Pou4f2‐GFP knock‐in mouse line: A model for studying retinal ganglion cell development

Pou4f2 acts as a key node in the comprehensive and step‐wise gene regulatory network (GRN) and regulates the development of retinal ganglion cells (RGCs). Accordingly, deletion of Pou4f2 results in RGC axon defects and apoptosis. To investigate the GRN involved in RGC regeneration, we generated a mouse line with a POU4F2‐green fluorescent protein (GFP) fusion protein expressed in RGCs. Co‐localization of POU4F2 and GFP in the retina and brain of Pou4f2‐GFP/+ heterozygote mice was confirmed using immunofluorescence analysis. Compared with those in wild‐type mice, the expression patterns of POU4F2 and POU4F1 and the co‐expression patterns of ISL1 and POU4F2 were unaffected in Pou4f2‐GFP/GFP homozygote mice. Moreover, the quantification of RGCs showed no significant difference between Pou4f2‐GFP/GFP homozygote and wild‐type mice. These results demonstrated that the development of RGCs in Pou4f2‐GFP/GFP homozygote mice was the same as in wild‐type mice. Thus, the present Pou4f2‐GFP knock‐in mouse line is a useful tool for further studies on the differentiation and regeneration of RGCs.     

Sodium houttuyfonate in vitro inhibits biofilm dispersion and expression of bdlA in Pseudomonas aeruginosa

Molecular Biology Reports - Biofilm dispersion is the last step in the development of biofilms, and allows bacteria to spawn novel biofilms in new locales. In the previous studies, we found that...     

化工废水生态毒性原因鉴别的实例研究

以南京市某一化工厂排出的废水为对象,对其生态毒性原因作了鉴别研究．结果表明,废水对Ｄａｐｈｎｉａｍａｇｎａ具有急性毒性,Ｃ１８固相提取可去除废水毒性,存在的主要毒物为非极性有机化合物．废水经Ｃ１８固相提取,发现废水中的主要可疑毒物为苯并吡喃酮和苯酚,是导致废水毒性的关键污染物,对废水毒性的贡献率分别为４４．６％和３２．９％．     

钩虾胆碱酯酶（ChE）和谷胱甘肽转硫酶（GST）的敏感性和特异性比较研究

研究了有机磷农药甲地基嘧啶流磷,有机氯农药林丹,菊酯类农药氯菊酯,表面活性剂直链苯磺酸钠和重金属Zn对钩虾（Gammarus pulex L.)胆碱酯酶（ChE）和谷胱甘肽转硫酶（GST）活性变化以及毒性影响,结果表明,在暴露24h和48h后,仅有机磷农药甲基嘧硫磷显著抑制胆碱酯的酶的活性,在暴露48h后,有机氯农药林丹和菊酯类农药氯菊酯能显著提高谷胱甘肽转硫酶活性,在暴露24h后,仅梵在导致谷胱甘肽转硫酶明显升高,作为生物标志物,胆碱酯酶比谷胱甘肽转硫酶具有更高的特异性,这两种生物标志物较毒性试验方法具有更高的敏感性。     

Metabolic profiling of isolated mitochondria and cytoplasm reveals compartment-specific metabolic responses

Introduction

Subcellular compartmentalization enables eukaryotic cells to carry out different reactions at the same time, resulting in different metabolite pools in the subcellular compartments. Thus, mutations affecting the mitochondrial energy metabolism could cause different metabolic alterations in mitochondria compared to the cytoplasm. Given that the metabolite pool in the cytosol is larger than that of other subcellular compartments, metabolic profiling of total cells could miss these compartment-specific metabolic alterations.

Objectives

To reveal compartment-specific metabolic differences, mitochondria and the cytoplasmic fraction of baker’s yeast Saccharomyces cerevisiae were isolated and subjected to metabolic profiling.

Methods

Mitochondria were isolated through differential centrifugation and were analyzed together with the remaining cytoplasm by gas chromatography–mass spectrometry (GC–MS) based metabolic profiling.

