pharmacokinetics. HM-3/HSA proteins were successfully expressed in Pichia pastoris and displayed improved pharmacokinetic properties and stability. Among them, the half-life of HM-3-HSA was longer than HSA-HM-3. In vitro, the IC50 values of HSA-HM-3 and HM-3-HSA were 0.38 ± 0.14 µM and 0.25 ± 0.08 µM in B16F10 cells, respectively. In vivo, the inhibition rates of B16F10 tumor growth were 36% (HSA-HM-3) and 56% (HM-3-HSA), respectively, indicating antitumor activity of HM-3-HSA was higher than HSA-HM-3. In conclusion, these results suggested that the HM-3/HSA fusion protein might be potential candidate HM-3 agent for treatment of melanoma and when HSA was fused at the C-terminus of HM-3, the fusion protein had a higher stability and activity.

Vulcaniibacterium gelatinicum sp. nov., a moderately thermophilic bacterium isolated from a hot spring.

The present study aimed to determine the taxonomic positions of strains designated R-5-52-3T, R-5-33-5-1-2, R-5-48-2 and R-5-51-4 isolated from hot spring water samples. Cells of these strains were Gram-stain-negative, non-motile and rod-shaped. The strains shared highest 16S rRNA gene sequence similarity with Vulcaniibacterium thermophilum KCTC 32020T (95.1%). Growth occurred at 28-55 °C, at pH 6-8 and with up to 3 % (w/v) NaCl. DNA fingerprinting, biochemical, phylogenetic and 16S rRNA gene sequence analyses suggested that R-5-52-3T, R-5-33-5-1-2, R-5-48-2 and R-5-51-4 were different strains but belonged to the same species. Hence, R-5-52-3T was chosen for further analysis and R-5-33-5-1-2, R-5-48-2 and R-5-51-4 were considered as additional strains of this species. R-5-52-3T possessed Q-8 as the only quinone and iso-C15:0, iso-C11:0, C16 : 0 and iso-C17 : 0 as major fatty acids. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, unidentified polar lipids and two unidentified phospholipids. The genomic G+C content was 71.6 mol%. Heat shock proteins (e.g. Hsp20, GroEL, DnaK and Clp ATPases) were noted in the R-5-52-3T genome, which could suggest its protection in the hot spring environment. Pan-genome analysis showed the number of singleton gene clusters among Vulcaniibacterium members varied. Average nucleotide identity (ANI) values between R-5-52-3T, Vulcaniibacterium tengchongense YIM 77520T and V. thermophilum KCTC 32020T were 80.1-85.8 %, which were below the cut-off level (95-96 %) recommended as the ANI criterion for interspecies identity. Thus, based on the above results, strain R-5-52-3T represents a novel species of the genus Vulcaniibacterium, for which the name Vulcaniibacterium gelatinicum sp. nov. is proposed. The type strain is R-5-52-3T (=KCTC 72061T=CGMCC 1.16678T).

Pseudomonas Saliphila sp. nov., a Bacterium Isolated from Oil-Well Production Water in Qinghai Oilfield of China.

Strain 16W4-4-3 T was isolated from the oil-well production water in Qinghai Oilfield, China. Cells were Gram-stain-negative, rod-shaped, catalase- and oxidase-positive, facultatively anaerobic and motile by single polar flagellum. The 16S rRNA gene sequences of strain 16W4-4-3 T showed the highest similarities with Pseudomonas profundi M5T (98.8%), P. pelagia CL-AP6T (98.0%), P. salina XCD-X85T (97.7%), and P. sabulinigri J64T (97.5%). The phylogenetic trees based on multilocus sequence analyses with concatenating 16S rRNA, gyrB, rpoD and rpoB genes suggested that this strain should be affiliated to the genus Pseudomonas but remotely related from other species. In addition, whole genome analyses revealed that the digital DNA-DNA hybridization values and average nucleotide identities of strain 16W4-4-3 T against its close relatives were all below 28.8% and 86.5%, respectively. Furthermore, the isolate had totally different whole cell protein profile as compared to those of other species. Major fatty acids were summed feature 8 (C18:1ω7c and/or C18:1ω6c), C16:0, summed feature 3 (C16:1ω7c and/or C16:1ω6c) and C17:0cyclo. Major isoprenoid quinone was ubiquinone (Q-9), and major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. The DNA G + C content was 58.5 mol%. Therefore, phenotypic, phylogenetic, genomic, chemotaxonomic, and proteomic traits showed that the isolate represented a novel species of the genus Pseudomonas, the name Pseudomonas saliphila sp. nov. is proposed. Type strain is 16W4-4-3 T (= CGMCC 1.13350 T = KCTC 72619 T).