Visualization of the spontaneous emergence of a complex, dynamic, and autocatalytic system.

Autocatalytic chemical reactions are widely studied as models of biological processes and to better understand the origins of life on Earth. Minimal self-reproducing amphiphiles have been developed in this context and as an approach to de novo "bottom-up" synthetic protocells. How chemicals come together to produce living systems, however, remains poorly understood, despite much experimentation and speculation. Here, we use ultrasensitive label-free optical microscopy to visualize the spontaneous emergence of an autocatalytic system from an aqueous mixture of two chemicals. Quantitative, in situ nanoscale imaging reveals heterogeneous self-reproducing aggregates and enables the real-time visualization of the synthesis of new aggregates at the reactive interface. The aggregates and reactivity patterns observed vary together with differences in the respective environment. This work demonstrates how imaging of chemistry at the nanoscale can provide direct insight into the dynamic evolution of nonequilibrium systems across molecular to microscopic length scales.

Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle.

To dissect the kinetics of structural transitions underlying the stepping cycle of kinesin-1 at physiological ATP, we used interferometric scattering microscopy to track the position of gold nanoparticles attached to individual motor domains in processively stepping dimers. Labeled heads resided stably at positions 16.4 nm apart, corresponding to a microtubule-bound state, and at a previously unseen intermediate position, corresponding to a tethered state. The chemical transitions underlying these structural transitions were identified by varying nucleotide conditions and carrying out parallel stopped-flow kinetics assays. At saturating ATP, kinesin-1 spends half of each stepping cycle with one head bound, specifying a structural state for each of two rate-limiting transitions. Analysis of stepping kinetics in varying nucleotides shows that ATP binding is required to properly enter the one-head-bound state, and hydrolysis is necessary to exit it at a physiological rate. These transitions differ from the standard model in which ATP binding drives full docking of the flexible neck linker domain of the motor. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily.

Label-free Imaging of Microtubules with Sub-nm Precision Using Interferometric Scattering Microscopy.

Current in vitro optical studies of microtubule dynamics tend to rely on fluorescent labeling of tubulin, with tracking accuracy thereby limited by the quantum yield of fluorophores and by photobleaching. Here, we demonstrate label-free tracking of microtubules with nanometer precision at kilohertz frame rates using interferometric scattering microscopy (iSCAT). With microtubules tethered to a glass substrate using low-density kinesin, we readily detect sequential 8 nm steps in the microtubule center of mass, characteristic of a single kinesin molecule moving a microtubule. iSCAT also permits dynamic changes in filament length to be measured with <5 nm precision. Using the arbitrarily long observation time enabled by label-free iSCAT imaging, we demonstrate continuous monitoring of microtubule disassembly over a 30 min period. The ability of iSCAT to track microtubules with nm precision together with its potential for label-free single protein detection and simultaneous single molecule fluorescence imaging represent a unique platform for novel approaches to studying microtubule dynamics.

Erratum: Label-free Imaging of Microtubules with Sub-nm Precision Using Interferometric Scattering Microscopy.

[This corrects the article DOI: 10.1016/j.bpj.2015.10.055.].

Reproductive seasonality in creole hair sheep in the tropic.

The objective of the present study was to evaluate the annual ovulatory activity of hair sheep at 15° N. Nineteen Creole ewes with body weight of 40.8 ± 0.3 kg were used. The ovulatory activity was monitored for a year by quantifying progesterone concentrations in blood samples obtained from all the ewes every 7 days. The differences in monthly proportions of ewes with ovulatory activity were analyzed by the chi-square test. Ovulatory activity decreased from May to July and in September, and 42% of ewes ovulated year round. It is concluded that at 15° N, a high proportion of ewes is capable of ovulating throughout the year in the tropical southeastern region of Mexico.

High-speed single-particle tracking of GM1 in model membranes reveals anomalous diffusion due to interleaflet coupling and molecular pinning.

