RS‐1 enhances CRISPR‐mediated targeted knock‐in in bovine embryos

Targeted knock‐in (KI) can be achieved in embryos by clustered regularly interspaced short palindromic repeats (CRISPR)‐assisted homology directed repair (HDR). However, HDR efficiency is constrained by the competition of nonhomologous end joining. The objective of this study was to explore whether CRISPR‐assisted targeted KI rates can be improved in bovine embryos by exposure to the HDR enhancer RS‐1. In vitro produced zygotes were injected with CRISPR components (300 ng/µl Cas9 messenger RNA and 100 ng/µl single guide RNA against a noncoding region) and a single‐stranded DNA (ssDNA) repair template (100 ng/µl). ssDNA template contained a 6 bp XbaI site insert, allowing targeted KI detection by restriction analysis, flanked by 50 bp homology arms. Following microinjection, zygotes were exposed to 0, 3.75, or 7.5 µM RS‐1 for 24 hr. No differences were noted between groups in terms of development or genome edition rates. However, targeted KI rates were doubled in the group exposed to 7.5 µM RS‐1 compared to the others (52.8% vs. 25% and 23.1%, for 7.5, 0, and 3.75 µM, respectively). In conclusion, transient exposure to 7.5 µM RS‐1 enhances targeted KI rates resulting in approximately half of the embryos containing the intended mutation, hence allowing direct KI generation in embryos.     

New triazole derivatives as antifungal agents: synthesis via click reaction, in vitro evaluation and molecular docking studies

A series of 1-(1H-1,2,4-triazol-1-yl)-2-(2,4-difluorophenyl)-3-substituted-2-propanols (5a-5y) which are analogues of fluconazole, have been designed and synthesized via Cu(I)-catalyzed azide-alkyne cycloaddition on the basis of computational docking experiments to the active site of the cytochrome P450 14α-demethylase (CYP51). The in vitro antifungal activities of all the target compounds were evaluated against eight human pathogenic fungi. Compound 5l showed the best antifungal activities.     

SOMPNN: an efficient non-parametric model for predicting transmembrane helices

Accurately predicting the transmembrane helices (TMH) in a helical membrane protein is an important but challenging task. Recent researches have demonstrated that statistics-based methods are promising routes to improve the TMH prediction accuracy. However, most of existing TMH predictors are parametric models and they have to make assumptions of several or even hundreds of adjustable parameters based on the underlying probability distribution, which is difficult when no a priori knowledge is available. Besides the performances of these parametric predictors significantly depend on the estimated parameters, some of them need to exploit the entire training dataset in the prediction stage, which will lead to low prediction efficiency and this problem will become even worse when dealing with large-scale dataset. In this paper, we propose a novel SOMPNN model for prediction of TMH that features by minimal parameter assumptions requirement and high computational efficiency. In the SOMPNN model, a self-organizing map (SOM) is used to adaptively learn the helices distribution knowledge hidden in the training data, and then a probabilistic neural network (PNN) is adopted to predict TMH segments based on the knowledge learned by SOM. Experimental results on two benchmark datasets show that the proposed SOMPNN outperforms most existing popular TMH predictors and is promising to be extended to deal with other complicated biological problems. The datasets and the source codes of SOMPNN are available at http://www.csbio.sjtu.edu.cn/bioinf/SOMPNN/.     

Analysis of the neutral polysaccharide fraction of MCP and its inhibitory activity on galectin-3

The pH-modified citrus pectin (MCP) has been demonstrated to inhibit galectin-3 in cancer progression. The components and structures of MCP related to this inhibition remained unknown. In this paper, we fractionated MCP on DEAE-cellulose column into a homogenous neutral fraction MCP-N (about 20?kDa) and a pectin mixture fraction MCP-A (wide molecular distribution on Sepharose CL-6B chromatography). Both MCP-N and MCP-A inhibited hemagglutination mediated by galectin-3 with minimum inhibition concentration (MIC) 625 and 0.5?μg/ml, respectively. MCP-N was identified to be a type I arabinogalactan (AG-I) with a main chain of β-1→4-galactan. MCP-N was digested by α-L-arabinofuranosidase to give its main chain structure fraction (M-galactan, around 18?kDa), which was more active than the original molecule, MIC 50?μg/ml. The acidic degradation of M-galactan increased the inhibitory activity, MIC about 5 times lower than M-galactan. These results above showed that the functional motif of the β-1→4-galactan fragment might lie in the terminal residues rather than in the internal region of the chain. Therefore, MCP-N and its degraded products might be developed to new potential galectin-3 inhibitors. This is the first report concerning the fractionation of MCP and its components on galectin-3 inhibition. The information provided in this paper is valuable for screening more active galectin-3 inhibitors from natural polysaccharides.     

Cost per QALY (Quality-Adjusted Life Year) and Lifetime Cost of Prolonged Mechanical Ventilation in Taiwan

Introduction

Patients who require prolonged mechanical ventilation (PMV) are increasing and producing financial burdens worldwide. This study determines the cost per QALY (quality-adjusted life year), out-of-pocket expenses, and lifetime costs for PMV patients stratified by underlying diseases and cognition levels.

Methods

A nationwide sample of 50,481 patients with continual mechanical ventilation for more than 21 days was collected during 1997–2007. After stratifying the patients according to specific diagnoses, a latent class analysis (LCA) was performed to categorise PMV patients with multiple co-morbidities into several homogeneous groups. The survival functions were estimated for individual groups using the Kaplan-Meier method and extrapolated to 300 months through a semi-parametric method. The survival functions were adjusted using an EQ-5D utility value derived from a convenience sample of 142 PMV patients to estimate quality-adjusted life expectancies (QALE). Another convenience sample of 165 patients was used to estimate the out-of-pocket expenses. The lifetime expenditures paid by the single-payer National Health Insurance (NHI) system and patients'' families were estimated by multiplying average monthly expenditures by the survival probabilities and summing the values over lifetime.

Results

PMV therapy costs more than 100,000 U.S. dollars (USD) per QALY for all patients with poor cognition. For patients with partial cognition, PMV therapy costs less than 56,000 USD per QALY for those with liver cirrhosis, intracranial or spinal cord injuries, and 57,000–69,000 USD for patients with multiple co-morbidities under age of 65. The average lifetime cost of PMV was usually below 56,000 USD. The out-of-pocket expenses were often more than one-third of the total cost of treatment.

Conclusions

PMV treatment for patients with poor cognition would cost more than 5 times Taiwan''s GDP (gross domestic products), or less cost-effective. The out-of-pocket expenses for PMV provision should also be considered in policy decision.