植物小RNA荧光原位杂交实验方法

小RNA是对植物生长发育十分重要的一类小分子核苷酸,在多种生命过程以及胁迫响应中发挥重要调控作用。对小RNA的定位研究有助于揭示它们的功能,而小RNA荧光原位杂交(sRNA-FISH)是一种通过荧光检测技术对生物体内小RNA进行定性或半定量分析的技术,目前该技术已经在动物体内被广泛应用,而在植物体内的应用还比较少。该文...     

Chemogenomic Approaches for Revealing Drug Target Interactions in Drug Discovery

The drug discovery process has been a crucial and cost-intensive process. This cost is not only monetary but also involves risks, time, and labour that are incurred while introducing a drug in the market. In order to reduce this cost and the risks associated with the drugs that may result in severe side effects, the in silico methods have gained popularity in recent years. These methods have had a significant impact on not only drug discovery but also the related areas such as drug repositioning, drug-target interaction prediction, drug side effect prediction, personalised medicine, etc. Amongst these research areas predicting interactions between drugs and targets forms the basis for drug discovery. The availability of big data in the form of bioinformatics, genetic databases, along with computational methods, have further supported data-driven decision-making. The results obtained through these methods may be further validated using in vitro or in vivo experiments. This validation step can further justify the predictions resulting from in silico approaches, further increasing the accuracy of the overall result in subsequent stages. A variety of approaches are used in predicting drug-target interactions, including ligand-based, molecular docking based and chemogenomic-based approaches. This paper discusses the chemogenomic methods, considering drug target interaction as a classification problem on whether or not an interaction between a particular drug and target would serve as a basis for understanding drug discovery/drug repositioning. We present the advantages and disadvantages associated with their application.     

Tick tock,tick tock: Mouse culture and tissue aging captured by an epigenetic clock

Aging is associated with dramatic changes to DNA methylation (DNAm), although the causes and consequences of such alterations are unknown. Our ability to experimentally uncover mechanisms of epigenetic aging will be greatly enhanced by our ability to study and manipulate these changes using in vitro models. However, it remains unclear whether the changes elicited by cells in culture can serve as a model of what is observed in aging tissues in vivo. To test this, we serially passaged mouse embryonic fibroblasts (MEFs) and assessed changes in DNAm at each time point via reduced representation bisulfite sequencing. By developing a measure that tracked cellular aging in vitro, we tested whether it tracked physiological aging in various mouse tissues and whether anti‐aging interventions modulate this measure. Our measure, termed CultureAGE, was shown to strongly increase with age when examined in multiple tissues (liver, lung, kidney, blood, and adipose). As a control, we confirmed that the measure was not a marker of cellular senescence, suggesting that it reflects a distinct yet progressive cellular aging phenomena that can be induced in vitro. Furthermore, we demonstrated slower epigenetic aging in animals undergoing caloric restriction and a resetting of our measure in lung and kidney fibroblasts when re‐programmed to iPSCs. Enrichment and clustering analysis implicated EED and Polycomb group (PcG) factors as potentially important chromatin regulators in translational culture aging phenotypes. Overall, this study supports the concept that physiologically relevant aging changes can be induced in vitro and used to uncover mechanistic insights into epigenetic aging.     

Tissue engineering strategies to bioengineer the ageing skin phenotype in vitro

Human skin ageing is a complex and heterogeneous process, which is influenced by genetically determined intrinsic factors and accelerated by cumulative exposure to extrinsic stressors. In the current world ageing demographic, there is a requirement for a bioengineered ageing skin model, to further the understanding of the intricate molecular mechanisms of skin ageing, and provide a distinct and biologically relevant platform for testing actives and formulations. There have been many recent advances in the development of skin models that recapitulate aspects of the ageing phenotype in vitro. This review encompasses the features of skin ageing, the molecular mechanisms that drive the ageing phenotype, and tissue engineering strategies that have been utilised to bioengineer ageing skin in vitro.     

