Direct measurement of DNA-mediated adhesion between lipid bilayers

Multivalent interactions between deformable mesoscopic units are ubiquitous in biology, where membrane macromolecules mediate the interactions between neighbouring living cells and between cells and solid substrates. Lately, analogous artificial materials have been synthesised by functionalising the outer surface of compliant Brownian units, for example emulsion droplets and lipid vesicles, with selective linkers, in particular short DNA sequences. This development extended the range of applicability of DNA as a selective glue, originally applied to solid nano and colloidal particles. On very deformable lipid vesicles, the coupling between statistical effects of multivalent interactions and mechanical deformation of the membranes gives rise to complex emergent behaviours, as we recently contributed to demonstrate [Parolini et al., Nature Communications, 2015, 6, 5948]. Several aspects of the complex phenomenology observed in these systems still lack a quantitative experimental characterisation and fundamental understanding. Here we focus on the DNA-mediated multivalent interactions of a single liposome adhering to a flat supported bilayer. This simplified geometry enables the estimate of the membrane tension induced by the DNA-mediated adhesive forces acting on the liposome. Our experimental investigation is completed by morphological measurements and the characterisation of the DNA-melting transition, probed by in-situ F\"{o}rster Resonant Energy Transfer spectroscopy. Experimental results are compared with the predictions of an analytical theory that couples the deformation of the vesicle to a full description of the statistical mechanics of mobile linkers. With at most one fitting parameter, our theory is capable of semi-quantitatively matching experimental data, confirming the quality of the underlying assumptions.Comment: 16 pages, 7 figure

Volume and porosity thermal regulation in lipid mesophases by coupling mobile ligands to soft membranes

Short DNA linkers are increasingly being exploited for driving specific self-assembly of Brownian objects. DNA-functionalised colloids can assemble into ordered or amorphous materials with tailored morphology. Recently, the same approach has been applied to compliant units, including emulsion droplets and lipid vesicles. The liquid structure of these substrates introduces new degrees of freedom: the tethers can diffuse and rearrange, radically changing the physics of the interactions. Unlike droplets, vesicles are extremely deformable and DNA-mediated adhesion causes significant shape adjustments. We investigate experimentally the thermal response of pairs and networks of DNA-tethered liposomes and observe two intriguing and possibly useful collective properties: negative thermal expansion and tuneable porosity of the liposome networks. A model providing a thorough understanding of this unexpected phenomenon is developed, explaining the emergent properties out of the interplay between the temperature-dependent deformability of the vesicles and the DNA-mediated adhesive forces.Funding was provided by the Ernest Oppenheimer Fund and Emmanuel College Cambridge (L.D.M.), EPSRC Programme Grant CAPITALS number EP/J017566/1 (L.P., J.K., P.C. and L.D.M.) and the Winton Fund for Physics of Sustainability (E.E.).This article was originally published in Nature Communications (L Parolini, BM Mognetti, J Kotar, E Eiser, P Cicuta, L Di Michele, Nature Communications 2015, 6, 5948

Monster black holes

A combination of ground-based and spacecraft observations has uncovered two black holes of 10 billion solar masses in the nearby Universe. The finding sheds light on how these cosmic monsters co-evolve with galaxies.Comment: 2 pages, 1 figure, LaTeX. Published in Nature "News & Views

Whole organic electronic synapses for dopamine detection

A whole organic artificial synapse has been fabricated by patterning PEDOT:PSS electrodes on PDMS that are biased in frequency to yield a STP response. The timescale of the STP response is shown to be sensitive to the concentration of dopamine, DA, a neurotransmitter relevant for monitoring the development of Parkinson's disease and potential locoregional therapies. The sensitivity of the sensor towards DA has been validated comparing signal variation in the presence of DA and its principal interfering agent, ascorbic acid, AA. The whole organic synapse is biocompatible, soft and flexible, and is attractive for implantable devices aimed to real-time monitoring of DA concentration in bodily fluids. This may open applications in chronic neurodegenerative diseases such as Parkinson's disease

Versatile multiplexed super-resolution imaging of nanostructures by Quencher-Exchange-PAINT

This is the final version of the article. Available from Springer Verlag via the DOI in this record.The optical super-resolution technique DNA-PAINT (Point Accumulation Imaging in Nanoscale Topography) provides a flexible way to achieve imaging of nanoscale structures at ∼10-nanometer resolution. In DNA-PAINT, fluorescently labeled DNA “imager” strands bind transiently and with high specificity to complementary target “docking” strands anchored to the structure of interest. The localization of single binding events enables the assembly of a super-resolution image, and this approach effectively circumvents photobleaching. The solution exchange of imager strands is the basis of Exchange-PAINT, which enables multiplexed imaging that avoids chromatic aberrations. Fluid exchange during imaging typically requires specialized chambers or washes, which can disturb the sample. Additionally, diffusional washout of imager strands is slow in thick samples such as biological tissue slices. Here, we introduce Quencher-Exchange-PAINT—a new approach to Exchange-PAINT in regular open-top imaging chambers—which overcomes the comparatively slow imager strand switching via diffusional imager washout. Quencher-Exchange-PAINT uses “quencher” strands, i.e., oligonucleotides that prevent the imager from binding to the targets, to rapidly reduce unwanted single-stranded imager concentrations to negligible levels, decoupled from the absolute imager concentration. The quencher strands contain an effective dye quencher that reduces the fluorescence of quenched imager strands to negligible levels. We characterized Quencher-Exchange-PAINT when applied to synthetic, cellular, and thick tissue samples. Quencher-Exchange-PAINT opens the way for efficient multiplexed imaging of complex nanostructures, e.g., in thick tissues, without the need for washing steps. [Figure not available: see fulltext.]The work was supported by funding from the Human Frontier Science Program (No. 0027/2013) and the Engineering and Physical Sciences Research Council of the UK (No. EP/N008235/1)

