Interleukin-17F attenuates H2O2-induced cell cycle arrest

Interleukin IL-17F was expressed in colon epithelial cells and showed multiple functions in colon tumorigenesis. However, the role of IL-17F in colon cancer cell cycle progression remains unclear. In this study, we analyzed the effects of IL-17F on oxidant-induced cell cycle shift in human colon cancer cells. IL-17F overexpressing and wildtype HCT116 cells were challenged with H₂O₂. Cell cycle distribution analysis showed IL-17F attenuated H₂O₂-induced G2/M phase arrest by inhibiting S to G2/M transition. We further checked expression levels of two critical cell cycle regulators p21 and p27. The results showed that IL-17F could inhibit H₂O₂ induced p27 up-regulation. Meanwhile, IL-17F could increase the phosphorylation of p38 after H₂O₂ treatment. The regulations of p27 level and p38 activity may contribute to the impaired G2/M phase arrest by IL-17F. Taken together, our findings extend IL-17F as an important factor in colon cancer development and provide new insight into the signaling pathway.     

High-throughput analysis of the protein sequence-stability landscape using a quantitative yeast surface two-hybrid system and fragment reconstitution

Stability evaluation of many mutants can lead to a better understanding of the sequence determinants of a structural motif and of factors governing protein stability and protein evolution. The traditional biophysical analysis of protein stability is low throughput, limiting our ability to widely explore sequence space in a quantitative manner. In this study, we have developed a high-throughput library screening method for quantifying stability changes, which is based on protein fragment reconstitution and yeast surface display. Our method exploits the thermodynamic linkage between protein stability and fragment reconstitution and the ability of the yeast surface display technique to quantitatively evaluate protein-protein interactions. The method was applied to a fibronectin type III (FN3) domain. Characterization of fragment reconstitution was facilitated by the co-expression of two FN3 fragments, thus establishing a yeast surface two-hybrid method. Importantly, our method does not rely on competition between clones and thus eliminates a common limitation of high-throughput selection methods in which the most stable variants are recovered predominantly. Thus, it allows for the isolation of sequences that exhibit a desired level of stability. We identified more than 100 unique sequences for a β-bulge motif, which was significantly more informative than natural sequences of the FN3 family in revealing the sequence determinants for the β-bulge. Our method provides a powerful means for the rapid assessment of the stability of many variants, for the systematic assessment of the contribution of different factors to protein stability, and for enhancement of the protein stability.     

Messenger RNA quantification after fluorescence activated cell sorting using intracellular antigens

Recent studies using stem cells or cancer stem cells have revealed the importance of detecting minor populations of cells in blood or tissue and analyzing their biological characteristics. The only possible method for carrying out such procedures is fluorescence activated cell sorting (FACS). However, FACS has the following limitations. First, cells without an appropriate cell surface marker cannot be sorted. Second, the cells have to be kept alive during the sorting process in order to analyze their biological characteristics. If an intracellular antigen that was specific to a particular cell type could be stained with a florescent dye and then the cells can be sorted without causing RNA degradation, a more simple and universal method for sorting and analyzing cells with a specific gene expression pattern could be established since the biological characteristics of the sorted cells could then be determined by analyzing their gene expression profile. In this study, we established a basic protocol for messenger RNA quantification after FACS (FACS-mQ) targeting intracellular antigens. This method can be used for the detection and analysis of stem cells or cancer stem cells in various tissues.     

The production of clonal and axenic cultures of microalgae using fluorescence-activated cell sorting

Unialgal cultures of the flagellate algae Cyanophora paradoxa, Haematococcus lacustris, Monomastix sp., Scherffelia dubia and Spermatozopsis similis which contained bacteria were sorted by flow cytometry to obtain axenic clonal cultures. The variables used for fluorescence-activated cell sorting (FACS) were chlorophyll autofluorescence, forward scatter and side scatter of the laser beam. To produce clonal cultures, a single cell was sorted into each culture flask. Depending on the species, about 20–30% of the sorted cultures grew successfully and at least 20% of these were axenic even if the numerical ratio betweeen bacteria and algae in the original cultures was as high as 300:1. FACS represents an effective and rapid method for the preparation of clonal and axenic cultures of microalgae.     

