荧光内参在校正Caspase-3／7活性检测偏差中的应用

目的改进Caspase-3／7活性检测方法。方法在顺铂诱导的HeLa细胞凋亡模型中,分别利用传统的和改良的实验方法检测Caspase-3／7的活性变化,并与Western印迹实验结果进行方法学比较。结果改良的实验方法显示不同剂量顺铂诱导下,HeLa细胞Caspase-3／7活性有剂量依赖性增高,与Western印迹实验结果相一致,但传统实验方法显示HeLa细胞Caspase-3／7活性呈现先增高后降低的趋势。结论由于参测细胞数不同,导致这种Caspase-3／7活性检测方法不能真实反映细胞凋亡程度。本研究成功建立了一种新的改良型Caspase-3／7活性检测方式,这种检测方法可以排除不同凋亡诱导方式诱导细胞凋亡时所产生的因参测细胞数不同所造成的Caspase-3／7活性检测的误差。     

Metabolic engineering of Bacillus subtilis for the co-production of uridine and acetoin

In this study, a uridine and acetoin co-production pathway was designed and engineered in Bacillus subtilis for the first time. A positive correlation between acetoin and uridine production was observed and investigated. By disrupting acetoin reductase/2,3-butanediol dehydrogenasegenebdhA, the acetoin and uridine yield was increased while 2,3-butanediol formation was markedly reduced. Subsequent overexpression of the alsSD operon further improved acetoin yield and abolished acetate formation. After optimization of fermentation medium, key supplementation strategies of yeast extract and soybean meal hydrolysate were identified and applied to improve the co-production of uridine and acetoin. With a consumption of 290.33 g/L glycerol, the recombinant strain can accumulate 40.62 g/L uridine and 60.48 g/L acetoin during 48 h of fed-batch fermentation. The results indicate that simultaneous production of uridine and acetoin is an efficient strategy for balancing the carbon metabolism in engineered Bacillus subtilis. More importantly, co-production of value-added products is a possible way to improve the economics of uridine fermentation.

     

Mandibular osteotomy-induced hypoxia enhances osteoclast activation and acid secretion by increasing glycolysis

The rapid bone remodeling after osteotomy has been reported for a long time. However, the underlying mechanism promoting the active bone reconstruction was still to be elucidated. Since not only the bone, blood vessels, and supportive tissues, but also the local microenvironment were destroyed, if the changes on the cell metabolism was contributed to the accelerated bone remodeling came into sight. In present study, we found that the mandibular osteotomy in rabbit activated osteoclasts, as well as the expression of hypoxia-inducible factor 1α (HIF-1α) in alveolar bone. Hypoxia or HIF-1α could enhanced osteoclastogenesis, bone absorption, and lactic acid concentration in receptor activator of nuclear factor κΒ ligand-induced RAW264.7 cells. Coincided with the upregulated HIF-1α expression, HIF-driven glycolytic enzymes, such as lactate dehydrogenase A (LDHA), glucokinase (GCK), pyruvate kinase M2 (PKM2), and phosphofructokinase1 (PFK1), were found massively increased in both hypoxic RAW264.7 cells and the alveolar HIF-1α-positive osteoclasts after mandibular osteotomy. Knockdown of HIF-1α suppressed not only the hypoxia-mediated glycolysis, but also the hypoxia-induced acid secretion and bone resorption in RAW264.7 cells. Application of inhibitor on glycolysis gave rise to the similar results as HIF-1α knockdown. Our findings suggested that hypoxia-driven glycolysis in osteoclasts was an adaptive mechanism to permit alveolar bone remodeling after mandibular osteotomy.     

