Deciphering how the regulatory DNA sequence of a gene dictates its expression in response to intra and extracellular cues is one of the leading challenges in modern genomics. The development of novel single-cell sequencing and imaging techniques, as well as a better exploitation of currently available single-molecule imaging techniques, provides an avenue to interrogate the process of transcription and its dynamics in cells by quantifying the number of RNA polymerases engaged in the transcription of a gene (or equivalently the number of nascent RNAs) at a given moment in time. In this paper, we propose that measurements of the cell-to-cell variability in the number of nascent RNAs provide a mostly unexplored method for deciphering mechanisms of transcription initiation in cells. We propose a simple kinetic model of transcription initiation and elongation from which we calculate nascent RNA copy-number fluctuations. To demonstrate the usefulness of this approach, we test our theory against published nascent RNA data for twelve constitutively expressed yeast genes. Rather than transcription being initiated through a single rate limiting step, as it had been previously proposed, our single-cell analysis reveals the presence of at least two rate limiting steps. Surprisingly, half of the genes analyzed have nearly identical rates of transcription initiation, suggesting a common mechanism. Our analytical framework can be used to extract quantitative information about dynamics of transcription from single-cell sequencing data, as well as from single-molecule imaging and electron micrographs of fixed cells, and provides the mathematical means to exploit the quantitative power of these technologies.
Microorganisms often exhibit a history-dependent phenotypic response after exposure to a stimulus which can be imperative for proper function. However, cells frequently experience unexpected environmental perturbations that might induce phenotypic switching. How cells maintain phenotypic states in the face of environmental fluctuations remains an open question. Here, we use environmental perturbations to characterize the resilience of phenotypic states in a synthetic gene network near a critical transition. We find that far from the critical transition an environmental perturbation may induce little to no phenotypic switching, whereas close to the critical transition the same perturbation can cause many cells to switch phenotypic states. This loss of resilience was observed for perturbations that interact directly with the gene circuit as well as for a variety of generic perturbations-such as salt, ethanol, or temperature shocks-that alter the state of the cell more broadly. We obtain qualitatively similar findings in natural gene circuits, such as the yeast GAL network. Our findings illustrate how phenotypic memory can become destabilized by environmental variability near a critical transition.
Roux-en-Y gastric bypass (RYGB) is highly effective in reversing obesity and associated diabetes. Recent observations in humans suggest a contributing role of increased circulating bile acids in mediating such effects. Here we use a diet-induced obesity (DIO) mouse model and compare metabolic remission when bile flow is diverted through a gallbladder anastomosis to jejunum, ileum or duodenum (sham control). We find that only bile diversion to the ileum results in physiologic changes similar to RYGB, including sustained improvements in weight, glucose tolerance and hepatic steatosis despite differential effects on hepatic gene expression. Circulating free fatty acids and triglycerides decrease while bile acids increase, particularly conjugated tauro-β-muricholic acid, an FXR antagonist. Activity of the hepatic FXR/FGF15 signalling axis is reduced and associated with altered gut microbiota. Thus bile diversion, independent of surgical rearrangement of the gastrointestinal tract, imparts significant weight loss accompanied by improved glucose and lipid homeostasis that are hallmarks of RYGB.
The Mauthner cell (M-cell) is a command-like neuron in teleost fish whose firing in response to aversive stimuli is correlated with short-latency escapes [1, 2 and 3]. M-cells have been proposed as evolutionary ancestors of startle response neurons of the mammalian reticular formation , and studies of this circuit have uncovered important principles in neurobiology that generalize to more complex vertebrate models . The main excitatory input was thought to originate from multisensory afferents synapsing directly onto the M-cell dendrites . Here, we describe an additional, convergent pathway that is essential for the M-cell-mediated startle behavior in larval zebrafish. It is composed of excitatory interneurons called spiral fiber neurons, which project to the M-cell axon hillock. By in vivo calcium imaging, we found that spiral fiber neurons are active in response to aversive stimuli capable of eliciting escapes. Like M-cell ablations, bilateral ablations of spiral fiber neurons largely eliminate short-latency escapes. Unilateral spiral fiber neuron ablations shift the directionality of escapes and indicate that spiral fiber neurons excite the M-cell in a lateralized manner. Their optogenetic activation increases the probability of short-latency escapes, supporting the notion that spiral fiber neurons help activate M-cell-mediated startle behavior. These results reveal that spiral fiber neurons are essential for the function of the M-cell in response to sensory cues and suggest that convergent excitatory inputs that differ in their input location and timing ensure reliable activation of the M-cell, a feedforward excitatory motif that may extend to other neural circuits.
