A two-photon fluorescent probe for lipid raft imaging: C-laurdan

The lipid-rafts hypothesis proposes that naturally occurring lipid aggregates exist in the plane of membrane that are involved in signal transduction, protein sorting, and membrane transport. To understand their roles in cell biology, a direct visualization of such domains in living cells is essential. For this purpose, 6-dodecanoyl-2-(dimethylamino)naphthalene (laurdan), a membrane probe that is sensitive to the polarity of the membrane, has often been used. We have synthesized and characterized 6-dodecanoyl-2-[N-methyl-N-(carboxymethyl)amino]naphthalene (C-laurdan), which has the advantages of greater sensitivity to the membrane polarity, a brighter two-photon fluorescence image, and reflecting the cell environment more accurately than laurdan. Lipid rafts can be visualized by two-photon microscopy by using C-laurdan as a probe. Our results show that the lipid rafts cover 38 % of the cell surface.     

Bioreducible Guanidinylated Polyethylenimine for Efficient Gene Delivery

Cationic polymers are known to afford efficient gene transfection. However, cytotoxicity remains a problem at the molecular weight for optimal DNA delivery. As such, optimized polymeric gene delivery systems are still a sought‐after research goal. A guanidinylated bioreducible branched polyethylenimine (GBPEI‐SS) was synthesized by using a disulfide bond to crosslink the guanidinylated BPEI (GBPEI). GBPEI‐SS showed sufficient plasmid DNA (pDNA) condensation ability. The physicochemical properties of GBPEI‐SS demonstrate that it has the appropriate size (～200 nm) and surface potential (～30 mV) at a nitrogen‐to‐phosphorus ratio of 10. No significant toxicity was observed, possibly due to bioreducibility and to the guanidine group delocalizing the positive charge of the primary amine in BPEI. Compared with the nonguanidinylated analogue, BPEI‐SS, GBPEI‐SS showed enhanced transfection efficiency owing to increased cellular uptake and efficient pDNA release by cleavage of disulfide bonds. This system is very efficient for delivering pDNA into cells, thereby achieving high transfection efficiency and low cytotoxicity.     

An integrated device for monitoring time-dependent in vitro expression from single genes in picolitre droplets

Microdroplets have great potential for high-throughput biochemical screening. We report the design of an integrated microfluidic device for droplet formation, incubation and screening. Picolitre water-in-oil droplets can be stored in a reservoir that contains approximately 10(6) droplets. In this reservoir droplets are stable for at least 6 h, which gives an extended timescale for biochemical experiments. We demonstrate the utility of the system by following the in vitro expression of green fluorescent protein. The high efficiency allows protein expression from a single molecule of DNA template, creating "monoclonal droplets" in which genotype and phenotype are combined in one emulsion compartment.     

Ssu72 Dual-Specific Protein Phosphatase: From Gene to Diseases

More than 70% of eukaryotic proteins are regulated by phosphorylation. However, the mechanism of dephosphorylation that counteracts phosphorylation is less studied. Phosphatases are classified into 104 distinct groups based on substrate-specific features and the sequence homologies in their catalytic domains. Among them, dual-specificity phosphatases (DUSPs) that dephosphorylate both phosphoserine/threonine and phosphotyrosine are important for cellular homeostasis. Ssu72 is a newly studied phosphatase with dual specificity that can dephosphorylate both phosphoserine/threonine and phosphotyrosine. It is important for cell-growth signaling, metabolism, and immune activation. Ssu72 was initially identified as a phosphatase for the Ser5 and Ser7 residues of the C-terminal domain of RNA polymerase II. It prefers the cis configuration of the serine–proline motif within its substrate and regulates Pin1, different from other phosphatases. It has recently been reported that Ssu72 can regulate sister chromatid cohesion and the separation of duplicated chromosomes during the cell cycle. Furthermore, Ssu72 appears to be involved in the regulation of T cell receptor signaling, telomere regulation, and even hepatocyte homeostasis in response to a variety of stress and damage signals. In this review, we aim to summarize various functions of the Ssu72 phosphatase, their implications in diseases, and potential therapeutic indications.     