Distinct acidic clusters and hydrophobic residues in the alternative splice domains X1 and X2 of alpha7 integrins define specificity for laminin isoforms

The binding specificity of alpha7beta1 integrins for different laminin isoforms is defined by the X1 and X2 splice domains located in the beta-propeller domain of the alpha7 subunit. In order to gain insight into the mechanism of specific laminin-integrin interactions, we defined laminin-binding epitopes of the alpha7X1 and -X2 domains by single amino acid substitutions and domain swapping between X1 and X2. The interaction of mutated, recombinantly prepared alpha7X1beta1 and alpha7X2beta1 heterodimers with various laminin isoforms was studied by surface plasmon resonance and solid phase binding assays. The data show that distinct clusters of surface-exposed acidic residues located in different positions of the X1 and the X2 loops are responsible for the specific recognition of laminins. These residues are conserved between the respective X1 or X2 splice domains of the alpha7 chains of different species, some also in the corresponding X1/X2 splice domains of alpha6 integrin. Interestingly, ligand binding was also modulated by mutating surface-exposed hydrophobic residues (alpha7X1L205, alpha7X2Y208) at positions corresponding to the fibronectin binding synergy site in alpha5beta1 integrin. Mutations in X1 that affected binding to laminin-1 also affected binding to laminin-8 and -10, but not to the same extent, thus allowing conclusions on the specific role of individual surface epitopes in the selective recognition of laminin-1 versus laminins -8 and -10. The role of the identified epitopes was confirmed by molecular dynamics simulations of wild-type integrins and several inactivating mutations. The analysis of laminin isoform interactions with various X1/X2 chimaera lend further support to the key role of negative surface charges and pointed to an essential contribution of the N-terminal TARVEL sequence of the X1 domain for recognition of laminin-8 and -10. In conclusion, specific surface epitopes containing charged and hydrophobic residues are essential for ligand binding and define specific interactions with laminin isoforms.     

Exploration of Alternate Catalytic Mechanisms and Optimization Strategies for Retroaldolase Design

Designed retroaldolases have utilized a nucleophilic lysine to promote carbon–carbon bond cleavage of β-hydroxy-ketones via a covalent Schiff base intermediate. Previous computational designs have incorporated a water molecule to facilitate formation and breakdown of the carbinolamine intermediate to give the Schiff base and to function as a general acid/base. Here we investigate an alternative active-site design in which the catalytic water molecule was replaced by the side chain of a glutamic acid. Five out of seven designs expressed solubly and exhibited catalytic efficiencies similar to previously designed retroaldolases for the conversion of 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone to 6-methoxy-2-naphthaldehyde and acetone. After one round of site-directed saturation mutagenesis, improved variants of the two best designs, RA114 and RA117, exhibited among the highest k_cat (> 10^− 3 s^− 1) and k_cat/K_M (11–25 M^− 1 s^− 1) values observed for retroaldolase designs prior to comprehensive directed evolution. In both cases, the > 10⁵-fold rate accelerations that were achieved are within 1–3 orders of magnitude of the rate enhancements reported for the best catalysts for related reactions, including catalytic antibodies (k_cat/k_uncat = 10⁶ to 10⁸) and an extensively evolved computational design (k_cat/k_uncat > 10⁷). The catalytic sites, revealed by X-ray structures of optimized versions of the two active designs, are in close agreement with the design models except for the catalytic lysine in RA114. We further improved the variants by computational remodeling of the loops and yeast display selection for reactivity of the catalytic lysine with a diketone probe, obtaining an additional order of magnitude enhancement in activity with both approaches.     

