Suppressing ROS-TFE3-dependent autophagy enhances ivermectin-induced apoptosis in human melanoma cells

Melanoma is an aggressive skin malignancy with a high mortality rate; however, successful treatment remains a clinical challenge. Ivermectin, a broad-spectrum antiparasitic drug, has recently been characterized as a potential anticancer agent due to its observed antitumor effects. However, the molecular mechanisms of ivermectin remain poorly understood. In the current study, we tested the involvement of autophagy in the ivermectin mechanism of action in human melanoma cells. We exposed SK-MEL-28 cells to different concentrations of ivermectin (2.5, 5, and 10 μM) for 24 hours. Here, ivermectin-induced apoptosis, as evidenced by the upregulation of cleaved poly (ADP-ribose) polymerase, BAX expression, and caspase-3 activity and downregulation of BCL-2 expression. In line with the apoptosis response, ivermectin triggered autophagy. Pharmacological or genetic inhibition of autophagy further sensitized SK-MEL-28 cells to ivermectin-induced apoptosis. Mechanistically, ivermectin-induced TFE3^(Ser321) dephosphorylation, activated TFE3 nuclear translocation and increased TFE3 reporter activity, which contributed to lysosomal biogenesis and the expression of autophagy-related genes, and subsequently, initiated autophagy in SK-MEL-28 cells. Moreover, N-acetyl-cysteine, an reactive oxygen species (ROS) scavenger, abrogated the effects of ivermectin on TFE3-dependent autophagy. Taken together, we demonstrated that ivermectin increases TFE3-dependent autophagy through ROS signaling pathways in human melanoma cells and that inhibiting autophagy enhances ivermectin-induced apoptosis in human melanoma cells.     

Factors Affecting the Tailing of Blunt End DNA with Fluorescent Pyrimidine dNTPs

The transferase activity of non-proofreading DNA polymerases is a well-known phenomenon that has been utilized in cloning and sequencing applications. The non-templated addition of modified nucleotides at DNA blunt ends is a potentially useful feature of DNA polymerases that can be used for selective transformation of DNA 3′ ends. In this paper, we characterized the tailing reaction at perfectly matched and mismatched duplex ends with Cy3- and Cy5-modified pyrimidine nucleotides. It was shown that the best DNA tailing substrate does not have a perfect Watson–Crick base pair at the end. Mismatched duplexes with a 3′ dC were the most efficient in the Taq DNA polymerase-catalysed tailing reaction with a Cy5-modified dUTP. We further demonstrated that the arrangement of the dye residue relative to the nucleobase notably affects the outcome of the tailing reaction. A comparative study of labelled deoxycytidine and deoxyuridine nucleotides showed higher efficiency for dUTP derivatives. The non-templated addition of modified nucleotides by Taq polymerase at a duplex blunt end was generally complicated by the pyrophosphorolysis and 5′ exonuclease activity of the enzyme.     

A Model for the Acrosome Reaction in Mammalian Sperm

The acrosome reaction is a complex, calcium-dependent reaction that results in an exocytotic event required for successful fertilization of the egg. It has long been thought that the acrosome reaction occurs upon sperm binding to the zona pellucida, a viscoelastic layer surrounding the oocyte. Recent studies have suggested that the reaction may even occur before the sperm encounters the zona, perhaps mediated by progesterone or some other agonist. It has been particularly difficult to understand differences between progesterone-induced and zona-induced reactions experimentally and whether one substance is the more biologically relevant trigger. Until this present work, there has been little effort to mathematically model the acrosome reaction in sperm as a whole. Instead, attention has been paid to modeling portions of the pathways involved in other cell types. Here we present a base model for the acrosome reaction which characterizes the known biochemical reactions and behaviors of the system. Our model allows us to analyze several pathways that may act as a stabilizing mechanism for avoiding sustained oscillatory calcium responses often observed in other cell types. Such an oscillatory regime might otherwise prevent acrosomal exocytosis and therefore inhibit fertilization. Results indicate that the acrosome reaction may rely upon multiple redundant mechanisms to avoid entering an oscillatory state and instead maintain a high resting level of calcium, known to be required for successful acrosomal exocytosis and, ultimately, fertilization of the oocyte.     

Structural analysis of <Emphasis Type="Italic">N</Emphasis>-/<Emphasis Type="Italic">O</Emphasis>-glycans assembled on proteins in yeasts

Protein glycosylation, the most universal and diverse post-translational modification, can affect protein secretion, stability, and immunogenicity. The structures of glycans attached to proteins are quite diverse among different organisms and even within yeast species. In yeast, protein glycosylation plays key roles in the quality control of secretory proteins, and particularly in maintaining cell wall integrity. Moreover, in pathogenic yeasts, glycans assembled on cell-surface glycoproteins can mediate their interactions with host cells. Thus, a comprehensive understanding of protein glycosylation in various yeast species and defining glycan structure characteristics can provide useful information for their biotechnological and clinical implications. Yeast-specific glycans are a target for glyco-engineering; implementing human-type glycosylation pathways in yeast can aid the production of recombinant glycoproteins with therapeutic potential. The virulenceassociated glycans of pathogenic yeasts could be exploited as novel targets for antifungal agents. Nowadays, several glycomics techniques facilitate the generation of species-and strain-specific glycome profiles and the delineation of modified glycan structures in mutant and engineered yeast cells. Here, we present the protocols employed in our laboratory to investigate the N-and O-glycan chains released from purified glycoproteins or cell wall mannoproteins in several yeast species.     