The biological functions of the cell membrane are influenced by the mobility of its constituents, which are thought to be strongly affected by nanoscale structure and organization. Interactions with the actin cytoskeleton have been proposed as a potential mechanism with the control of mobility imparted through transmembrane "pickets" or GPI-anchored lipid nanodomains. This hypothesis is based on observations of molecular mobility using various methods, although many of these lack the spatiotemporal resolution required to fully capture all the details of the interaction dynamics. In addition, the validity of certain experimental approaches, particularly single-particle tracking, has been questioned due to a number of potential experimental artifacts. Here, we use interferometric scattering microscopy to track molecules labeled with 20-40 nm scattering gold beads with simultaneous <2 nm spatial and 20 µs temporal precision to investigate the existence and mechanistic origin of anomalous diffusion in bilayer membranes. We use supported lipid bilayers as a model system and demonstrate that the label does not influence time-dependent diffusion in the small particle limit (≤40 nm). By tracking the motion of the ganglioside lipid GM1 bound to the cholera toxin B subunit for different substrates and lipid tail properties, we show that molecular pinning and interleaflet coupling between lipid tail domains on a nanoscopic scale suffice to induce transient immobilization and thereby anomalous subdiffusion on the millisecond time scale.

Non-steady state thermometry with optical diffraction tomography.

Label-free thermometry is a pivotal tool for many disciplines. However, most current approaches are only suitable for planar heat sources in steady state, thereby restricting the range of systems that can be reliably studied. Here, we introduce pump probe-based optical diffraction tomography (ODT) as a method to map temperature precisely and accurately in three dimensions (3D) at the single-particle level. To do so, we first systematically characterize the thermal landscape in a model system consisting of gold nanorods in a microchamber and then benchmark the results against simulations and quantitative phase imaging thermometry. We then apply ODT thermometry to resolve thermal landscapes inaccessible to other label-free approaches in the form of nonplanar heat sources embedded in complex environments and freely diffusing gold nanorods in a microchamber. Last, we foresee that our approach will find many applications where routine thermal characterization of heterogeneous nanoparticles samples in 3D or in non-steady state is required.

Molecular fingerprinting of biological nanoparticles with a label-free optofluidic platform.

Label-free detection of multiple analytes in a high-throughput fashion has been one of the long-sought goals in biosensing applications. Yet, for all-optical approaches, interfacing state-of-the-art label-free techniques with microfluidics tools that can process small volumes of sample with high throughput, and with surface chemistry that grants analyte specificity, poses a critical challenge to date. Here, we introduce an optofluidic platform that brings together state-of-the-art digital holography with PDMS microfluidics by using supported lipid bilayers as a surface chemistry building block to integrate both technologies. Specifically, this platform fingerprints heterogeneous biological nanoparticle populations via a multiplexed label-free immunoaffinity assay with single particle sensitivity. First, we characterise the robustness and performance of the platform, and then apply it to profile four distinct ovarian cell-derived extracellular vesicle populations over a panel of surface protein biomarkers, thus developing a unique biomarker fingerprint for each cell line. We foresee that our approach will find many applications where routine and multiplexed characterisation of biological nanoparticles are required.

Simultaneous Sizing and Refractive Index Analysis of Heterogeneous Nanoparticle Suspensions.

Rapid and reliable characterization of heterogeneous nanoparticle suspensions is a key technology across the nanosciences. Although approaches exist for homogeneous samples, they are often unsuitable for polydisperse suspensions, as particles of different sizes and compositions can lead to indistinguishable signals at the detector. Here, we introduce holographic nanoparticle tracking analysis, holoNTA, as a straightforward methodology that decouples size and material refractive index contributions. HoloNTA is applicable to any heterogeneous nanoparticle sample and has the sensitivity to measure the intrinsic heterogeneity of the sample. Specifically, we combined high dynamic range k-space imaging with holographic 3D single-particle tracking. This strategy enables long-term tracking by extending the imaging volume and delivers precise and accurate estimates of both scattering amplitude and diffusion coefficient of individual nanoparticles, from which particle refractive index and hydrodynamic size are determined. We specifically demonstrate, by simulations and experiments, that irrespective of localization uncertainty and size, the sizing sensitivity is improved as our extended detection volume yields considerably longer particle trajectories than previously reported by comparable technologies. As validation, we measured both homogeneous and heterogeneous suspensions of nanoparticles in the 40-250 nm size range and further monitored protein corona formation, where we identified subtle differences between the nanoparticle-protein complexes derived from avidin, bovine serum albumin, and streptavidin. We foresee that our approach will find many applications of both fundamental and applied nature where routine quantification and sizing of nanoparticles are required.

Molecular fingerprinting of biological nanoparticles with a label-free optofluidic platform.

