In silico methods in enzyme screening and gene expression

INTMSAlign is a software to assign consensus residues of target protein utilizing large amount of their family sequences. We generated three protein sequences with S-selective hydroxynitrile lyase (S-HNL) activity, which we call designed S-HNLs; these proteins folded as efficiently as the native S-HNL (1). a-Amino-e-caprolactam (ACL) racemase from Achromobacter obae has been shown to be an effective catalyst for the dynamic kinetic resolution of amino acid amide and a-aminonitriles to form chiral amino acids. We searched for ACL racemase in silico with INTMSAlign software. By fixing Lys 241 as one of the key residues, we discovered thirteen ACL racemase genes from 413 fold type-I PLP genes (2). Insolubility of proteins expressed in Escherichia coli expression hinders the progress of both basic and applied research. Insoluble proteins contain residues that decrease their solubility (aggregation hotspots). We discovered a phenomenon of soluble expression of HNL from Manihot esculenta, in E. coli. By random mutagenesis, we found that a single point mutation H103L, and mutation with alterations at three positions (Lys-Pro mutations at positions 176, 199 and 224) cause total solubility in E. coli even when grown at 37°C (3). If a relationship between soluble expression and mutation points could be established, it will become very easy to generate a mutant for correctly folded expression in E. coli. Using a combination of approaches involving directed evolution and primary sequence analysis, we found two rules of thumb to help identify hotspots: one focuses on the hydrophobicity of amino acids in the a-helix structure, and another one focuses the difference in hydrophobicity relative to the corresponding amino acid in the consensus protein. Using these two relationships together, we succeeded in developing methods to improve the solubility of expressed proteins in E. coli (4). References: (1) S. Nakano and Y. Asano, Sci. Rep., 5, 8193 (2015). (2) W. Payoungkiattikun, S. Okazaki, S. Nakano, A. Ina, A. H-Kittikun, and Y. Asano, Appl. Biochem. Biotechnol., 176 (5), 1303-1314 (2015). (3) Y. Asano, M. Dadashipour, M. Yamazaki, N. Doi, and H. Komeda. Prot. Eng. Des. Sel., 24 (8), 607-616 (2011). (4) D. Matsui, S. Nakano, M. Dadashipour, and Y. Asano, submitted

Reconstruction of ancestral L-amino acid oxidases to broaden substrate selectivity

Characteristic functions of enzymes, such as high thermal stability and substrate specificity, are attained during the evolutionary process. Ancestral sequence reconstruction (ASR) is applied to infer the process by designing artificial enzymes which are located on ancestral node of phylogenetic tree; here, the inferred enzymes called ancestral enzymes. Ancestral enzymes often exhibit substrate promiscuity and high thermal stability of which functions are suitable to perform enzyme engineering. In addition, applicability of the ASR is high because the method requires only sequence data to design ancestral enzymes. Thus, we believe that artificial enzymes contributing to progress in enzyme engineering can be designed by ASR. Please click Additional Files below to see the full abstract

Application of CRISPR/Cas9 genome editing system for molecular breeding of orchids

Orchid is an important ornamental plant in Indonesia due to their natural beauty of flowers. In the tropical forest, orchids are being acquired for trading and commercial market. Thus, the effort is required to proliferate orchid in large quantities for conservation and improve the floral variation for plant breeding. The purpose of this study is to develop a firmed methodology of molecular breeding of orchids using CRISPR/Cas9 KO system. The plant material used was Phalaenopsis amabilis protocorms growth on NP medium+pepton (2 g/L). Protocorm were submerged in the culture of Agrobacterium tumefaciens that Ti‐plasmid had been filled with a T‐DNA construct of a pRGEB32 vector harboring sgRNA with PDS3 sequence. Detection for transformants was confirmed by PCR using HPT primers (545 bp), Cas9 primers (402 bp), PDS primers (280 bp) and trnL‐F (1200 bp) as an internal control. The results showed that 0.96% PDS transformants were obtained from PDS3T2 lines. Several transformant showed pale leaf color compared to non‐transformant plants. This study suggests that the target gene has successfully edited by CRISPR/Cas9 system and could be applied for that functional gene editing in orchids

Treatment-related damage in elderly-onset ANCA-associated vasculitis: safety outcome analysis of two nationwide prospective cohort studies

Background It is not elucidated that there is treatment-related damage in elderly patients with antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis (AAV). Methods Elderly (≥ 75 years of age) patients were enrolled from two nationwide prospective inception cohort studies. The primary outcome was 12-month treatment-related Vasculitis Damage Index (VDI) score. Secondary outcomes included serious infections within 6 months, total VDI score, remission, and relapse. Patient characteristics and outcomes were compared across three different initial glucocorticoid (GC) dose groups: high-dose, prednisolone (PSL) ≥ 0.8 mg/kg/day; medium-dose, 0.6 ≤ PSL  Results Of the 179 eligible patients, the mean age was 80.0 years; 111 (62%) were female. The mean Birmingham Vasculitis Activity Score was 16.1. Myeloperoxidase-ANCA findings were positive in 168 (94%) patients, while proteinase 3-ANCA findings were positive in 11 (6%). The low-dose group was older and had higher serum creatinine levels than the other groups. There were no statistically significant intergroup differences in remission or relapse, whereas serious infection developed more frequently in the high-dose (29 patients [43%]) than the low-dose (13 patients [22%]) or medium-dose (10 patients [19%]) groups (p = 0.0007). Frequent VDI items at 12 months included hypertension (19%), diabetes (13%), atrophy and weakness (13%), osteoporosis (8%), and cataracts (8%). Logistic regression analysis revealed that GC dose at 12 months (odds ratio, 1.14; 95% confidence interval, 1.00–1.35) was a predictor for diabetes. Conclusion A reduced initial GC dose with rapid reduction might be required to ensure the safe treatment of elderly AAV patients

Local microbleeding facilitates IL-6– and IL-17–dependent arthritis in the absence of tissue antigen recognition by activated T cells

Local microbleeding induces the accumulation of Th17 cells and the development of IL-17– and IL-6–dependent arthritis in the absence of cognate antigen recognition by CD4+ T cells

Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure < 100 mmHg (n = 1127), estimated glomerular filtration rate < 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation