ssDNA Aptamer Specifically Targets and Selectively Delivers Cytotoxic Drug Doxorubicin to HepG2 Cells

Hepatocellular carcinoma (HCC) is the third leading cause of death due to cancer worldwide with over 500,000 people affected annually. Although chemotherapy has been widely used to treat patients with HCC, alternate modalities to specifically deliver therapeutic cargos to cancer cells have been sought in recent years due to the severe side effects of chemotherapy. In this respect, aptamer-based tumor targeted drug delivery has emerged as a promising approach to increase the efficacy of chemotherapy and reduce or eliminate drug toxicity. In this study, we developed a new HepG2-specific aptamer (HCA#3) by a procedure known as systematic evolution of ligands by exponential enrichment (SELEX) and exploited its role as a targeting ligand to deliver doxorubicin (Dox) to HepG2 cells in vitro. The selected 76-base nucleotide aptamer preferentially bound to HepG2 hepatocellular carcinoma cells but not to control cells. The aptamer HCA#3 was modified with paired CG repeats at the 5′-end to carry and deliver a high payload of intercalated Dox molecules at the CG sites. Four Dox molecules (mol/mol) were fully intercalated in each conjugate aptamer-Dox (ApDC) molecule. Biostability analysis showed that the ApDC molecules are stable in serum. Functional analysis showed that ApDC specifically targeted and released Dox within HepG2 cells but not in control cells, and treatment with HCA#3 ApDC induced HepG2 cell apoptosis but had minimal effect on control cells. Our study demonstrated that HCA#3 ApDC is a promising aptamer-targeted therapeutic that can specifically deliver and release a high doxorubicin payload in HCC cells.     

The Potential Biomarkers to Identify the Development of Steatosis in Hyperuricemia

Hyperuricemia (HU) often progresses to combine with non-alcoholic fatty liver disease (NAFLD) in the clinical scenario, which further exacerbates metabolic disorders; early detection of biomarkers, if obtained during the HU progression, may be beneficial for preventing its combination with NAFLD. This study aimed to decipher the biomarkers and mechanisms of the development of steatosis in HU. Four groups of subjects undergoing health screening, including healthy subjects, subjects with HU, subjects with HU combined with NAFLD (HU+NAFLD) and subjects with HU initially and then with HU+NAFLD one year later (HU→HU+NAFLD), were recruited in this study. The metabolic profiles of all subjects’ serum were analyzed by liquid chromatography quadruple time-of-flight mass spectrometry. The metabolomic data from subjects with HU and HU+NAFLD were compared, and the biomarkers for the progression from HU to HU+NAFLD were predicted. The metabolomic data from HU→HU+NAFLD subjects were collected for further verification. The results showed that the progression was associated with disturbances of phospholipase metabolism, purine nucleotide degradation and Liver X receptor/retinoic X receptor activation as characterized by up-regulated phosphatidic acid, cholesterol ester (18:0) and down-regulated inosine. These metabolic alterations may be at least partially responsible for the development of steatosis in HU. This study provides a new paradigm for better understanding and further prevention of disease progression.     

Generation and Characterization of an Immortalized Human Esophageal Myofibroblast Line

Stromal cells with a myofibroblast phenotype present in the normal human esophagus are increased in individuals with gastro-esophageal reflux disease (GERD). We have previously demonstrated that myofibroblasts stimulated with acid and TLR4 agonists increase IL-6 and IL-8 secretion using primary cultures of myofibroblasts established from normal human esophagus. While primary cultures have the advantage of reflecting the in vivo environment, a short life span and unavoidable heterogeneity limits the usefulness of this model in larger scale in vitro cellular signaling studies. The major aim of this paper therefore was to generate a human esophageal myofibroblast line with an extended lifespan. In the work presented here we have generated and characterized an immortalized human esophageal myofibroblast line by transfection with a commercially available GFP-hTERT lentivirus. Immortalized human esophageal myofibroblasts demonstrate phenotypic, genotypic and functional similarity to primary cultures of esophageal myofibroblasts we have previously described. We found that immortalized esophageal myofibroblasts retain myofibroblast spindle-shaped morphology at low and high confluence beyond passage 80, and express α-SMA, vimentin, and CD90 myofibroblast markers. Immortalized human esophageal myofibroblasts also express the putative acid receptor TRPV1 and TLR4 and retain the functional capacity to respond to stimuli encountered in GERD with secretion of IL-6. Finally, immortalized human esophageal myofibroblasts also support the stratified growth of squamous esophageal epithelial cells in 3D organotypic cultures. This newly characterized immortalized human esophageal myofibroblast cell line can be used in future cellular signaling and co-culture studies.     