Well-to-wheel GHG emissions and mitigation potential from light-duty vehicles in Macau

Purpose

The rapid growth of vehicle sales and usage has highlighted the need for greenhouse gas (GHG) emission reduction in Macau, a special administrative region (SAR) of China. As the most primary vehicle type, light-duty vehicles (LDV, including light-duty gasoline vehicles (LDGVs) and light-duty diesel vehicles (LDDVs)) play a key role in promoting the GHG reduction and development of green transportation system in Macau.

Methods

This study, on the basis of real-world tested and statistical data, firstly performed a streamlined life-cycle assessment (SLCA) on LDVs, to evaluate the potential GHG emissions and reduction through shifting to hybrid electric vehicles (HEVs) and electric vehicles (EVs).

Results and discussion

The results show that the mean GHG emissions from the LDGVs, LDDVs, and HEVs per 100 km were 25.16, 20.30, and 15.00 kg CO₂ eq, respectively. Under the current electricity mix in Macau, EVs with the emissions of 12.39 kg CO₂ eq/100 km can achieve a significant GHG emission reduction of LDVs in Macau. The total GHG emissions from LDVs increased from 124.99 to 247.82 thousand metric tons over the periods 2001–2014, with a 5.42% annual growth rate. A scenario analysis indicated that the development of HEVs and EVs—especially EVs—has the potential to control the GHG emissions from LDVs. Under the electricity mix of natural gas (NG) and solar energy (SE), the GHG emissions from EVs would drop by about 22 and 28%, respectively, by 2030.

Conclusions

This study develops a useful approach to evaluate the potential GHG emissions and its reduction strategies in Macau. All the obtained results could be useful for decision makers, providing robust support for drawing up an appropriate plan for improving green transportation systems in Macau.

     

黄瓜Ri T—DNA转化根的植株再生

本文利用发根农杆菌感染黄瓜植体得到的转化根,在不同激素配比的分分培养基上培养,进行再生植株的诱导试验,结果表明,ＮＡＡ和ＢＡ的合适配比,有利于黄瓜植株的再生,转化根及其再生植株检测到冠瘿碱的存在,说明农杆菌Ｒｉ质粒的Ｔ－ＤＮＡ已转移到黄瓜基因组中。     

缺失数据下蛋白质多结构叠加的迭代方法

蛋白质三维结构叠加面临的主要问题是,参与叠加的目标蛋白质的氨基酸残基存在某些缺失,但是多结构叠加方法却大多数需要完整的氨基酸序列,而目前通用的方法是直接删去缺失的氨基酸序列,导致叠加结果不准确。由于同源蛋白质间结构的相似性,因此,一个蛋白质结构中缺失的某个区域,可能存在于另一个同源蛋白质结构中。基于此,本文提出一种新的、简单、有效的缺失数据下的蛋白质结构叠加方法（ITEMDM）。该方法采用缺失数据的迭代思想计算蛋白质的结构叠加,采用优化的最小二乘算法结合矩阵SVD分解方法,求旋转矩阵和平移向量。用该方法成功叠加了细胞色素C家族的蛋白质和标准Fischer’s 数据库的蛋白质（67对蛋白质）,并且与其他方法进行了比较。数值实验表明,本算法有如下优点：①与THESEUS算法相比较,运行时间快,迭代次数少;②与PSSM算法相比较,结果准确,运算时间少。结果表明,该方法可以更好地叠加缺失数据的蛋白质三维结构。     

Responses of two kidney bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>) cultivars to the combined stress of sulfur deficiency and cadmium toxicity

Plants suffer from combined stress of sulfur deficiency and cadmium toxicity in some agricultural lands. However, little is known about the reaction in plants, such as responses in antioxidant enzymes and non-protein thiol compounds, to such combined stress. Therefore, in this study, four treatments, S-sufficiency (T_S?Cd), S-deficiency (T_?S?Cd), Cd stress (T_S+Cd) and combined stress of S-deficiency and Cd stress (T_?S+Cd), were set up to investigate (1) the effects of sulfur deficiency or sulfur sufficiency on Cd toxicity to kidney bean cultivar seedlings and the related mechanisms, and (2) the responses of two kidney bean cultivars to combined stress of S-deficiency and Cd-tolerance. The results showed significant increases in hydrogen peroxide (H₂O₂) and malondialdehyde contents and significant increases in antioxidant enzyme (superoxide dismutase, catalase, peroxidase, and glutathione S-transferase) activities and non-protein thiol compounds (non-protein thiols, reduced glutathione, phytochelatins) synthesis in the plants in T_S+Cd and T_?S+Cd. On the tissue level, higher proportion of Cd was found to be immobilized/deposited in roots, while on the sub-cell level, higher proportion of Cd was located in cell walls and vacuole fractions with lower in cell organelles. Taken together, the results indicated that Cd detoxification was achieved by the two kidney bean cultivars through antioxidant enzyme activation, non-protein thiol compound synthesis and sub-cellular compartmentalization. In addition, the results indicated that sufficient S supply helped to relieve Cd toxicity, which is of special significance for remediation or utilization of Cd-contaminated soils as S is a plant essential nutrient.