SCOPE-Seq: a scalable technology for linking live cell imaging and single-cell RNA sequencing

Optically decodable beads link the identity of a sample to a measurement through an optical barcode, enabling libraries of biomolecules to be captured on beads in solution and decoded by fluorescence. This approach has been foundational to microarray, sequencing, and flow-based expression profiling technologies. We combine microfluidics with optically decodable beads and show that phenotypic analysis of living cells can be linked to single-cell sequencing. As a proof-of-concept, we demonstrate the accuracy and scalability of our tool called Single Cell Optical Phenotyping and Expression sequencing (SCOPE-Seq) to combine live cell imaging with single-cell RNA sequencing.     

Ameboid cell motility: a model and inverse problem, with an application to live cell imaging data

In this article a mathematical model for ameboid cell movement is developed using a spring-dashpot system with Newtonian dynamics. The model is based on the facts that the cytoskeleton plays a primary role for cell motility and that the cytoplasm is viscoelastic. Based on the model, the inverse problem can be posed: if a structure like a spring-dashpot system is embedded into the living cell, what kind of characteristic properties must the structure have in order to reproduce a given movement of the cell? This inverse problem is the primary topic of this paper. On one side the model mimics some features of the movement, and on the other side, the solution to the inverse problem provides model parameters that give some insight, principally into the mechanical aspect, but also, through qualitative reasoning, into chemical and biophysical aspects of the cell. Moreover, this analysis can be done locally or globally and in different media by using the simplest possible information: positions of the cell and nuclear membranes. It is shown that the model and solution to the inverse problem for simulated data sets are highly accurate. An application to a set of live cell imaging data obtained from random movements of a human brain tumor cell (U87-MG human glioblastoma cell line) then provides an example of the efficiency of the model, through the solution of its inverse problem, as a way of understanding experimental data.     

Deuterium oxide effects upon three parameters characterizing the activity of glycerol-extracted muscle: phosphate release, ATP-induced shortening, and 22Na+ distribution

ATP splitting activity is progressively reduced with increasing heavy water (D2)) concentration. In contrast, sarcomere shorteining inhibition produced by D2O does not significantly depend on its concentration. Even at low concentration, the presence of D2O does reduce the excessive accumulation of radioactive sodium within glycerinated frog muscle. These heavy water effects on muscular contraction and soidum distribution can be interpreted to indicate adsorbed water within the cells. Evaluation of these experimental results in terms of Gibbs free enthalpy of binding at the adsorption sites of D20 or H20 is in good agreement with the data in the literature.     

A protocol for non-radioactive DNA labelling and detection in the RFLP analysis of rice and tomato using single-copy probes

An improved protocol for non-radioactive labelling and detection of rice and tomato DNA is described. The procedure includes the use of polymerase chain reaction for the incorporation of digoxigenin-dUTP in the DNA molecule and the use of a chemiluminescent compound (AMPPD) for the signal detection.     

Vaccinia virus proteins A36 and F12/E2 show strong preferences for different kinesin light chain isoforms

Vaccinia virus (VACV) utilizes microtubule‐mediated trafficking at several stages of its life cycle, of which virus egress is the most intensely studied. During egress VACV proteins A36, F12 and E2 are involved in kinesin‐1 interactions; however, the roles of these proteins remain poorly understood. A36 forms a direct link between virions and kinesin‐1, yet in its absence VACV egress still occurs on microtubules. During a co‐immunoprecipitation screen to seek an alternative link between virions and kinesin, A36 was found to bind isoform KLC1 rather than KLC2. The F12/E2 complex associates preferentially with the C‐terminal tail of KLC2, to a region that overlaps the binding site of cellular 14‐3‐3 proteins. F12/E2 displaces 14‐3‐3 from KLC and, unlike 14‐3‐3, does not require phosphorylation of KLC for its binding. The region determining the KLC1 specificity of A36 was mapped to the KLC N‐terminal heptad repeat region that is responsible for its association with kinesin heavy chain. Despite these differing binding properties F12/E2 can co‐operatively enhance A36 association with KLC, particularly when using a KLC1‐KLC2 chimaera that resembles several KLC1 spliceforms and can bind A36 and F12/E2 efficiently. This is the first example of a pathogen encoding multiple proteins that co‐operatively associate with kinesin‐1.      

