Asymmetric biocatalysis of S-3-amino-3-phenylpropionic acid with new isolated Methylobacterium Y1-6

β-amino acids are widely used in drug research, and S-3-amino-3-phenylpropionic acid (S-APA) is an important pharmaceutical intermediate of S-dapoxetine, which has been approved for the treatment of premature ejaculation. Chiral catalysis is an excellent method for the preparation of enantiopure compounds. In this study, we used (±)-ethyl-3-amino-3-phenylpropanoate (EAP) as the sole carbon source. Three hundred thirty one microorganisms were isolated from 30 soil samples, and 17 strains could produce S-APA. After three rounds of cultivation and identification, the strain Y1-6 exhibiting the highest enantioselective activity of S-APA was identified as Methylobacterium oryzae. The optimal medium composition contained methanol (2.5 g/L), 1,2-propanediol (7.5 g/L), soluble starch (2.5 g/L), and peptone (10 g/L); it was shaken at 220 rpm for 4–5 days at 30 °C. The optimum condition for biotransformation of EAP involved cultivation at 37 °C for 48 h with 120 mg of wet cells and 0.64 mg of EAP in 1 ml of transfer solution. Under this condition, substrate ee was 92.1% and yield was 48.6%. We then attempted to use Methylobacterium Y1-6 to catalyze the hydrolytic reaction with substrates containing 3-amino-3-phenyl-propanoate ester, N-substituted-β-ethyl-3-amino-3-phenyl-propanoate, and γ-lactam. It was found that 5 compounds with ester bonds could be stereoselectively hydrolyzed to S-acid, and 2 compounds with γ-lactam bonds could be stereoselectively hydrolyzed to (-)-γ-lactam.     

Trehalose Maintains Bioactivity and Promotes Sustained Release of BMP-2 from Lyophilized CDHA Scaffolds for Enhanced Osteogenesis In Vitro and In Vivo

Calcium phosphate (Ca-P) scaffolds have been widely employed as a supportive matrix and delivery system for bone tissue engineering. Previous studies using osteoinductive growth factors loaded Ca-P scaffolds via passive adsorption often experience issues associated with easy inactivation and uncontrolled release. In present study, a new delivery system was fabricated using bone morphogenetic protein-2 (BMP-2) loaded calcium-deficient hydroxyapatite (CDHA) scaffold by lyophilization with addition of trehalose. The in vitro osteogenesis effects of this formulation were compared with lyophilized BMP-2/CDHA construct without trehalose and absorbed BMP-2/CDHA constructs with or without trehalose. The release characteristics and alkaline phosphatase (ALP) activity analyses showed that addition of trehalose could sufficiently protect BMP-2 bioactivity during lyophilization and achieve sustained BMP-2 release from lyophilized CDHA construct in vitro and in vivo. However, absorbed BMP-2/CDHA constructs with or without trehalose showed similar BMP-2 bioactivity and presented a burst release. Quantitative real-time PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) demonstrated that lyophilized BMP-2/CDHA construct with trehalose (lyo-tre-BMP-2) promoted osteogenic differentiation of bone marrow stromal cells (bMSCs) significantly and this formulation could preserve over 70% protein bioactivity after 5 weeks storage at 25°C. Micro-computed tomography, histological and fluorescent labeling analyses further demonstrated that lyo-tre-BMP-2 formulation combined with bMSCs led to the most percentage of new bone volume (38.79% ±5.32%) and area (40.71% ±7.14%) as well as the most percentage of fluorochrome stained bone area (alizarin red S: 2.64% ±0.44%, calcein: 6.08% ±1.37%) and mineral apposition rate (4.13±0.62 µm/day) in critical-sized rat cranial defects healing. Biomechanical tests also indicated the maximum stiffness (118.17±15.02 Mpa) and load of fracture (144.67±16.13 N). These results lay a potential framework for future study by using trehalose to preserve growth factor bioactivity and optimize release profile of Ca-P based delivery system for enhanced bone regeneration.     

Fluorescent-Based Methods for Gene Knockdown and Functional Cardiac Imaging in Zebrafish

A notable advantage of zebrafish as a model organism is the ease of gene knockdown using morpholino antisense oligonucleotide (MO). However, zebrafish morphants injected with MO for a target protein often show heterogeneous phenotypes, despite controlling the injection volume of the MO solution in all embryos. We developed a method for estimating the quantity of MO injected into each living morphant, based on the co-injection of a control MO labeled with the fluorophore lissamine. By applying this method for knockdown of cardiac troponin T (tnnt2a) in zebrafish, we could efficiently select the partial tnnt2a-depleted zebrafish with a decreased heart rate and impairment of cardiac contraction. To investigate cardiac impairment of the tnnt2a morphant, we performed fluorescent cardiac imaging using Bodipy-ceramide. Cardiac image analysis showed moderate reduction of tnnt2a impaired diastolic distensibility and decreased contraction and relaxation velocities. To the best of our knowledge, this is the first report to analyze the role of tnnt2a in cardiac function in tnnt2a-depleted living animals. Our combinatorial approach can be applied for analyzing the molecular function of any protein associated with human cardiac diseases.     