应用iTRAQ定量蛋白质组学研究海分枝杆菌mkl的基因功能

【目的】通过分离海分枝杆菌野生株和mkl突变株的全菌蛋白,并进行差异蛋白质组分析,以期为探索分枝杆菌重要毒力基因mkl的功能提供新思路。【方法】以海分枝杆菌野生株和mkl突变株为研究材料,提取全菌蛋白,i TRAQ试剂标记后进行质谱鉴定和定量分析,并利用Uni Prot数据库对差异蛋白进行生物信息学分析。【结果】共鉴定出在野生株和mkl突变株中差异表达蛋白566个,其中在突变株中上调表达蛋白232个(比值≥1.4),下调表达蛋白334个(比值≤0.7)。生物信息学预测这些蛋白主要参与细菌脂质代谢、细胞壁和细胞进程、中间代谢、呼吸作用等生物学功能。其中Des A3下调最显著,其功能为脂肪酸去饱和酶,与油酸合成相关,进一步验证发现mkl突变株在不含油酸的固体培养基中生长受限,提示mkl可能在油酸的生物合成通路中发挥功能。【结论】通过i TRAQ分析了海分枝杆菌mkl突变株和野生株的差异表达蛋白谱,发现可能影响分枝杆菌油酸、脂质等合成代谢通路,为进一步研究mkl基因在分枝杆菌致病中发挥作用的相关机制奠定了基础。     

Insights into functional genes and taxonomical/phylogenetic diversity of microbial communities in biological heap leaching system and their correlation with functions

Applied Microbiology and Biotechnology - Although the taxonomical/phylogenetic diversity of microbial communities in biological heap leaching systems has been investigated, the diversity of...     

Approximate parameter inference in systems biology using gradient matching: a comparative evaluation

Background

A challenging problem in current systems biology is that of parameter inference in biological pathways expressed as coupled ordinary differential equations (ODEs). Conventional methods that repeatedly numerically solve the ODEs have large associated computational costs. Aimed at reducing this cost, new concepts using gradient matching have been proposed, which bypass the need for numerical integration. This paper presents a recently established adaptive gradient matching approach, using Gaussian processes (GPs), combined with a parallel tempering scheme, and conducts a comparative evaluation with current state-of-the-art methods used for parameter inference in ODEs. Among these contemporary methods is a technique based on reproducing kernel Hilbert spaces (RKHS). This has previously shown promising results for parameter estimation, but under lax experimental settings. We look at a range of scenarios to test the robustness of this method. We also change the approach of inferring the penalty parameter from AIC to cross validation to improve the stability of the method.

Methods

Methodology for the recently proposed adaptive gradient matching method using GPs, upon which we build our new method, is provided. Details of a competing method using RKHS are also described here.

Results

We conduct a comparative analysis for the methods described in this paper, using two benchmark ODE systems. The analyses are repeated under different experimental settings, to observe the sensitivity of the techniques.

Conclusions

Our study reveals that for known noise variance, our proposed method based on GPs and parallel tempering achieves overall the best performance. When the noise variance is unknown, the RKHS method proves to be more robust.

     

The autophagy–lysosomal system in subarachnoid haemorrhage

The autophagy–lysosomal pathway is a self‐catabolic process by which dysfunctional or unnecessary intracellular components are degraded by lysosomal enzymes. Proper function of this pathway is critical for maintaining cell homeostasis and survival. Subarachnoid haemorrhage (SAH) is one of the most devastating forms of stroke. Multiple pathogenic mechanisms, such as inflammation, apoptosis, and oxidative stress, are all responsible for brain injury and poor outcome after SAH. Most recently, accumulating evidence has demonstrated that the autophagy–lysosomal pathway plays a crucial role in the pathophysiological process after SAH. Appropriate activity of autophagy–lysosomal pathway acts as a pro‐survival mechanism in SAH, while excessive self‐digestion results in cell death after SAH. Consequently, in this review article, we will give an overview of the pathophysiological roles of autophagy–lysosomal pathway in the pathogenesis of SAH. And approaching the molecular mechanisms underlying this pathway in SAH pathology is anticipated, which may ultimately allow development of effective therapeutic strategies for SAH patients through regulating the autophagy–lysosomal machinery.