Translocation of cell‐penetrating peptides into Candida fungal pathogens

Cell‐penetrating peptides (CPPs) are small peptides capable of crossing cellular membranes while carrying molecular cargo. Although they have been widely studied for their ability to translocate nucleic acids, small molecules, and proteins into mammalian cells, studies of their interaction with fungal cells are limited. In this work, we evaluated the translocation of eleven fluorescently labeled peptides into the important human fungal pathogens Candida albicans and C. glabrata and explored the mechanisms of translocation. Seven of these peptides (cecropin B, penetratin, pVEC, MAP, SynB, (KFF)₃K, and MPG) exhibited substantial translocation (>80% of cells) into both species in a concentration‐dependent manner, and an additional peptide (TP‐10) exhibiting strong translocation into only C. glabrata. Vacuoles were involved in translocation and intracellular trafficking of the peptides in the fungal cells and, for some peptides, escape from the vacuoles and localization in the cytosol were correlated to toxicity toward the fungal cells. Endocytosis was involved in the translocation of cecropin B, MAP, SynB, MPG, (KFF)₃K, and TP‐10, and cecropin B, penetratin, pVEC, and MAP caused membrane permeabilization during translocation. These results indicate the involvement of multiple translocation mechanisms for some CPPs. Although high levels of translocation were typically associated with toxicity of the peptides toward the fungal cells, SynB was translocated efficiently into Candida cells at concentrations that led to minimal toxicity. Our work highlights the potential of CPPs in delivering antifungal molecules and other bioactive cargo to Candida pathogens.     

Downregulation of tumor suppressor QKI in gastric cancer and its implication in cancer prognosis

Gastric cancer (GC) is the fourth most common cancer and second leading cause of cancer-related death worldwide. RNA-binding protein Quaking (QKI) is a newly identified tumor suppressor in multiple cancers, while its role in GC is largely unknown. Our study here aimed to clarify the relationship between QKI expression with the clinicopathologic characteristics and the prognosis of GC. In the 222 GC patients' specimens, QKI expression was found to be significantly decreased in most of the GC tissues, which was largely due to promoter hypermethylation. QKI overexpression reduced the proliferation ability of GC cell line in vitro study. In addition, the reduced QKI expression correlated well with poor differentiation status, depth of invasion, gastric lymph node metastasis, distant metastasis, advanced TNM stage, and poor survival. Multivariate analysis showed QKI expression was an independent prognostic factor for patient survival.     

Chemotherapy not only enriches but also induces cancer stem cells

Cancer stem cells are regarded as the hurdle of cancer therapy at least partially due to their intrinsic resistance to therapy. To this end, chemotherapy is widely used for enrichment of cancer stem cells. In contrast to the dogma, we hypothesized that besides enrichment, cancer stem cells could also be induced by chemotherapy in those regions without sufficient drug delivery. Due to the imbalance of the angiogenesis and insufficient blood supply in certain regions of the tumor mass, chemotherapy delivery is compromised in these regions. The insufficient drug delivery in turn transforms the bulk cancer cells to stem cells rather than kill them through NFkappaB-HIF, NFkappaB-Wnt and other signals. Detection of the induction of cancer stem cells from the chemotherapy treated non-stem cancer cells would shed light on our hypothesis, which in turn would broad our understanding of clinical cancer chemotherapy.     

质量平衡法及其在标准物质定值中的应用进展

为保证不同地区、不同时间测量结果的可比性，测量结果需溯源至适当的、规定的参考标准。对于化学、生物、工程、物理学领域的材料和样品测量，该参考标准为标准物质。由此可见，标准物质的定值对物质的检测及定量是十分重要的。标准物质（reference material，RM）是一种足够均匀的、具有一种或多种相对容易确定的特性值的材料或物质，可用于给材料赋值、评价测量方法及校准测量仪器等。质量平衡法作为标准物质的定量方法之一，是一种常用的纯度测量方法，将水分、灰分、挥发组分、无机元素等杂质的含量从100%中扣除，再根据主要组分在有机组分中的百分比来确定物质纯度。质量平衡法具有较高准确度，能够溯源到国际单位制中的质量单位，且若使用基准方法测量样品中的主成分及各部分杂质以完成整个质量平衡法的测量，质量平衡法则有望成为新的基准方法。基于此，对质量平衡法原理及质量平衡法在标准物质的研制中的应用进行了介绍，并对近期质量平衡法在标准物质中的最新应用进行了总结，以期探索质量平衡法在标准物质研制中的更多可能。