Microsatellite development for the endangered Yangtze sturgeon (Acipenser dabryanus Duméril, 1869) using 454 sequencing

The Yangtze sturgeon (Acipenser dabryanus) is an endemic species in China. Using 454 sequencing, eight polymorphic tri‐, tetra‐, penta‐, and hexanucleotide microsatellite loci were isolated in this study. The raw sequence data from a one‐eighth run of 454 sequencings were 38.0 Mbp containing 94 222 reads/sequences. Of 80 microsatellite loci, only eight loci were polymorphic in a population of 30 individuals. The number of alleles per locus ranged from 4 to 14 (mean 7.62), and the observed heterozygosities varied between 0.46 and 0.88 (mean 0.74). Cross amplification was tested in congeneric species Acipenser sturio and Acipenser sinensis. These new microsatellite markers will be useful for further studies on genetic variation, parentage analysis, and conservation management for this critically endangered species.     

Jeotgalibacillus aurantiacus sp. nov., a novel orange-pigmented species with a carotenoid biosynthetic gene cluster,isolated from wetland soil

A Gram-stain-positive, orange-pigmented, rod-shaped and flagellated bacterial strain T12^T was isolated from wetland soil in Kunyu Mountain Wetland in Yantai, China. The strain was able to grow at 15–40 °C (optimum 37 °C), at 0.0–9.0% NaCl (optimum 2%, w/v) and at pH 5.5–9.0 (optimum 8.5). A phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain T12^T is a member of the family Planococcaceae, sharing 97.6% and 97.1% sequence similarity with the type strains of Jeotgalibacillus salarius and Jeotgalibacillus marinus, respectively. Genome-based analyses revealed a genome size of 3,506,682 bp and a DNA G?+?C content of 43.7%. Besides, the genome sequence led to 55.0–74.6% average amino acid identity values and 67.8–74.7% average nucleotide identity values between strain T12^T and the current closest relatives. Digital DNA-DNA hybridization of strain T12^T with the type strains of Jeotgalibacillus proteolyticus and J. marinus demonstrated 19.0% and 20.3% relatedness, respectively. The chemotaxonomic analysis showed that the sole quinone was MK-7. The predominant cellular fatty acids were iso-C_15:0, anteiso-C_15:0, C_16:1ω7c alcohol and iso-C_14:0. The polar lipids consisted of an unidentified aminolipid, phosphatidylglycerol, diphosphatidylglycerol and two unidentified phospholipids. Based on the polyphasic characterization, strain T12^T is considered to represent a novel species, for which the name Jeotgalibacillus aurantiacus sp. nov. is proposed. The type strain is T12^T (=?KCTC 43296 ^T?=?MCCC 1K07171^T).

     

The Effect of High-Temperature Stress on the Physiological Indexes,Chloroplast Ultrastructure,and Photosystems of two Herbaceous Peony Cultivars

In this study, two herbaceous peony cultivars with different heat tolerances (‘Fenyunu’ FYN low sensitivity and ‘Qiaoling’ QL high sensitivity) were used as research materials. An integrated view of the factors underlying the decrease in photosynthetic rate under high-temperature (HT) stress was provided by analyzing the biochemical parameters, chloroplast ultrastructure, gas-exchange parameters, chlorophyll fluorescence, and modulated 820 nm reflection of herbaceous peony leaves. The results showed that hydrogen peroxide, superoxide anion, malondialdehyde, and electrical conductivity increased significantly, while the photosynthetic pigments content and photosynthetic capacity decreased significantly in QL than in FYN under HT. The contents of soluble sugars and proline increased greatly in FYN than in QL, while the activity of SOD decreased markedly in QL than in FYN after HT. Compared with FYN, the ultrastructure of QL was more seriously disrupted under HT. Chlorophyll fluorescence analysis showed that HT changed the shapes of OJIP curve, resulting in the increase of K phase and J phase. The PSII acceptor side was more damaged than the donor side, and the electron transfer was seriously blocked. The energy flow in the process of light energy absorption, capture, and electron transfer were significantly changed after HT stress. Meanwhile, PSI was also significantly inhibited, and the coordination of both photosystems decreased. The variation of these parameters in FYN was less than that in QL. These results suggested that FYN featured a more heat-tolerance ability as evidenced by the good performances on the antioxidant system, osmoregulatory capacity, and the thermostability of membranes and photosystems.

     

Development of a refined ex vivo model of peritoneal adhesion formation,and a role for connexin 43 in their development

Molecular and Cellular Biochemistry - Despite many advances across the surgical sciences, post-surgical peritoneal adhesions still pose a considerable risk in modern-day procedures and are highly...