Single-cell isolation and cloning of Tetrahymena thermophila cells with a fluorescence-activated cell sorter

We developed a method for cloning cells of the ciliate Tetrahymena thermophila in chemically defined medium (CDM) using a fluorescence-activated cell sorter (FACS). Although T. thermophila is a model unicellular eukaryote, two major technical difficulties remain in its cloning. First, T. thermophila fails to proliferate from low density in CDM, particularly if the inoculum contains single cells. Second, general cloning methods are time consuming and have low throughput. Here, we modified the CDM by addition of bovine serum albumin that helped growth from an inoculum with a density of 10 cell/ml (1 cell/100 μl). In addition, we applied a FACS for isolation of single cells. We showed that it is possible to separate cell populations based on the presence or absence of phagocytosed fluorescent beads and to isolate single cells in a modified CDM by FACS. Our techniques allow the direct isolation of single cells and facilitate the establishment of clonal strains.     

Flow cytometry and FACS applied to filamentous fungi

Flow cytometry is an automated, laser- or impedance-based, high throughput method that allows very rapid analysis of multiple chemical and physical characteristics of single cells within a cell population. It is an extremely powerful technology that has been used for over four decades with filamentous fungi. Although single cells within a cell population are normally analysed rapidly on a cell-by-cell basis using the technique, flow cytometry can also be used to analyse cell (e.g. spore) aggregates or entire microcolonies. Living or fixed cells can be stained with a wide range of fluorescent reporters to label different cell components or measure different physiological processes. Flow cytometry is also suited for measurements of cell size, interaction, aggregation or shape using non-labelled cells by means of analysing their light scattering characteristics. Fluorescence-activated cell sorting (FACS) is a specialized form of flow cytometry that provides a method for sorting a heterogeneous mixture of cells into two or more containers based upon the fluorescence and/or light scattering properties of each cell. The major advantage of analysing cells by flow cytometry over microscopy is the speed of analysis: thousands of cells can be analysed per second or sorted in minutes. Drawbacks of flow cytometry are that specific cells cannot be followed in time and normally spatial information relating to individual cells is lacking. A big advantage over microscopy is when using FACS, cells with desired characteristics can be sorted for downstream experimentation (e.g. for growth, infection, enzyme production, gene expression assays or ‘omics’ approaches). In this review, we explain the basic concepts of flow cytometry and FACS, define its advantages and disadvantages in comparison with microscopy, and describe the wide range of applications in which these powerful technologies have been used with filamentous fungi.     

Quantification of plasmid loss in Escherichia coli cells by use of flow cytometry

A method was developed to study plasmid stability in Escherichia coli cells, which utilised the high speed analysis properties of flow cytometry. To discriminate between plasmid-harbouring cells and plasmid-free cells a plasmid-encoded Lac repressor protein was used to regulate the expression of a chromosomally inserted green fluorescent protein gene in the host cells. Flow cytometric analysis enabled detection and quantification of plasmid-free cells due to their green fluorescent phenotype. The reported system offers real-time analysis in combination with a very low detection level of plasmid loss in bacterial populations. This could be useful in future investigations of plasmid stability and population selection in bacterial communities.     

Recovery of a bacterial sub-population from sewage using immunofluorescent flow cytometry and cell sorting

Abstract The effectiveness of immunofluorescence flow cytometry and cell sorting to detect, quantify and separate indigenous bacterial populations present in low concentrations in sewage outflow was investigated. Preparatory experiments for targeted recovery revealed indigenous, immunoglobulin-G-binding particles present at low levels in sewage outflow samples taken from Coniston Water. Fluorescence-activated cell sorting of this population was employed to enrich for these particles, which were confirmed as bacterial cells. This cell population comprised approximately 23% of the total plate count on MacConkey agar before cell sorting, rising to approximately 95% after sorting. These results corresponded to cell densities of less than 5% of the total plate count on R2A agar. Taxonomic tests suggested the bacterium to be Ochrobactrum anthropi .     