Results

Seventy-two metabolites were identified, of which eight were found exclusively in mitochondria and sixteen exclusively in the cytoplasm. Based on the metabolic signature of mitochondria and of the cytoplasm, mutants of the succinate dehydrogenase (respiratory chain complex II) and of the F_OF₁-ATP-synthase (complex V) can be discriminated in both compartments by principal component analysis from wild-type and each other. These mitochondrial oxidative phosphorylation machinery mutants altered not only citric acid cycle related metabolites but also amino acids, fatty acids, purine and pyrimidine intermediates and others.

Conclusion

By applying metabolomics to isolated mitochondria and the corresponding cytoplasm, compartment-specific metabolic signatures can be identified. This subcellular metabolomics analysis is a powerful tool to study the molecular mechanism of compartment-specific metabolic homeostasis in response to mutations affecting the mitochondrial metabolism.

     

An in vitro propagation protocol of two submerged macrophytes for lake revegetation in east China

Thirty-six programs have been set up to revegetate the degraded lake wetlands in east China since 2002. Most projects however faced deficiency of submerged macrophyte propagules. To solve the problem, alternative seedling sources must be found besides traditional field collection. This paper deals with an in vitro propagation protocol for two popularly used submerged macrophytes, Myriophyllum spicatum L. and Potamogeton crispus L. Full strength Murashige and Skoog-based liquid media (MS) plus 3% sucrose in addition to 0–2.0 mg l⁻¹ 6-benzylaminopurine (BA) and 0–1.0 mg l⁻¹ indoleacetic acid (IAA) were tried for shoot regeneration. Meanwhile, full, half or quarter strength MS in addition to 0, 0.1 or 0.2 mg l⁻¹ naphthaleneacetic acid (NAA) were tested for root induction, respectively. Results indicated that both species had the ability of regeneration from stem fragments in MS without further regulators. However, the addition of 2.0 mg l⁻¹ BA with 0.2 or 1.0 mg l⁻¹ IAA in MS drastically stimulated the regeneration efficiency of M. spicatum, while the addition of 2.0 mg l⁻¹ BA with 0.2 or 0.5 mg l⁻¹ IAA in MS significantly stimulated that of P. crispus. For root induction, full strength MS in combination with 0.1or 0.2 mg l⁻¹ NAA was preferred by M. spicatum, and the same MS without or with 0.1 mg l⁻¹ NAA was preferred by P. crispus. Seedlings of each species produced from tissue culture room had a 100% survival rate on clay, sandy loam or their mixture (1:1) in an artificial pond, and phenotypic plasticity was exhibited when the nutrient levels varied among the three types of sediments. This acclimation of seedlings helped develop the shoot and root systems, which ensured seedling quality and facilitated the transplantation. Our study has established an effective protocol to produce high quality seedlings for lake revegetation programs at a larger scale. Since the two species we tested represent different regeneration performances in nature but shared similar in vitro propagation conditions, this study has indicated a potentially wide use of the common media for preparing seedlings of other submerged macrophytes.     

Allelopathic activity of volatile substance from submerged macrophytes on Microcystin aeruginosa

Considerable attention has been paid on the allelopathic potential of aquatic macrophytes against blue-green algae. This study compared inhibitory activity of volatile compounds that were contained in two submerged macrophytes, Ceratophyllum demersum and Vallisneria spiralis, on Microcystin aeruginosa. When the concentration of essential oils was 100 mg/L, the antialgal activities for fresh macrophytes were similar between C. demersum and V. spiralis but stronger than those for dry macrophytes. Essential oils from C. demersum in dry macrophytes showed stronger antialgal activity than those from V. spiralis. There exists a positive correlation between the concentration of the essential oils and the inhibitory activity. The essential oils were composed of fatty compounds, terpenoids, phenolic compounds, phthalates, and some unknown compounds. More than 40% of the essential oils from fresh macrophytes were composed of phthalates, whereas approximately 70% of those from dry macrophytes were composed of lipid compounds and terpenoids. However, approximately 20% of the essential oils from fresh macrophytes were composed of unknown compounds, which were unstable and undetectable in dry macrophytes. Therefore, their chemical structures and bioactivities need further study.     

