Quantitative and sensitive detection of rare mutations using droplet-based microfluidics

Somatic mutations within tumoral DNA can be used as highly specific biomarkers to distinguish cancer cells from their normal counterparts. These DNA biomarkers are potentially useful for the diagnosis, prognosis, treatment and follow-up of patients. In order to have the required sensitivity and specificity to detect rare tumoral DNA in stool, blood, lymph and other patient samples, a simple, sensitive and quantitative procedure to measure the ratio of mutant to wild-type genes is required. However, techniques such as dual probe TaqMan(?) assays and pyrosequencing, while quantitative, cannot detect less than ～1% mutant genes in a background of non-mutated DNA from normal cells. Here we describe a procedure allowing the highly sensitive detection of mutated DNA in a quantitative manner within complex mixtures of DNA. The method is based on using a droplet-based microfluidic system to perform digital PCR in millions of picolitre droplets. Genomic DNA (gDNA) is compartmentalized in droplets at a concentration of less than one genome equivalent per droplet together with two TaqMan(?) probes, one specific for the mutant and the other for the wild-type DNA, which generate green and red fluorescent signals, respectively. After thermocycling, the ratio of mutant to wild-type genes is determined by counting the ratio of green to red droplets. We demonstrate the accurate and sensitive quantification of mutated KRAS oncogene in gDNA. The technique enabled the determination of mutant allelic specific imbalance (MASI) in several cancer cell-lines and the precise quantification of a mutated KRAS gene in the presence of a 200,000-fold excess of unmutated KRAS genes. The sensitivity is only limited by the number of droplets analyzed. Furthermore, by one-to-one fusion of drops containing gDNA with any one of seven different types of droplets, each containing a TaqMan(?) probe specific for a different KRAS mutation, or wild-type KRAS, and an optical code, it was possible to screen the six common mutations in KRAS codon 12 in parallel in a single experiment.     

Fast and stable photochromic oxazines for fluorescence switching

The stringent limitations imposed by diffraction on the spatial resolution of fluorescence microscopes demand the identification of viable strategies to switch fluorescence under optical control. In this context, the photoinduced and reversible transformations of photochromic compounds are particularly valuable. In fact, these molecules can be engineered to regulate the emission intensities of complementary fluorophores in response to optical stimulations. On the basis of this general design logic, we assembled a functional molecular construct consisting of a borondipyrromethene fluorophore and a nitrospiropyran photochrome and demonstrated that the emission of the former can be modulated with the interconversion of the latter. This fluorophore-photochrome dyad, however, has a slow switching speed and poor fatigue resistance. To improve both parameters, we developed a new family of photochromic switches based on the photoinduced opening and thermal closing of an oxazine ring. These compounds switch back and forth between ring-closed and -open isomers on nanosecond-microsecond timescales and tolerate thousands of switching cycles with no sign of degradation. In addition, the attachment of appropriate chromophoric fragments to their switchable oxazine ring can be exploited to either deactivate or activate fluorescence reversibly in response to illumination with a pair of exciting beams. Specifically, we assembled three dyads, each based on either a borondipyrromethene or a coumarin fluorophore and an oxazine photochrome, and modulated their fluorescence in a few microseconds with outstanding fatigue resistance. The unique photochemical and photophysical properties of our fluorophore-photochrome dyads can facilitate the development of switchable fluorophores for superresolution imaging and, ultimately, provide valuable molecular probes for the visualization of biological samples on the nanometer level.     

In vitro antiprotozoal activity of extracts of five Turkish Lamiaceae species

The in vitro antiprotozoal activities of crude methanolic extracts from the aerial parts of five Lamiaceae plants (Salvia tomentosa, S. sclarea, S. dichroantha, Nepeta nuda subsp. nuda and Marrubium astracanicum subsp. macrodon) were evaluated against four parasitic protozoa, i.e. Trypanosoma brucei rhodesiense, T. cruzi, Leishmania donovani and Plasmodium falciparum. The cytotoxic potentials of the extracts on L6 cells were also evaluated. Melarsoprol, benznidazole, miltefosine, chloroquine and podophyllotoxin were used as reference drugs. All crude MeOH extracts showed antiprotozoal potential against at least three parasites, so they were dispersed in water and partitioned against n-hexane and chloroform to yield three subextracts that were screened in the same test systems. The n-hexane extract of N. nuda was the most active against T. brucei rhodesiense while the CHCl3 extracts of S. tomentosa and S. dichroantha showed significant activity against L. donovani. All organic extracts displayed in vitro antimalarial and moderate trypanocidal activities against T. cruzi with the n-hexane extract of S. sclarea being the most active against the latter. The extracts displayed low or no cytotoxicity towards mammalian L6 cells.     

