Thermal unfolding studies show the disease causing F508del mutation in CFTR thermodynamically destabilizes nucleotide-binding domain 1

Misfolding and degradation of CFTR is the cause of disease in patients with the most prevalent CFTR mutation, an in-frame deletion of phenylalanine (F508del), located in the first nucleotide-binding domain of human CFTR (hNBD1). Studies of (F508del)CFTR cellular folding suggest that both intra- and inter-domain folding is impaired. (F508del)CFTR is a temperature-sensitive mutant, that is, lowering growth temperature, improves both export, and plasma membrane residence times. Yet, paradoxically, F508del does not alter the fold of isolated hNBD1 nor did it seem to perturb its unfolding transition in previous isothermal chemical denaturation studies. We therefore studied the in vitro thermal unfolding of matched hNBD1 constructs ±F508del to shed light on the defective folding mechanism and the basis for the thermal instability of (F508del)CFTR. Using primarily differential scanning calorimetry (DSC) and circular dichroism, we show for all hNBD1 pairs studied, that F508del lowers the unfolding transition temperature (T_m) by 6–7°C and that unfolding occurs via a kinetically-controlled, irreversible transition in isolated monomers. A thermal unfolding mechanism is derived from nonlinear least squares fitting of comprehensive DSC data sets. All data are consistent with a simple three-state thermal unfolding mechanism for hNBD1 ± F508del: N(±MgATP) ⇄ I_T(±MgATP) → A_T → (A_T)_n. The equilibrium unfolding to intermediate, I_T, is followed by the rate-determining, irreversible formation of a partially folded, aggregation-prone, monomeric state, A_T, for which aggregation to (A_T)_n and further unfolding occur with no detectable heat change. Fitted parameters indicate that F508del thermodynamically destabilizes the native state, N, and accelerates the formation of A_T.     

MSI4/FVE is required for accumulation of 24-nt siRNAs and DNA methylation at a subset of target regions of RNA-directed DNA methylation

DNA methylation is an important epigenetic mark. In plants, de novo DNA methylation occurs mainly through the RNA-directed DNA methylation (RdDM) pathway. Researchers have previously inferred that a flowering regulator, MULTICOPY SUPPRESSOR OF IRA1 4 (MSI4)/FVE, is involved in non-CG methylation at several RdDM targets, suggesting a role of FVE in RdDM. However, whether and how FVE affects RdDM genome-wide is not known. Here, we report that FVE is required for DNA methylation at thousands of RdDM target regions. In addition, dysfunction of FVE significantly reduces 24-nucleotide siRNA accumulation that is dependent on factors downstream in the RdDM pathway. By using chromatin immunoprecipitation and sequencing (ChIP-seq), we show that FVE directly binds to FVE-dependent 24-nucleotide siRNA cluster regions. Our results also indicate that FVE may function in RdDM by physically interacting with RDM15, a downstream factor in the RdDM pathway. Our study has therefore revealed that FVE, by associating with RDM15, directly regulates DNA methylation and siRNA accumulation at a subset of RdDM targets.     

Construction of a rAAV-SaCas9 system expressing eGFP and its application to improve muscle mass

Biotechnology Letters - An ideal rAAV gene editing system not only effectively edits genes at specific site, but also prevents the spread of the virus from occurring off-target or carcinogenic...     

双歧杆菌影响肌醇三磷酸的实验研究

本实验首次发现双歧杆菌可降低大肠癌细胞内肌醇三磷酸水平，且存在时间依赖关系，提示双歧杆影响脂代谢通路的信息传递过程，这种影响是通过关闭亚细胞器上钙离子通道实现的。     

A comparative study on physiological activities of lager and ale brewing yeasts under different gravity conditions

High gravity (HG) or very high gravity (VHG) brewing has become popular in modern breweries due to its economic and product quality advantages. However, there are the negative impacts such as the fermentation performance of brewer??s yeast in HG or VHG wort, which are closely related to changes in cell physiological activity. In the present study, 3 kinds of worts, with different gravities, were used to examine the systematic effects on fermentation performance and physiological activity of lager yeast FBY009505 (Saccharomyces pastorianus) and ale yeast FBY0099 (Saccharomyces cerevisiae), as well as the resulting beer flavor. Results showed that the responses of FBY009505 and FBY0099 to the HG or VHG worts were similar. The specific fermentation rate and viability of cropped yeast of FBY009505 and FBY0099 were decreased with increasing wort gravity. The increased wort gravity resulted in the increase of energy charge and the decrease of ??-glucosides transport rate and glycolytic enzyme activities. Moreover, the environmental stresses in the HG or VHG wort showed a higher inhibitory activity against ??-glucoside transport than glycolytic enzymes. The content of intracellular trehalose and glycerol of FBY009505 and FBY0099 increased with the increase in wort gravity. The results from this study provided a potential means to systematically understand the physiology of brewer??s yeast under HG or VHG conditions.     

Mechanisms of Dihydroartemisinin and Dihydroartemisinin/Holotransferrin Cytotoxicity in T-Cell Lymphoma Cells

The validated therapeutic effects of dihydroartemisinin (DHA) in solid tumors have encouraged us to explore its potential in treating T-cell lymphoma. We found that Jurkat cells (a T-cell lyphoma cell line) were sensitive to DHA treatment with a IC50 of dihydroartemisinin. The cytotoxic effect of DHA in Jurkat cells showed a dose- and time- dependent manner. Interestingly, the cytotoxic effect of DHA was further enhanced by holotransferrin (HTF) due to the high expression of transferrin receptors in T-cell lymphoma. Mechanistically, DHA significantly increased the production of intracellular reactive oxygen species, which led to cell cycle arrest and apoptosis. The DHA treatment also inhibited the expression of protumorgenic factors including VEGF and telomerase catalytic subunit. Our results have proved the therapeutic effect of DHA in T-cell lymphoma. Especially in combination with HTF, DHA may provide a novel efficient approach in combating the deadly disease.     

Self-assembly of Large-scale Two-dimensional Plasmonic Superlattices Based on Single-Crystal Au Nanospheres and the FDTD Simulation of Its Optical Properties

Plasmonics - Large-scale ordered two-dimensional (2D) superlattices at oil/water interface were fabricated using single-crystal Au nanospheres (NSs) with different diameters as building blocks. A...     

Application of the phosphomannose-isomerase/mannose selection system in the Agrobacterium-mediated transformation of Lonicera hypoglauca Miq.

Journal of Plant Biochemistry and Biotechnology - The dried buds of Lonicera hypoglauca Miq. have antipyretic, antidotal and anti-inflammatory properties and as Flos lonicerae are widely used in...