Py3-FITC: a new fluorescent probe for live cell imaging of collagen-rich tissues and ionocytes

Polypyrrole-based polyamides are used as sequence-specific DNA probes. However, their cellular uptake and distribution are affected by several factors and have not been extensively studied in vivo. Here, we generated a series of fluorescence-conjugated polypyrrole compounds and examined their cellular distribution using live zebrafish and cultured human cells. Among the evaluated compounds, Py₃-FITC was able to visualize collagen-rich tissues, such as the jaw cartilage, opercle and bulbus arteriosus, in early-stage living zebrafish embryos. Then, we stained cultured human cells with Py₃-FITC and found that the staining became more intense as the amount of collagen was increased. In addition, Py₃-FITC-stained HR cells, which represent a type of ionocyte on the body surface of living zebrafish embryos. Py₃-FITC has low toxicity, and collagen-rich tissues and ionocytes can be visualized when soaked in Py₃-FITC solution. Therefore, Py₃-FITC may be a useful live imaging tool for detecting changes in collagen-rich tissue and ionocytes, including their mammalian analogues, during both normal development and disease progression.     

A multichamber fluidic device for 3D cultures under interstitial flow with live imaging: Development,characterization, and applications

Interstitial flow is an important biophysical cue that can affect capillary morphogenesis, tumor cell migration, and fibroblast remodeling of the extracellular matrix, among others. Current models that incorporate interstitial flow and that are suitable for live imaging lack the ability to perform multiple simultaneous experiments, for example, to compare effects of growth factors, extracellular matrix composition, etc. We present a nine‐chamber radial flow device that allows simultaneous 3D fluidic experiments for relatively long‐term culture with live imaging capabilities. Flow velocity profiles were characterized by fluorescence recovery after photobleaching (FRAP) for flow uniformity and estimating the hydraulic conductivity. We demonstrate lymphatic and blood capillary morphogenesis in fibrin gels over 10 days, comparing flow with static conditions as well as the effects of an engineered variant of VEGF that binds fibrin via Factor XIII. We also demonstrate the culture of contractile fibroblasts and co‐cultures with tumor cells for modeling the tumor microenvironment. Therefore, this device is useful for studies of capillary morphogenesis, cell migration, contractile cells like fibroblasts, and multicellular cultures, all under interstitial flow. Biotechnol. Bioeng. 2010;105: 982–991. © 2009 Wiley Periodicals, Inc.     

A multimodal metabolomics approach using imaging mass spectrometry and liquid chromatography-tandem mass spectrometry for spatially characterizing monoterpene indole alkaloids secreted from roots

Plants release specialized (secondary) metabolites from their roots to communicate with other organisms, including soil microorganisms. The spatial behavior of such metabolites around these roots can help us understand roles for the communication; however, currently, they are unclear because soil-based studies are complex. Here, we established a multimodal metabolomics approach using imaging mass spectrometry (IMS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to spatially assign metabolites under laboratory conditions using agar. In a case study using Catharanthus roseus, we showed that 58 nitrogen (N)-containing metabolites are released from the roots into the agar. For the metabolite assignment, we used ¹⁵N-labeled and non-labeled LC-MS/MS data, previously reported. Four metabolite ions were identified using authentic standard compounds as derived from monoterpene indole alkaloids (MIAs) such as ajmalicine, catharanthine, serpentine, and yohimbine. An alkaloid network analysis using dot products and spinglass methods characterized five clusters to which the 58 ions belong. The analysis clustered ions from the indolic skeleton-type MIAs to a cluster, suggesting that other communities may represent distinct metabolite groups. For future chemical assignments of the serpentine community, key fragmentation patterns were characterized using the ¹⁵N-labeled and non-labeled MS/MS spectra.     

Going live: A comparative analysis of the suitability of the RFP derivatives RedStar,mCherry and tdTomato for intravital and in vitro live imaging of Plasmodium parasites

Fluorescent proteins have proven to be important tools for in vitro live imaging of parasites and for imaging of parasites within the living host by intravital microscopy. We observed that a red fluorescent transgenic malaria parasite of rodents, Plasmodium berghei-RedStar, is suitable for in vitro live imaging experiments but bleaches rapidly upon illumination in intravital imaging experiments using mice. We have therefore generated two additional transgenic parasite lines expressing the novel red fluorescent proteins tdTomato and mCherry, which have been reported to be much more photostable than first- and second-generation red fluorescent proteins including RedStar. We have compared all three red fluorescent parasite lines for their use in in vitro live and intravital imaging of P. berghei blood and liver parasite stages, using both confocal and wide-field microscopy. While tdTomato bleached almost as rapidly as RedStar, mCherry showed improved photostability and was bright in all experiments performed.     