Label-free detecting multiple analytes in a high-throughput fashion has been one of the long-sought goals in biosensing applications. Yet, for all-optical approaches, interfacing state-of-the-art label-free techniques with microfluidics tools that can process small volumes of sample with high throughput, and with surface chemistry that grants analyte specificity, poses a critical challenge to date. Here, we introduce an optofluidic platform that brings together state-of-the-art digital holography with PDMS microfluidics by using supported lipid bilayers as a surface chemistry building block to integrate both technologies. Specifically, this platform fingerprints heterogeneous biological nanoparticle populations via a multiplexed label-free immunoaffinity assay with single particle sensitivity. Herein, we first thoroughly characterise the robustness and performance of the platform, and then apply it to profile four distinct ovarian cell-derived extracellular vesicle populations over a panel of surface protein biomarkers, thus developing a unique biomarker fingerprint for each cell line. We foresee that our approach will find many applications where routine and multiplexed characterisation of biological nanoparticles is required.

Molecular fingerprinting of biological nanoparticles with a label-free optofluidic platform.

Label-free detecting multiple analytes in a high-throughput fashion has been one of the long-sought goals in biosensing applications. Yet, for all-optical approaches, interfacing state-of-the-art label-free techniques with microfluidics tools that can process small volumes of sample with high throughput, and with surface chemistry that grants analyte specificity, poses a critical challenge to date. Here, we introduce an optofluidic platform that brings together state-of-the-art digital holography with PDMS microfluidics by using supported lipid bilayers as a surface chemistry building block to integrate both technologies. Specifically, this platform fingerprints heterogeneous biological nanoparticle populations via a multiplexed label-free immunoaffinity assay with single particle sensitivity. Herein, we first thoroughly characterise the robustness and performance of the platform, and then apply it to profile four distinct ovarian cell-derived extracellular vesicle populations over a panel of surface protein biomarkers, thus developing a unique biomarker fingerprint for each cell line. We foresee that our approach will find many applications where routine and multiplexed characterisation of biological nanoparticles is required.

Interferometric scattering microscopy (iSCAT): new frontiers in ultrafast and ultrasensitive optical microscopy.

Optical microscopes have for centuries been our window to the microscopic world. The advent of single-molecule optics over the past few decades has ushered in a new era in optical imaging, partly because it has enabled the observation of motion and more recently structure on the nanoscopic scale through the development of super-resolution techniques. The large majority of these studies have relied on the efficient detection of fluorescence as the basis of single-molecule sensitivity. Despite the many advantages of using single emitters as light sources, the intensity and duration of their emission impose fundamental limits on the imaging speed and precision for tracking studies. Here, we discuss the potential of a novel imaging technique based on interferometric scattering (iSCAT) that pushes both the sensitivity and time resolution far beyond what is currently achievable by single-emitter-based approaches. We present recent results that demonstrate single-molecule sensitivity and imaging speeds on the microsecond timescale.

Ionic Species Affect the Self-Propulsion of Urease-Powered Micromotors.

Enzyme-powered motors self-propel through the catalysis of in situ bioavailable fuels, which makes them excellent candidates for biomedical applications. However, fundamental issues like their motion in biological fluids and the understanding of the propulsion mechanism are critical aspects to be tackled before a future application in biomedicine. Herein, we investigated the physicochemical effects of ionic species on the self-propulsion of urease-powered micromotors. Results showed that the presence of PBS, NaOH, NaCl, and HEPES reduced self-propulsion of urease-powered micromotors pointing towards ion-dependent mechanisms of motion. We studied the 3D motion of urease micromotors using digital holographic microscopy to rule out any motor-surface interaction as the cause of motion decay when salts are present in the media. In order to protect and minimize the negative effect of ionic species on micromotors' performance, we coated the motors with methoxypolyethylene glycol amine (mPEG) showing higher speed compared to noncoated motors at intermediate ionic concentrations. These results provide new insights into the mechanism of urease-powered micromotors, study the effect of ionic media, and contribute with potential solutions to mitigate the reduction of mobility of enzyme-powered micromotors.

3D tracking of extracellular vesicles by holographic fluorescence imaging.

Fluorescence microscopy is the method of choice in biology for its molecular specificity and super-resolution capabilities. However, it is limited to a narrow z range around one observation plane. Here, we report an imaging approach that recovers the full electric field of fluorescent light with single-molecule sensitivity. We expand the principle of digital holography to fast fluorescent detection by eliminating the need for phase cycling and enable three-dimensional (3D) tracking of individual nanoparticles with an in-plane resolution of 15 nm and a z-range of 8 mm. As a proof-of-concept biological application, we image the 3D motion of extracellular vesicles (EVs) inside live cells. At short time scales (<4 s), we resolve near-isotropic 3D diffusion and directional transport. For longer lag times, we observe a transition toward anisotropic motion with the EVs being transported over long distances in the axial plane while being confined in the horizontal dimension.