The Puzzle of RNAs that Target Gene Promoters

Setting the right target : Most researchers who use small RNAs in mammalian cells assume that mRNA will be the target. Recent studies suggest that small RNAs can also target chromosomal DNA.

     

酵母中表达基因工程重组蛋白药物的研究进展

本文主要从酵母载体系统的选择、酵母表达宿主的选择、多拷贝菌株的构建、高密度发酵培养酵母细胞、应用酵母系统表达外源基因的研究现状、酵母表达系统的缺陷及对策等几个方面综述了近年来酵母表达体系在基因工程重组蛋白药物开发方面的研究进展。     

Genes and Gene Networks Involved in Sodium Fluoride-Elicited Cell Death Accompanying Endoplasmic Reticulum Stress in Oral Epithelial Cells

Here, to understand the molecular mechanisms underlying cell death induced by sodium fluoride (NaF), we analyzed gene expression patterns in rat oral epithelial ROE2 cells exposed to NaF using global-scale microarrays and bioinformatics tools. A relatively high concentration of NaF (2 mM) induced cell death concomitant with decreases in mitochondrial membrane potential, chromatin condensation and caspase-3 activation. Using 980 probe sets, we identified 432 up-regulated and 548 down-regulated genes, that were differentially expressed by >2.5-fold in the cells treated with 2 mM of NaF and categorized them into 4 groups by K-means clustering. Ingenuity^® pathway analysis revealed several gene networks from gene clusters. The gene networks Up-I and Up-II included many up-regulated genes that were mainly associated with the biological function of induction or prevention of cell death, respectively, such as Atf3, Ddit3 and Fos (for Up-I) and Atf4 and Hspa5 (for Up-II). Interestingly, knockdown of Ddit3 and Hspa5 significantly increased and decreased the number of viable cells, respectively. Moreover, several endoplasmic reticulum (ER) stress-related genes including, Ddit3, Atf4 and Hapa5, were observed in these gene networks. These findings will provide further insight into the molecular mechanisms of NaF-induced cell death accompanying ER stress in oral epithelial cells.     

葡萄球菌肠毒素B基因真核表达系统的构建及目的蛋白的鉴定

目的　克隆葡萄球菌肠毒素B(SEB)基因 ,构建其真核表达系统并表达目的蛋白。方法　采用高保真PCR从金黄色葡萄球菌FRIS6B株中扩增全长SEB ,目的片段T A克隆后测序。重组质粒pUCm T SEB经酶切获得的pUCm T SEB基因片段与真核表达载体pPIC9K连接。将重组真核载体pPIC9K SEB转化入P .pastorisGS115株 ,经PCR筛选并鉴定pPIC9K SEB P .pastorisGS115。在 0 5 % (v v)甲醇诱导下 ,采用SDS PAGE检测重组SEB(rSEB)的表达 ,并对rSEB的N端氨基酸序列和诱导人脐静脉内皮EVC 30 4细胞分泌IL 1α和TNFα的作用进行了检测。结果　所克隆的SEB基因核苷酸及氨基酸序列的同源性分别高达 98 84 %～ 10 0 %和 10 0 %。表达的SEB在SDS PAGE图谱的位置与预期相符。rSEB氨基末端氨基酸序列与预期完全相符。rSEB与SEB商品有相似的促人脐静脉内皮细胞EVC 30 4分泌IL 1α和TNFα的活性。结论　已成功地构建了SEB的真核表达系统 ,为进一步分析SEB分子中毒性相关活性位点、定位突变获得减毒或无毒的突变体奠定了基础。     