Detecting nanoscale distribution of protein pairs by proximity dependent super-resolution microscopy

Interactions between biomolecules such as proteins underlie most cellular processes. It is crucial to visualize these molecular-interaction complexes directly within the cell, to show precisely where these interactions occur and thus improve our understanding of cellular regulation. Currently available proximity-sensitive assays for in situ imaging of such interactions produce diffraction-limited signals and therefore preclude information on the nanometer-scale distribution of interaction complexes. By contrast, optical super-resolution imaging provides information about molecular distributions with nanometer resolution, which has greatly advanced our understanding of cell biology. However, current co-localization analysis of super-resolution fluorescence imaging is prone to false positive signals as the detection of protein proximity is directly dependent on the local optical resolution. Here we present proximity-dependent PAINT (PD-PAINT), a method for subdiffraction imaging of protein pairs, in which proximity detection is decoupled from optical resolution. Proximity is detected via the highly distance-dependent interaction of two DNA constructs anchored to the target species. Labeled protein pairs are then imaged with high-contrast and nanoscale resolution using the super-resolution approach of DNA-PAINT. The mechanisms underlying the new technique are analyzed by means of coarse-grained molecular simulations and experimentally demonstrated by imaging DNA-origami tiles and epitopes of cardiac proteins in isolated cardiomyocytes. We show that PD-PAINT can be straightforwardly integrated in a multiplexed super-resolution imaging protocol and benefits from advantages of DNA-based super-resolution localization microscopy, such as high specificity, high resolution, and the ability to image quantitatively

Ataxia with oculomotor apraxia type 2: a clinical, pathologic, and genetic study

BACKGROUND: Ataxia with oculomotor apraxia type 2 (AOA2) is characterized by onset between age 10 and 22 years, cerebellar atrophy, peripheral neuropathy, oculomotor apraxia (OMA), and elevated serum alpha-fetoprotein (AFP) levels. Recessive mutations in SETX have been described in AOA2 patients. OBJECTIVE: To describe the clinical features of AOA2 and to identify the SETX mutations in 10 patients from four Italian families. METHODS: The patients underwent clinical examination, routine laboratory tests, nerve conduction studies, sural nerve biopsy, and brain MRI. All were screened for SETX mutations. RESULTS: All the patients had cerebellar features, including limb and truncal ataxia, and slurred speech. OMA was observed in two patients, extrapyramidal symptoms in two, and mental impairment in three. High serum AFP levels, motor and sensory axonal neuropathy, and marked cerebellar atrophy on MRI were detected in all the patients who underwent these examinations. Sural nerve biopsy revealed a severe depletion of large myelinated fibers in one patient, and both large and small myelinated fibers in another. Postmortem findings are also reported in one of the patients. Four different homozygous SETX mutations were found (a large-scale deletion, a missense change, a single-base deletion, and a splice-site mutation). CONCLUSIONS: The clinical phenotype of oculomotor apraxia type 2 is fairly homogeneous, showing only subtle intrafamilial variability. OMA is an inconstant finding. The identification of new mutations expands the array of SETX variants, and the finding of a missense change outside the helicase domain suggests the existence of at least one more functional region in the N-terminus of senataxin

Daily Distribution of Macronutrient Intakes of Professional Soccer Players From the English Premier League.

The daily distribution of macronutrient intake can modulate aspects of training adaptations, performance and recovery. We therefore assessed the daily distribution of macronutrient intake (as assessed using food diaries supported by the remote food photographic method and 24 h recalls) of professional soccer players (n=6) of the English Premier League during a 7-day period consisting of two match days and five training days. On match days, average carbohydrate (CHO) content of the pre-match (0.05) though such intakes were lower than contemporary guidelines considered optimal for pre-match CHO intake and post-match recovery. On training days, we observed a skewed and hierarchical approach (Plunch (0.6 g.kg(-1))>breakfast (0.3 g.kg(-1))>evening snacks (0.1 g.kg(-1)). We conclude players may benefit from consuming greater amounts of CHO in both the pre-match and post-match meals so as to increase CHO availability and maximize rates of muscle glycogen re-synthesis, respectively. Furthermore, attention should also be given to ensuring even daily distribution of protein intake so as to potentially promote components of training adaptation

Crossover between Equilibrium and Shear-controlled Dynamics in Sheared Liquids

We present a numerical simulation study of a simple monatomic Lennard-Jones liquid under shear flow, as a function of both temperature and shear rate. By investigating different observables we find that i) It exists a line in the (temperature-shear) plane that sharply marks the boarder between an ``equilibrium'' and a ``shear-controlled'' region for both the dynamic and the thermodynamic quantities; and ii) Along this line the structural relaxation time, is proportional to the inverse shear rate, i.e. to the typical time-scale introduced by the shear flow. Above the line the liquid dynamics is unaffected by the shear flow, while below it both temperature and shear rate control the particle motion.Comment: 14 pages, 5 figure