Recently developed lab-on-a-chip technologies integrate multiple traditional assays on a single chip with higher sensitivity, faster assay time, and more streamlined sample operation. We discuss the prospects of the lab-on-a-tip platform, where assays can be integrated on a miniaturized tip for in situ and in vivo analysis. It will resolve some of the limitations of available lab-on-a-chip platforms and enable next generation multifunctional in vivo sensors, as well as analytical techniques at the single cell or even sub-cellular levels
Optical cavities that are capable for detecting single nanoparticles could lead to great progress in early stage disease diagnostics and the study of biological interactions on the single-molecule level. In particular, photonic crystal (PhC) cavities are excellent platforms for label-free single-nanoparticle detection, owing to their high quality (Q) factors and wavelength-scale modal volumes. Here, we demonstrate the design and fabrication of a high-Q (>104) slot-mode PhC nanobeam cavity, which is able to strongly confine light in the slotted regions. The enhanced light-matter interaction results in an order of magnitude improvement in both refractive index sensitivity (439 nm/RIU) and single-nanoparticle sensitivity compared with conventional dielectric-mode PhC cavities. Detection of single polystyrene nanoparticles with radii of 20 nm and 30 nm is demonstrated in aqueous environments (D2O), without additional laser and temperature stabilization techniques.
We report exceptionally low thresholds (9.1 μJ/cm2) for room temperature lasing at ∼450 nm in optically pumped Gallium Nitride (GaN) nanobeam cavity structures. The nanobeam cavity geometry provides high theoretical Q (>100 000) with small modal volume, leading to a high spontaneous emission factor, β = 0.94. The active layer materials are Indium Gallium Nitride (InGaN) fragmented quantum wells (fQWs), a critical factor in achieving the low thresholds, which are an order-of-magnitude lower than obtainable with continuous QW active layers. We suggest that the extra confinement of photo-generated carriers for fQWs (compared to QWs) is responsible for the excellent performance.
Book description: At long last, here is the thoroughly revised and updated, and long-anticipated, third edition of the hugely successful Art of Electronics. Widely accepted as the best single authoritative text on electronic circuit design, it will be an indispensable reference and the gold standard for anyone in the field.
Single-crystal diamond, with its unique optical, mechanical and thermal properties, has emerged as a promising material with applications in classical and quantum optics. However, the lack of heteroepitaxial growth and scalable fabrication techniques remains the major limiting factors preventing more wide-spread development and application of diamond photonics. In this work, we overcome this difficulty by adapting angled-etching techniques, previously developed for realization of diamond nanomechanical resonators, to fabricate racetrack resonators and photonic crystal cavities in bulk single-crystal diamond. Our devices feature large optical quality factors, in excess of 105, and operate over a wide wavelength range, spanning visible and telecom. These newly developed high-Q diamond optical nanocavities open the door for a wealth of applications, ranging from nonlinear optics and chemical sensing, to quantum information processing and cavity optomechanics.
We experimentally demonstrate a label-free sensor based on nanoslotted parallel quadrabeam photonic crystal cavity (NPQC). The NPQC possesses both high sensitivity and high Q-factor. We achieved sensitivity (S) of 451 nm/refractive index unit and Q-factor >7000 in water at telecom wavelength range, featuring a sensor figure of merit >2000, an order of magnitude improvement over the previous photonic crystal sensors. In addition, we measured the streptavidin-biotin binding affinity and detected 10 ag/mL concentrated streptavidin in the phosphate buffered saline solution.