丙型肝炎病毒全基因重组质粒的构建及其转染细胞系的建立

目的构建丙型肝炎病毒(HCV)全基因重组质粒,并建立其转染细胞系。方法双酶切质粒JFH1,回收HCVDNA,分别与真核表达载体pIRESneo和逆转录病毒载体pLNCX连接,构建重组质粒pIRESneo-HCV和pLNCX-HCV。将重组质粒pIRESneo-HCV转染BHK细胞,pLNCX-HCV转染pA317细胞,通过PCR、间接ELISA和免疫荧光试验鉴定转染细胞。结果重组质粒PCR和酶切鉴定证明构建正确。转染细胞上清经PCR检测均可见目的基因条带;ELISA结果表明,转染细胞冻融上清均可与抗HCV小鼠血清发生强的结合反应;转染细胞均可与抗HCV小鼠血清反应,产生荧光,但荧光不多且斑点不亮。结论已成功构建HCV全基因重组质粒,并建立了可表达HCV蛋白的转染细胞系,为转基因动物模型的建立奠定了基础。     

Human Primary Dermal Fibroblasts Interacting with 3-Dimensional Matrices for Surgical Application Show Specific Growth and Gene Expression Programs

Several types of 3-dimensional (3D) biological matrices are employed for clinical and surgical applications, but few indications are available to guide surgeons in the choice among these materials. Here we compare the in vitro growth of human primary fibroblasts on different biological matrices commonly used for clinical and surgical applications and the activation of specific molecular pathways over 30 days of growth. Morphological analyses by Scanning Electron Microscopy and proliferation curves showed that fibroblasts have different ability to attach and proliferate on the different biological matrices. They activated similar gene expression programs, reducing the expression of collagen genes and myofibroblast differentiation markers compared to fibroblasts grown in 2D. However, differences among 3D matrices were observed in the expression of specific metalloproteinases and interleukin-6. Indeed, cell proliferation and expression of matrix degrading enzymes occur in the initial steps of interaction between fibroblast and the investigated meshes, whereas collagen and interleukin-6 expression appear to start later. The data reported here highlight features of fibroblasts grown on different 3D biological matrices and warrant further studies to understand how these findings may be used to help the clinicians choose the correct material for specific applications.     

Melatonin Enhances Drought Tolerance in Rice Seedlings by Modulating Antioxidant Systems,Osmoregulation, and Corresponding Gene Expression

Rice is the third largest food crop in the world, especially in Asia. Its production in various regions is affected to different degrees by drought stress. Melatonin (MT), a novel growth regulator, plays an essential role in enhancing stress resistance in crops. Nevertheless, the underlying mechanism by which melatonin helps mitigate drought damage in rice remains unclear. Therefore, in the present study, rice seedlings pretreated with melatonin (200 μM) were stressed with drought (water potential of −0.5 MPa). These rice seedlings were subsequently examined for their phenotypes and physiological and molecular properties, including metabolite contents, enzyme activities, and the corresponding gene expression levels. The findings demonstrated that drought stress induced an increase in malondialdehyde (MDA) levels, lipoxygenase (LOX) activity, and reactive oxygen species (ROS, e.g., O₂⁻ and H₂O₂) in rice seedlings. However, the melatonin application significantly reduced LOX activity and the MDA and ROS contents (O₂⁻ production rate and H₂O₂ content), with a decrease of 29.35%, 47.23%, and (45.54% and 49.33%), respectively. It activated the expression of ALM1, OsPOX1, OsCATC, and OsAPX2, which increased the activity of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX), respectively. Meanwhile, the melatonin pretreatment enhanced the proline, fructose, and sucrose content by inducing OsP5CS, OsSUS7, and OsSPS1 gene expression levels. Moreover, the melatonin pretreatment considerably up-regulated the expression levels of the melatonin synthesis genes TDC2 and ASMT1 under drought stress by 7-fold and 5-fold, approximately. These improvements were reflected by an increase in the relative water content (RWC) and the root-shoot ratio in the drought-stressed rice seedlings that received a melatonin application. Consequently, melatonin considerably reduced the adverse effects of drought stress on rice seedlings and improved rice’s ability to tolerate drought by primarily boosting endogenous antioxidant enzymes and osmoregulation abilities.     