A molecular protocol using quenched FRET probes for the quarantine surveillance of Tilletia indica, the causal agent of Karnal bunt of wheat

The current surveillance protocol for Karnal bunt of wheat in most countries, including the USA, European Union (EU), and Australia, involves the tentative identification of the spores based on morphology followed by a molecular analysis. Germination of spores is required for confirmation which incurs a delay of about two weeks, which is highly unsatisfactory in a quarantine situation. A two-step PCR protocol using FRET probes for the direct detection and identification of Tilletia indica from a very few number of spores (≤10) is presented. The protocol involves amplification of the ITS1 DNA segment in the highly repeated rDNA unit from any Tilletia species, followed by FRET analysis to detect and unequivocably distinguish T. indica and the closely related T. walkeri. This rapid, highly sensitive, fluorescent molecular tool is species-specific, and could supersede the conventional microscopic diagnosis used in a quarantine surveillance protocol for Karnal bunt which is often confounded by overlapping morphological characters of closely related species.     

E4F and ATF, two transcription factors that recognize the same site, can be distinguished both physically and functionally: a role for E4F in E1A trans activation.

Previous experiments have identified an element in the adenovirus E4 promoter that is critical for E1A-dependent trans activation and that can confer inducibility to a heterologous promoter. This DNA element is a recognition site for multiple nuclear factors, including ATF, which is likely a family of DNA-binding factors with similar DNA recognition properties. However, ATF activity was found not to be altered in any demonstrable way as a result of adenovirus infection. In contrast, another factor that recognizes this element, termed E4F, was found at only very low levels in uninfected cells but was increased markedly upon adenovirus infection, as measured in DNA-binding assays. Although both the ATF activity and the E4F activity recognized and bound to the same two sites in the E4 promoter, they differed in their sequence recognition of these sites. Furthermore, E4F bound only to a small subset of the ATF recognition sites; for instance, E4F did not recognize the ATF sites in the E2 or E3 promoters. Various E4F and ATF binding sites were inserted into an expression vector and tested by cotransfection assays for responsiveness to E1A. We found that a sequence capable of binding E4F could confer E1A inducibility. In contrast, a sequence that could bind ATF but not E4F did not confer E1A inducibility. We also found that E4F formed a stable complex with the E4 promoter, whereas the ATF DNA complex was unstable and rapidly dissociated. We conclude that the DNA-binding specificity of E4F as well as the alterations in DNA-binding activity of E4F closely correlates with E1A stimulation of the E4 promoter.     

A new mode of cell cycle stimulation: Cyclin E and CDK2-mediated cytoplasmic retention of repressive E2F complexes

Comment on: Kolupaeva V, et al. Cell Cycle 2012; 11:2557-66.     

IS231 D, E and F, three new insertion sequences in Bacillus thuringiensis: extension of the IS231 family

IS231 constitutes a family of insertion sequences widespread among Bacillus thuringiensis subspecies. Three new IS231 variants have been isolated from B. thuringiensis subspecies finitimus (IS231 D and E) and israelensis (IS231F). Like the previously described IS231A, B and C, these 1.7 kb elements display single open reading frames encoding 477/478-amino-acid proteins which share between 72% and 88% identity with those of the other members of the family. Sequence comparisons also reveal that all the iso-IS231 terminal inverted repeats are strongly conserved 20 bp sequences. A region susceptible to forming a stable hairpin structure is found just upstream of the open reading frame. Nucleotide substitutions occurring on one strand of the hairpin stems are compensated for by complementary changes at facing positions, giving credence to the hypothesis that this secondary structure plays a role in the regulation of transposition. Examination of IS231 D, E and F flanking sequences reveals that IS231F is bordered by a 12 bp direct repeat. No direct repeats were found flanking IS231D or IS231E.     

Synthetic DNA probes for detection of genes for enterotoxins A, B, C, D, E and for TSST-1 in staphylococcal strains

B. JAULHAC, M. BES, N. BORNSTEIN, Y. PIÉMONTY. BRUN AND J. FLEURETTE. 1992. A dot blot hybridization technique with oligonucleotide probes was developed for the specific detection of the TSST-1 gene and the staphylococcal enterotoxin (SE) genes A, B, C, D and E. For each toxin gene a probe sequence was chosen from the previously determined sequence. A total of 145 staphylococcal strains (133 Staphylococcus aureus and 12 coagulase-negative staphylococci (CNS)) were studied by this genotypic method and by two phenotypic assays (gel immunodiffusion and ELISA). An excellent correlation (96%) was observed between the genotypic and phenotypic assays. DNA from two CNS strains hybridized with a probe without detection of the corresponding toxin (SEB for one strain and SEC for the other strain). One Staph. aureus strain was shown to be an SEC producer, but was not detected by the corresponding probe. Gene probe and immunological assays seem to be complementary methods for studies of staphylococcal strains producing (or potentially producing) TSST-1 or enterotoxins.     