Rapid directed molecular evolution of fluorescent proteins in mammalian cells

In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracellular brightness. Here we describe a practical method for rapid optimization of fluorescent proteins via directed molecular evolution in cultured mammalian cells. Using this method, we were able to perform screening of large gene libraries containing up to 2 × 10⁷ independent random genes of fluorescent proteins expressed in HEK cells, completing one iteration of directed evolution in a course of 8 days. We employed this approach to develop a set of green and near‐infrared fluorescent proteins with enhanced intracellular brightness. The developed near‐infrared fluorescent proteins demonstrated high performance for fluorescent labeling of neurons in culture and in vivo in model organisms such as Caenorhabditis elegans, Drosophila, zebrafish, and mice. Spectral properties of the optimized near‐infrared fluorescent proteins enabled crosstalk‐free multicolor imaging in combination with common green and red fluorescent proteins, as well as dual‐color near‐infrared fluorescence imaging. The described method has a great potential to be adopted by protein engineers due to its simplicity and practicality. We also believe that the new enhanced fluorescent proteins will find wide application for in vivo multicolor imaging of small model organisms.     

Culture of an epithelial cell line (MDCK) with a serum substitute

This paper reports the suitability of culturing a line of dog kidney epithelial cells, MDCK, in the presence of a serum substitute, Ultroser G. Serial subcultivation with this product was possible for at least 10 passages without any change in cell shape and size, saturation density, dome-forming ability, transepithelial resistance, and growth curve. Adhesion of newly plated cells to plastic was somewhat lower than in fetal calf serum but the trypsin-harvesting kinetics were essentially the same. However, the membrane ion transport systems was alterd: cell sodium influx was greatly diminished, suggesting a deep change in the amiloride-sensitive Na⁺ channels: sodium efflux was highly enhanced (both active and passive).     

PRIMORDIAL GERM CELL-DERIVED MOUSE EMBRYONIC GERM (EG) CELLSIN VITRORESEMBLE UNDIFFERENTIATED STEM CELLS WITH RESPECT TO DIFFERENTIATION CAPACITY AND CELL CYCLE DISTRIBUTION

Embryonic germ (EG) cells of line EG-1 derived from mouse primordial germ cells were investigated for theirin vitrodifferentiation capacity. By cultivation as embryo-like aggregates EG-1 cells differentiated into cardiac, skeletal muscle and neuronal cells accompanied by the expression of tissue-specific genes and proteins as shown by RT-PCR analysis and indirect immunofluorescence. In comparison to embryonic stem (ES) cells of line D3 the efficiency of differentiation into cardiac and muscle cells was comparatively low, whereas spontaneous neuronal differentiation was more efficient than in D3 cells. Furthermore, the distribution of cell cycle phases as a parameter for the differentiation state was analysed in undifferentiated EG cells and ES cells and compared to data obtained for embryonic carcinoma (EC) cells of line P19 and differentiated, epithelioid EPI-7 cells. Flow cytometric analysis revealed similar cell cycle phase distributions in EG, EC and ES cells. In contrast, the somatic differentiated EPI-7 cells showed a longer G₁-phase and shorter S- and G₂/M-phases. Together, our results demonstrate that the differentiation state and capacity of EG cellsin vitroresemble that of totipotent ES cells.     

In vitro anticryptosporidial activity of dinitroaniline herbicides

Abstract Despite the evaluation of over 100 antimicrobial drugs, the diarrheal disease cryptosporidiosis has remained refractory to treatment. We report the evaluation of five dinitroaniline herbicides including trifluralin, profluralin, nitralin, pendimethalin, and fluchloralin for anticryptosporidial activity in an in vitro cultivation model of Cryptosporidium parvum . All five compounds exhibited significant anticryptosporidial activities with no corresponding evidence of toxicity. The most active compound was pendimethalin with an IC₅₀ of 0.19 μM while nitralin was the least active with an IC₅₀ of 4.5 μM. These compounds should be evaluated further in an animal model of cryptosporidiosis.     

Some aspects of the immune response of koalas (Phascolarctos cinereus) and in vitro neutralization of chlamydia psittaci (koala strains)

Abstract Western-blot analysis was used to study the reaction of koala antisera, two specific polyclonal antibodies and one monoclonal antibody, with chlamydial antigens in koalas infected with Chlamydia psittaci . The koala sera recognized four C. psittaci surface antigens, corresponding to the major outer membrane protein (39.5 kDa), 31 kDa protein, 18 kDa protein and lipopolysaccharide. The S25-23 LPS specific monoclonal antibody inhibited chlamydial infection (55–67%) with both koala strains (type I and type II). Both koala antiserum and rabbit polyclonal antibodies against either type of chlamydia significantly reduced the number of infected cells resulting from type II infections at a dilution of 1 in 20. Rabbit antiserum against type II was effective in neutralizing infection by type II elementary bodies, but was less effective against type I infection. In addition, no koala antiserum was effective in neutralizing type I infection.