Stable expression of soluble therapeutic peptides in eukaryotic cells by multimerisation: application to the HIV-1 fusion inhibitory peptide C46

A major drawback of therapeutic peptides is their short half-life, which results in the need for multiple applications and high synthesis costs. To overcome this, we established a eukaryotic expression system that allows the stable expression of small therapeutic peptides by multimerisation. By inserting the sequence encoding the therapeutic peptide between a signal peptide and the multimerising domain of the alpha-chain from the human C4bp plasma protein, therapeutic peptides as small as 5 kDa are secreted as multimers from transfected cells; this allows easy purification. As proof of principle, we show that the T20-derived HIV-1 fusion inhibitory peptide C46 in its multimeric form: i) was efficiently secreted, ii) was more stable than the current antiviral drug T20 in vitro and in vivo, and iii) inihibited HIV-1 entry with similar efficiency in vitro. Besides the gain in stability, multimerisation also leads to increased valency and allows the combination of several therapeutic functions. Furthermore, by expressing the multimers from cells, post-translational modifications could easily be introduced.     

Gene Expression Profiling as a Tool to Investigate the Molecular Machinery Activated during Hippocampal Neurodegeneration Induced by Trimethyltin (TMT) Administration

Trimethyltin (TMT) is an organotin compound exhibiting neurotoxicant effects selectively localized in the limbic system and especially marked in the hippocampus, in both experimental animal models and accidentally exposed humans. TMT administration causes selective neuronal death involving either the granular neurons of the dentate gyrus or the pyramidal cells of the Cornu Ammonis, with a different pattern of localization depending on the different species studied or the dosage schedule. TMT is broadly used to realize experimental models of hippocampal neurodegeneration associated with cognitive impairment and temporal lobe epilepsy, though the molecular mechanisms underlying the associated selective neuronal death are still not conclusively clarified. Experimental evidence indicates that TMT-induced neurodegeneration is a complex event involving different pathogenetic mechanisms, probably acting differently in animal and cell models, which include neuroinflammation, intracellular calcium overload, and oxidative stress. Microarray-based, genome-wide expression analysis has been used to investigate the molecular scenario occurring in the TMT-injured brain in different in vivo and in vitro models, producing an overwhelming amount of data. The aim of this review is to discuss and rationalize the state-of-the-art on TMT-associated genome wide expression profiles in order to identify comparable and reproducible data that may allow focusing on significantly involved pathways.     

Development and Characterization of Indole-Responsive Whole-Cell Biosensor Based on the Inducible Gene Expression System from Pseudomonas putida KT2440

Indole is a biologically active compound naturally occurring in plants and some bacteria. It is an important specialty chemical that is used as a precursor by the pharmaceutical and chemical industries, as well as in agriculture. Recently, indole has been identified as an important signaling molecule for bacteria in the mammalian gut. The regulation of indole biosynthesis has been studied in several bacterial species. However, this has been limited by the lack of in vivo tools suitable for indole-producing species identification and monitoring. The genetically encoded biosensors have been shown to be useful for real-time quantitative metabolite analysis. This paper describes the identification and characterization of the indole-inducible system PpTrpI/P_{PP_RS00425} from Pseudomonas putida KT2440. Indole whole-cell biosensors based on Escherichia coli and Cupriavidus necator strains are developed and validated. The specificity and dynamics of biosensors in response to indole and its structurally similar derivatives are investigated. The gene expression system PpTrpI/P_{PP_RS00425} is shown to be specifically induced up to 639.6-fold by indole, exhibiting a linear response in the concentration range from approximately 0.4 to 5 mM. The results of this study form the basis for the use of whole-cell biosensors in indole metabolism-relevant bacterial species screening and characterization.     