Interferometric scattering microscopy and its combination with single-molecule fluorescence imaging.

Interferometric scattering microscopy (iSCAT) is a light scattering-based imaging modality that offers a unique combination of imaging speed and precision for tracking nanoscopic labels and enables label-free optical sensing down to the single-molecule level. In contrast to fluorescence, iSCAT does not suffer from limitations associated with dye photochemistry and photophysics, or the requirement for fluorescent labeling. Here we present a protocol for constructing an iSCAT microscope from commercially available optical components and demonstrate its compatibility with simultaneously operating single-molecule, objective-type, total internal reflection fluorescence microscopy. Given an intermediate level of experience with optics and microscopy, for instance graduate-level familiarity with laser beam steering and optical components, this protocol can be completed in a time frame of 2 weeks.

[Management of type 2 diabetic patients in primary care in Spain]. / Tratamiento de los pacientes con diabetes mellitus tipo 2 en atención primaria en España.

BACKGROUND AND OBJECTIVE: We assess the metabolic control, complications, quality of life related to health (QLRH) and the type and amount of medical resource consumption (MRC) in type 2 diabetic patients (2DMp) followed by primary care physicians (PCP) in Spain. PATIENTS AND METHOD: We studied 628 2DMp divided in 4 cohorts: 1. Either newly diagnosed 2DMp who required pharmacological treatment or failed to non-pharmacological measures; 2. Patients pharmacologically treated for less than 1 year; 3. Patients with pharmacological treatment for more than 1 year; 4. Patients with impaired fasting glucose (control group). RESULTS: Eighty percent of the subjects were overweight. At baseline, 27.9, 23.5 and 36.9% of patients from cohorts 1, 2 and 3, respectively, had HbA1C < 8%. After 6 months of follow-up, 14.6, 21.3 and 22.8% of patients from cohorts 1, 2 and 3, respectively, still had "bad control". At baseline, 38.0%, 21.2% and 20.7% of patients from cohorts 1, 2, and 3, respectively, had "bad lipid profile". After 6 months, 57.4%, 54.2% and 45.3% of cohorts 1, 2 and 3, respectively, still had plasma cLDL levels > 130 mg/dl. Complications were more frequent in cohort 3. During the 6-month period, MRC was higher among 2DMp than controls (p < 0.05) and higher among patients from cohort 3 (p < 0.05) compared with all the other patients. More diabetic than control patients and more patients from cohort 3 than patients from cohort 1 and 2 reported that their expected quality of life would be better without diabetes. CONCLUSIONS: One out of four of diabetic patients studied had HbA1C and lipids higher than the limits suggested by guidelines. Type 2 diabetes is associated with higher MRC and worse QLRH. This situation is worse among long-term diabetic patients.

Bullous hemorrhagic dermatosis at distant sites / Dermatosis ampollosa hemorrágica a distancia

Un hombre de 72 años, es hospitalizado por falla cardiaca aguda. Tiene historia de hipertensión arterial en manejo con enalapril e hidroclorotiazida. Desde su ingreso, se inició trom-boprofilaxis con enoxaparina 40 mg diarios por vía subcutánea en abdomen. Tres días después aparecen lesiones cutáneas ampollosas, tensas, de contenido hemático y diámetro entre 5-15 mm. Las lesiones se localizan en miembro superior derecho (Figura 1) y pie izquierdo (Figura 2), tienen haloeritematoso y no son dolorosas. Las pruebas de coagulación, proteína C reactiva, VSG y hemograma son normales. Se realizó biopsia de piel que mostró lesión subepidérmica con material hemorrágico en dermis papilar y reticular con neutrófilos escasos, sin vasculitis o trombosis. Estos hallazgos, confirman el diagnóstico de dermatosis ampo-llosa hemorrágica a distancia asociada a enoxaparina. Este constituye un efecto adverso infrecuente y autorresolutivo de las heparinas, no relacionado con la dosificación y en sitios distantes a su aplicación. Este paciente se trató con el retiro del medicamento, resolviendo las lesiones a los sietedías y sin recurrencia de las mismas luego de tres meses de seguimiento