Community shifts of actively growing lake bacteria after N-acetyl-glucosamine addition: improving the BrdU-FACS method

In aquatic environments, community dynamics of bacteria, especially actively growing bacteria (AGB), are tightly linked with dissolved organic matter (DOM) quantity and quality. We analyzed the community dynamics of DNA-synthesizing and accordingly AGB by linking an improved bromodeoxyuridine immunocytochemistry approach with fluorescence-activated cell sorting (BrdU-FACS). FACS-sorted cells of even oligotrophic ecosystems in winter were characterized by 16S rRNA gene analysis. In incubation experiments, we examined community shifts of AGB in response to the addition of N-acetyl-glucosamine (NAG), one of the most abundant aminosugars in aquatic systems. Our improved BrdU-FACS analysis revealed that AGB winter communities of oligotrophic Lake Stechlin (northeastern Germany) substantially differ from those of total bacteria and consist of Alpha-, Beta-, Gamma-, Deltaproteobacteria, Actinobacteria, Candidatus OP10 and Chloroflexi. AGB populations with different BrdU-fluorescence intensities and cell sizes represented different phylotypes suggesting that single-cell growth potential varies at the taxon level. NAG incubation experiments demonstrated that a variety of widespread taxa related to Alpha-, Beta-, Gammaproteobacteria, Bacteroidetes, Actinobacteria, Firmicutes, Planctomycetes, Spirochaetes, Verrucomicrobia and Chloroflexi actively grow in the presence of NAG. The BrdU-FACS approach enables detailed phylogenetic studies of AGB and, thus, to identify those phylotypes which are potential key players in aquatic DOM cycling.     

微生物流式分选的单细胞样品制备及存活率提高的方法优化

【背景】以流式细胞技术为代表的高通量筛选技术能够高效筛选具有目标性状的微生物工程菌株。在流式分选中微生物的粘连会造成分析数据不准确,分选纯度降低,因此快速简便的单细胞样品制备是流式检测的关键。优势菌大多是通过筛选偶联荧光蛋白的随机突变库获得,阳性率低,杂质和死细胞的自发荧光较强,容易混入分选门内造成存活率降低,亟须提高分选存活率的方法。【目的】建立一种简便的微生物流式分选的单细胞样品制备方法,并通过碘化丙啶(propidium iodide, PI)染色提高分选样品存活率。【方法】分别在大肠杆菌、枯草芽孢杆菌、谷氨酸棒状杆菌和酵母菌4种底盘细胞中探索超声波、消化酶、表面活性剂及超声-表面活性剂联合作用4种方式对单细胞制备效率的影响。提高微生物流式分选存活率,用常压室温等离子诱变(atmospheric and room temperature plasma, ARTP)技术处理含有绿色荧光蛋白(green fluorescent protein, GFP)的酿酒酵母HZ848 (简称HZ848-GFP),形成不同强度GFP文库后,按照GFP强度分选全细胞和PI染色阴性细胞的前0.5%,统计单细胞存活率。【结果】酵母细胞分散条件为：0.01% Tween-80联合超声1 min,单细胞率达到88%以上,PI染色细胞破损率＜1.4%。谷氨酸棒状杆菌单细胞分散条件为：0.01% Tween-80联合超声5 min,单细胞率达到97%以上,PI染色细胞破损率＜1%。分选存活率结果表明,未用PI染色的酿酒酵母分选后单细胞存活率是4.3%,用PI染色去除死细胞后再分选单细胞存活率是18.3%,后者是前者的4.3倍,且具有显著性差异。【结论】本研究为微生物流式分选建立了一套简单快捷的单细胞样品制备方法,证实了PI染色法能够显著提高分选样品存活率。     

A Cre-loxP based system for studying horizontal gene transfer

We have previously shown that DNA can be transferred to phagocytosing cells via the uptake of apoptotic cells. We report a model system that facilitates study of antigen presentation of genes transferred specifically via horizontal gene transfer. Constructs were generated encoding the LacZ gene or the influenza A nucleoprotein silenced by a STOP sequence flanked by two loxP sites. These reporter genes were demonstrated to be silent in donor cells and become activated after phagocytosis of Cre-expressing fibroblasts or macrophages. These results provide a model system for studying the influence of horizontally transferred antigens on activation of the immune system.