A fluorescent version of the bradykinin B2 receptor antagonist B-9430: pharmacological characterization and use in live cell imaging

B-9430 (d-Arg-[Hyp(3), Igl(5), D-Igl(7), Oic(8)]-bradykinin), where Hyp is trans-4-hydroxyproline, Igl is alpha-(2-indanyl)glycine and Oic is (3as, 7as)-octahydroindol-2-yl-carbonyl is a high affinity bradykinin B(2) receptor antagonist with effects extended to the B(1) receptors at high concentrations. The N-terminus of B-9430 has been extended with d-biotinyl (B-10330) or 5(6)-carboxyfluorescein-varepsilon-aminocaproyl (B-10380) to derive fluorescent receptor probes. The pharmacological profile of B-10380 was similar to that of B-9430 with a minor loss of potency (a competitive antagonist of bradykinin at the B(2) receptors of the human isolated umbilical vein, pA(2) 6.83; an insurmountable antagonist at the B(2) receptors in the rabbit jugular vein; a weak competitive antagonist of the B(1) receptors in the rabbit aorta, pA(2) 5.95). B-10330 and B-10380 displaced the binding of [(3)H]bradykinin from rabbit B(2) receptors with a potency slightly inferior to that of B-9430 (larger gap at the rat B(2) receptor). Treatment with B-10330 and fluorescent streptavidin did not support imaging of recombinant B(2) receptors. However, the plasma membrane of HEK 293a cells that transiently expressed recombinant rabbit B(2) receptors, but not B(1) receptors, was labeled with 5-50nM B-10380 (epifluorescence microscopy). B-10380 staining was not observed in nontransfected cells and was abolished by co-treating receptor-expressing cells with a nonpeptide antagonist. The N-terminal extension of a potent peptide antagonist of the bradykinin B(2) receptor with a fluorophore produced a fluorescent probe suitable for live cell imaging and other applications at the expense of a minor loss of affinity.     

Bioactive apocarotenoids annuionones F and G: structural revision of annuionones A, B and E

The polar bioactive fractions of Helianthus annuus cv. Stella and SH-222 have yielded eight apocarotenoids, two of them isolated for the first time as natural products (annuionones F and G). The isolation of higher amounts of annuionones A and E allowed us to realize a more comprehensive spectroscopical study. We propose a revised structure for annuionone A, B and E based on careful re-analyses of new spectroscopical data.     

A simple and accurate protocol for absolute polar metabolite quantification in cell cultures using quantitative nuclear magnetic resonance

Absolute analyte quantification by nuclear magnetic resonance (NMR) spectroscopy is rarely pursued in metabolomics, even though this would allow researchers to compare results obtained using different techniques. Here we report on a new protocol that permits, after pH-controlled serum protein removal, the sensitive quantification (limit of detection [LOD] = 5−25 μM) of hydrophilic nutrients and metabolites in the extracellular medium of cells in cultures. The method does not require the use of databases and uses PULCON (pulse length-based concentration determination) quantitative NMR to obtain results that are significantly more accurate and reproducible than those obtained by CPMG (Carr–Purcell–Meiboom–Gill) sequence or post-processing filtering approaches. Three practical applications of the method highlight its flexibility under different cell culture conditions. We identified and quantified (i) metabolic differences between genetically engineered human cell lines, (ii) alterations in cellular metabolism induced by differentiation of mouse myoblasts into myotubes, and (iii) metabolic changes caused by activation of neurotransmitter receptors in mouse myoblasts. Thus, the new protocol offers an easily implementable, efficient, and versatile tool for the investigation of cellular metabolism and signal transduction.     

Synthetic DNA probes for detection of genes for enterotoxins A, B, C, D, E and for TSST-1 in staphylococcal strains.

A dot blot hybridization technique with oligonucleotide probes was developed for the specific detection of the TSST-1 gene and the staphylococcal enterotoxin (SE) genes A, B, C, D and E. For each toxin gene a probe sequence was chosen from the previously determined sequence. A total of 145 staphylococcal strains (133 Staphylococcus aureus and 12 coagulase-negative staphylococci (CNS) were studied by this genotypic method and by two phenotypic assays (gel immunodiffusion and ELISA). An excellent correlation (96%) was observed between the genotypic and phenotypic assays. DNA from two CNS strains hybridized with a probe without detection of the corresponding toxin (SEB for one strain and SEC for the other strain). One Staph. aureus strain was shown to be an SEC producer, but was not detected by the corresponding probe. Gene probe and immunological assays seem to be complementary methods for studies of staphylococcal strains producing (or potentially producing) TSST-1 or enterotoxins.