A 72-year-old man was hospitalized for acute heart failure. He had a history of arterial hypertension being treated with enalapril and hydrochlorothiazide. Thromboprophylaxis was begun at admission with 40 mg of subcutaneous enoxaparin daily, in the abdomen. Three days later, tense, blood-filled bullous skin lesions with a 5-15 mm diameter appeared. The lesions were on the right arm (Figure 1) and the left foot (Fig-ure 2); they had an erythematous halo and were not painful Clotting, C-reactive protein, ESR and complete blood count were normal. A skin biopsy was performed, which showed a subepidermal lesion with hemorrhagic material in the papillary and reticular dermis with scant neutrophils and no vasculitis or thrombosis. These findings confirm the diagnosis of bullous hemorrhagic dermatosis at distant sites related to enoxaparin. This is an uncommon and self-resolving adverse effect of heparins, unrelated to the dose and at sites distant from their application. This patient was treated by discontinu-ing the medication, and the lesions resolved seven days later with no recurrence after three months of follow-up.

Structural dynamics of myosin 5 during processive motion revealed by interferometric scattering microscopy.

Myosin 5a is a dual-headed molecular motor that transports cargo along actin filaments. By following the motion of individual heads with interferometric scattering microscopy at nm spatial and ms temporal precision we found that the detached head occupies a loosely fixed position to one side of actin from which it rebinds in a controlled manner while executing a step. Improving the spatial precision to the sub-nm regime provided evidence for an ångstrom-level structural transition in the motor domain associated with the power stroke. Simultaneous tracking of both heads revealed that consecutive steps follow identical paths to the same side of actin in a compass-like spinning motion demonstrating a symmetrical walking pattern. These results visualize many of the critical unknown aspects of the stepping mechanism of myosin 5 including head-head coordination, the origin of lever-arm motion and the spatiotemporal dynamics of the translocating head during individual steps.

Direct observation and control of supported lipid bilayer formation with interferometric scattering microscopy.

Supported lipid bilayers (SLB) are frequently used to study processes associated with or mediated by lipid membranes. The mechanism by which SLBs form is a matter of debate, largely due to the experimental difficulty associated with observing the adsorption and rupture of individual vesicles. Here, we used interferometric scattering microscopy (iSCAT) to directly visualize membrane formation from nanoscopic vesicles in real time. We observed a number of previously proposed phenomena such as vesicle adsorption, rupture, movement, and a wave-like bilayer spreading. By varying the vesicle size and the lipid-surface interaction strength, we rationalized and tuned the relative contributions of these phenomena to bilayer formation. Our results support a model where the interplay between bilayer edge tension and the overall interaction energy with the surface determine the mechanism of SLB formation. The unique combination of sensitivity, speed, and label-free imaging capability of iSCAT provides exciting prospects not only for investigations of SLB formation, but also for studies of assembly and disassembly processes on the nanoscale with previously unattainable accuracy and sensitivity.

How to build your own coronary anastomosis simulator from scratch.

OBJECTIVES: Gaining cardiac surgical competence is a complex, multifactorial process that may take years of experience and on-the-job training. It is critical to provide suitable educational opportunities to gain the necessary knowledge, judgment and skills. In response to the multitude of factors (e.g. European Working Time Directive) currently influencing cardiac surgical training, there have been concerted efforts to reform training practices. Simulation plays an increasingly important role in the educational process and serves to fill the most important gap in the current training model, i.e. operative exposure. Therefore, a contest has been written out for cardiac surgical trainees to construct their own coronary anastomosis simulator using everyday materials. METHODS: Cardiac surgical trainees were invited to construct their own coronary anastomosis simulator. An international jury of cardiac surgeons assessed the simulator and its presentation according to preset developmental criteria (low fidelity concept, innovative character, general presentation and description, general attractiveness to the scholar, ergonomical issues, perceived haptics, number of applicable components, transportability, ease of construction, repeatability and overall costs of the simulator). RESULTS: Six prototypes of simulators built by cardiac surgical trainees were generated. A general evaluation of each simulator prototype is provided according to the preset developmental criteria. CONCLUSIONS: All simulator prototypes have provided a considerable contribution to the field of surgical simulation. By designing simulator prototypes, the trainees have demonstrated their 'out of the box' thinking capability, which is of paramount importance for the development of future innovative surgical techniques and procedures. The Valladolid cardiac team coronary anastomosis simulator box was selected for the EACTS Ethicon Simulation Award 2011. This project will be mass produced and distributed to the participants of structured simulation sessions for coronary anastomoses.