An Efficient and Universal Protoplast Isolation Protocol Suitable for Transient Gene Expression Analysis and Single-Cell RNA Sequencing

The recent advent of single-cell RNA sequencing (scRNA-seq) has enabled access to the developmental landscape of a complex organ by monitoring the differentiation trajectory of every specialized cell type at the single-cell level. A main challenge in this endeavor is dissociating plant cells from the rigid cell walls and some species are recalcitrant to such cellular isolation. Here, we describe the establishment of a simple and efficient protocol for protoplast preparation in Chirita pumila, which includes two consecutive digestion processes with different enzymatic buffers. Using this protocol, we generated viable cell suspensions suitable for an array of expression analyses, including scRNA-seq. The universal application of this protocol was further tested by successfully isolating high-quality protoplasts from multiple organs (petals, fruits, tuberous roots, and gynophores) from representative species on the key branches of the angiosperm lineage. This work provides a robust method in plant science, overcoming barriers to isolating protoplasts in diverse plant species and opens a new avenue to study cell type specification, tissue function, and organ diversification in plants.     

Gene Therapy in HIV‐Infected Cells to Decrease Viral Impact by Using an Alternative Delivery Method

The ability of dendrimer 2G‐[Si{O(CH₂)₂N(Me)₂⁺(CH₂)₂NMe₃⁺(I^?)₂}]₈ (NN16) to transfect a wide range of cell types, as well as the possible biomedical application in direct or indirect inhibition of HIV replication, was investigated. Cells implicated in HIV infection such as primary peripheral blood mononuclear cells (PBMC) and immortalized suspension cells (lymphocytes), primary macrophages and dendritic cells, and immortalized adherent cells (astrocytes and trophoblasts) were analyzed. Dendrimer toxicity was evaluated by mitochondrial activity, cell membrane rupture, release of lactate dehydrogenase, erythrocyte hemolysis, and the effect on global gene expression profiles using whole‐genome human microarrays. Cellular uptake of genetic material was determined using flow cytometry and confocal microscopy. Transfection efficiency and gene knockdown was investigated using dendrimer‐delivered antisense oligonucleotides and small interfering RNA (siRNA). Very little cytotoxicity was detected in a variety of cells relevant to HIV infection and erythrocytes after NN16 dendrimer treatment. Imaging of cellular uptake showed high transfection efficiency of genetic material in all cells tested. Interestingly, NN16 further enhanced the reduction of HIV protein 24 antigen release by antisense oligonucleotides due to improved transfection efficiency. Finally, the dendrimer complexed with siRNA exhibited therapeutic potential by specifically inhibiting cyclooxygenase‐2 gene expression in HIV‐infected nervous system cells. NN16 dendrimers demonstrated the ability to transfect genetic material into a vast array of cells relevant to HIV pathology, combining high efficacy with low toxicity. These results suggest that NN16 dendrimers have the potential to be used as a versatile non‐viral vector for gene therapy against HIV infection.     

羊布氏菌vjbR基因的克隆及原核表达

目的克隆并原核表达羊布氏菌强毒株16M vjbR基因。方法 PCR扩增羊布氏杆菌16M株vjbR基因,亚克隆至原核表达载体pET-28a中,构建重组原核表达质粒pET-28a-vjbR,转化感受态大肠杆菌BL21(DE3),IPTG诱导表达。表达的重组蛋白经His Trap FF纯化后,进行Western blot分析。结果扩增的vjbR基因大小为708 bp,与预期一致;重组原核表达质粒pET-28a-vjbR经双酶切及测序证明构建正确;表达的重组蛋白相对分子质量约为29 000,诱导4 h表达量可达6.84 mg/ml;纯化的重组蛋白纯度为65.7%,能被羊布氏杆菌阳性血清所识别。结论成功在大肠杆菌中表达了羊布氏菌VJBR蛋白,为其功能的研究、羊布病诊断试剂盒的研制及亚单位疫苗的研发奠定了基础。