Sodium houttuyfonate, a potential phytoanticipin derivative of antibacterial agent, inhibits bacterial attachment and pyocyanine secretion of Pseudomonas aeruginosa by attenuating flagella-mediated swimming motility

Pseudomonas aeruginosa is a well-known clinical pathogen for its recalcitrant infection caused by biofilm formation which are initiated by flagella-mediated attachment. Sodium houttuyfonate (SH) is a natural phytoanticipin derivative of houttuynin and has anti-pathogenic effect on P. aeruginosa biofilm formation. In this paper, when using 1/2 × MIC SH, the diameter of P. aeruginosa swimming motility was sharply shortened to 36 % in 24 h incubation, and the fold changes of fliC required for swimming motility was 0.36 in 24 h cultivation, the adherence inhibition accounted for about 46 %, and the pyocyanin production decreased to 47 % after 1-day treatment and 56 % after 3-day treatment with obvious visual changes from dark green to light green, compared with the negative control. With the help of mass spectra and scanning electronic microscope, 1/2 × MIC SH was further testified to be enough to eradicate flagella and inhibit pyocyanin secretion of P. aeruginosa. The results do not only re-affirm the close interplay of attachment and virulence (i.e. swimming motility and pyocyanin), but also unravel the potential mechanism of SH on anti-biofilm of P. aeruginosa.     

Pentachlorophenol treatment in vivo elevates point mutation rate in zebrafish p53 gene

Pentachlorophenol (PCP), a probable human carcinogen, has been heavily used as an aseptic and a biocide throughout the world, and is widely present in the environment. Recent survey in Germany revealed that the average PCP amount in the urine of general German populations was 1.04 microg/L while the peak concentration could reach up to 19.1 microg/L. PCP was reported to cause DNA damage, but whether it can be involved in inducing point mutations in genome is unknown. To determine the genotoxicity of PCP on vertebrate, we first performed acute toxicity test on zebrafish for the effect of PCP exposure. The LC50 values of zebrafish exposed to PCP at 24, 48, 72 and 96 h were determined to be 0.196, 0.130, 0.130 and 0.130 mg/L, respectively. We then treated zebrafish with PCP for 10 days at 0 (control), 0.5, 5 and 50 microg/L, respectively, to determine whether PCP could be involved in inducing point mutations. Employing denaturing high-performance liquid chromatography analysis and DNA sequencing, we demonstrated that exposure of PCP to zebrafish at a concentration as low as 5 microg/L for 10 days elevates point mutation rate in p53 gene in liver cells. This is the first direct evidence revealing that PCP can elevate point mutation rate in the vertebrate genomes. The result implies PCP might be involved in carcinogenesis by elevating point mutation rate in the somatic genomes.     

Reactivation of tumour suppressor in breast cancer by enhancer switching through NamiRNA network

Dysfunction of Tumour Suppressor Genes (TSGs) is a common feature in carcinogenesis. Epigenetic abnormalities including DNA hypermethylation or aberrant histone modifications in promoter regions have been described for interpreting TSG inactivation. However, in many instances, how TSGs are silenced in tumours are largely unknown. Given that miRNA with low expression in tumours is another recognized signature, we hypothesize that low expression of miRNA may reduce the activity of TSG related enhancers and further lead to inactivation of TSG during cancer development. Here, we reported that low expression of miRNA in cancer as a recognized signature leads to loss of function of TSGs in breast cancer. In 157 paired breast cancer and adjacent normal samples, tumour suppressor gene GPER1 and miR-339 are both downregulated in Luminal A/B and Triple Negative Breast Cancer subtypes. Mechanistic investigations revealed that miR-339 upregulates GPER1 expression in breast cancer cells by switching on the GPER1 enhancer, which can be blocked by enhancer deletion through the CRISPR/Cas9 system. Collectively, our findings reveal novel mechanistic insights into TSG dysfunction in cancer development, and provide evidence that reactivation of TSG by enhancer switching may be a promising alternative strategy for clinical breast cancer treatment.