Acanthifoliosides, minor steroidal saponins from the Caribbean sponge Pandaros acanthifolium

Seven novel steroid glycosides, acanthifoliosides A-F (1-6), and the methyl ester of 6 (7), were isolated from the marine sponge Pandaros acanthifolium as minor components. Acanthifoliosides are characterized by a rare C-15 and C-16 oxidized D ring, which was previously found in saponins produced by starfishes. Very uncommon is the presence of additional sugar residues at C-15 or C-16. Their structures were determined on the basis of extensive spectroscopic analyses, including two-dimensional NMR and HRESIMS data. The absolute configurations of the aglycones were assigned by comparison between experimental and TDDFT calculated CD spectra of 1, whereas the absolute configurations of the monosaccharide units were determined by chiral GC analyses of the acid methanolysates. Some of the acanthifoliosides exhibit moderate antiprotozoal activity but to a lesser extent than the most potent pandarosides.     

Forced Folding of a Disordered Protein Accesses an Alternative Folding Landscape

Intrinsically disordered proteins (IDPs) are involved in diverse cellular functions. Many IDPs can interact with multiple binding partners, resulting in their folding into alternative ligand‐specific functional structures. For such multi‐structural IDPs, a key question is whether these multiple structures are fully encoded in the protein sequence, as is the case in many globular proteins. To answer this question, here we employed a combination of single‐molecule and ensemble techniques to compare ligand‐induced and osmolyte‐forced folding of α‐synuclein. Our results reveal context‐dependent modulation of the protein′s folding landscape, suggesting that the codes for the protein′s native folds are partially encoded in its primary sequence, and are completed only upon interaction with binding partners. Our findings suggest a critical role for cellular interactions in expanding the repertoire of folds and functions available to disordered proteins.     

Triazine herbicide imprinted monolithic column for capillary electrochromatography

Trietazine was selectively separated from aqueous solution containing the competitor molecule cyanazine, which is similar in size and shape to the template molecule. Structural features of the molecularly imprinted column were figured out by SEM. The influence of the mobile‐phase composition, applied electrical field, and pH of the mobile phase on the recognition of trietazine by the imprinted monolithic polymer has been evaluated, and the imprint effect in the trietazine‐imprinted monolithic polymer was demonstrated by an imprinting factor. The optimized monolithic column resulted in separation of trietazine from a structurally related competitor molecule, cyanazine. In addition, fast separation was obtained within 6 min by applying higher electrical field, with the electrophoretic mobility of 2.97 × 10^?8 m²V^?1s^?1 at pH 11.0.     

Facile intramolecular Diels–Alder reaction of 4-(o-propargyloxy)styrylcoumarins leading to highly fluorescent coumarin-containing polycyclic compounds

The 4-(o-propargyloxy)styrylcoumarins are prepared by the condensation of O-propargylated salicylaldehyde with substituted coumarin-4-acetic acids. The intramolecular Diels–Alder reaction of 4-(o-propargyloxy)styrylcoumarins, without any catalyst gives fused-ring coumarins. The reaction in boiling nitrobenzene leads to aromatization of the initial Diels–Alder adduct and these aromatized products are highly fluorescent.     

Photochemistry of desonide, a non-fluorinated steroidal anti-inflammatory drug

The photochemistry of anti-inflammatory drug desonide (De, 1) was studied in aerobic as well as in anaerobic condition with different irradiation wavelengths (254, 310 nm) in acetonitrile and 2-propanol. All photoproducts obtained were isolated and characterized on the basis of IR, (1)H-, (13)C-NMR spectroscopy and elemental analysis study. The products were: 11beta,21-dihydroxy-16alpha,17alpha-(1-methylethylidenedioxy)-1,5-cyclopregn-3-ene-2,20-dione 2 (254 nm), 11beta-hydroxy-16alpha,17alpha-(1-methylethylidenedioxy)androsta-1,4-diene-3-one 3 (310 nm/2-propanol), 17beta-hydroperoxy-11beta-hydroxy-16alpha,17alpha-(1-methylethylidenedioxy)androsta-1,4-diene-3-one 4 (310 nm/O(2)/2-propanol). Cyclohexadienone moiety in ring A and keto group at C(17) were found to be deeply modified by UV light therefore, loss of biological activity both during storage and in vivo can not be ruled out.     

Ion specific surface forces between membrane surfaces

Entities such as ion distributions and forces between lipid membranes depend on effects due to the intervening salt solution that have not been recognized previously. These specific ion or Hofmeister effects influence membrane fusion. A typical illustrative example is this: measurements of forces between double-chained cationic bilayers adsorbed onto molecularly smooth mica surfaces across different 0.6-2 mM salt solutions have revealed a large degree of ion specificity [Pashley et al. J. Phys. Chem. 1986, 90, 1637]. This has been interpreted in terms of very specific anion "binding" to the adsorbed bilayers, as it would too for micelles and other self-assembled systems. However, we show here that inclusion of nonelectrostatic (NES) or ionic dispersion potentials acting between ions and the two surfaces explains such "ion binding". The observed Hofmeister sequence for the calculated pressure without any direct ion binding is given correctly. This demonstrates the importance of a source of ion specificity that has been ignored. It is due to ionic physisorption caused by attractive NES ionic dispersion potentials. There appear to be some far reaching consequences for interpretations of membrane intermolecular interactions in salt solutions.