Fluorescent labeling techniques have been widely used in live cell studies; however, the labeling processes can be laborious and challenging for use in non-transfectable cells, and labels can interfere with protein functions. While label-free biosensors have been realized by nanofabrication, a method to track intracellular protein dynamics in real-time, in situ and in living cells has not been found. Here we present the first demonstration of label-free detection of intracellular p53 protein dynamics through a nanoscale surface plasmon-polariton fiber-tip-probe (FTP).
Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of techniques have been developed to better visualize these phase objects; phase contrast and DIC are among the most popular methods for enhancing contrast. However, recording position and shape in the out-of-imaging-plane direction remains challenging. This report introduces a simple experimental method to accurately determine the location and geometry of objects in three dimensions, using digital inline holographic microscopy (DIHM). Broadly speaking, the accessible sample volume is defined by the camera sensor size in the lateral direction, and the illumination coherence in the axial direction. Typical sample volumes range from 200 µm x 200 µm x 200 µm using LED illumination, to 5 mm x 5 mm x 5 mm or larger using laser illumination. This illumination light is configured so that plane waves are incident on the sample. Objects in the sample volume then scatter light, which interferes with the unscattered light to form interference patterns perpendicular to the illumination direction. This image (the hologram) contains the depth information required for three-dimensional reconstruction, and can be captured on a standard imaging device such as a CMOS or CCD camera. The Rayleigh-Sommerfeld back propagation method is employed to numerically refocus microscope images, and a simple imaging heuristic based on the Gouy phase anomaly is used to identify scattering objects within the reconstructed volume. This simple but robust method results in an unambiguous, model-free measurement of the location and shape of objects in microscopic samples.
Swimming bacteria explore their environment by performing a random walk, which is biased in response to, for example, chemical stimuli, resulting in a collective drift of bacterial populations towards ‘a better life’. This phenomenon, called chemotaxis, is one of the best known forms of collective behaviour in bacteria, crucial for bacterial survival and virulence. Both single-cell and macroscopic assays have investigated bacterial behaviours. However, theories that relate the two scales have previously been difficult to test directly. We present an image analysis method, inspired by light scattering, which measures the average collective motion of thousands of bacteria simultaneously. Using this method, a time-varying collective drift as small as 50 nm s−1 can be measured. The method, validated using simulations, was applied to chemotactic Escherichia colibacteria in linear gradients of the attractant α-methylaspartate. This enabled us to test a coarse-grained minimal model of chemotaxis. Our results clearly map the onset of receptor methylation, and the transition from linear to logarithmic sensing in the bacterial response to an external chemoeffector. Our method is broadly applicable to problems involving the measurement of collective drift with high time resolution, such as cell migration and fluid flows measurements, and enables fast screening of tactic behaviours.
It is widely believed that the swimming speed, v, of many flagellated bacteria is a nonmonotonic function of the concentration, c, of high-molecular-weight linear polymers in aqueous solution, showing peaked v(c) curves. Pores in the polymer solution were suggested as the explanation. Quantifying this picture led to a theory that predicted peaked v(c) curves. Using high-throughput methods for characterizing motility, we measured v and the angular frequency of cell body rotation, Ω, of motile Escherichia coli as a function of polymer concentration in polyvinylpyrrolidone (PVP) and Ficoll solutions of different molecular weights. We find that nonmonotonic v(c) curves are typically due to low-molecular-weight impurities. After purification by dialysis, the measured v(c) and Ω(c) relations for all but the highest-molecular-weight PVP can be described in detail by Newtonian hydrodynamics. There is clear evidence for non-Newtonian effects in the highest-molecular-weight PVP solution. Calculations suggest that this is due to the fast-rotating flagella seeing a lower viscosity than the cell body, so that flagella can be seen as nano-rheometers for probing the non-Newtonian behavior of high polymer solutions on a molecular scale.
We demonstrate an innovative multifunctional artificial material that combines exotic metamaterial properties and the environmentally responsive nature of phase change media. The tunable metamaterial is designed with the aid of two interwoven coordinate-transformation equations and implemented with a network of thin film resistors and vanadium dioxide (VO2). The strong temperature dependence of VO2 electrical conductivity results in a relevant modification of the resistor network behavior, and we provide experimental evidence for a reconfigurable metamaterial electric circuit (MMEC) that not only mimics a continuous medium but is also capable of responding to thermal stimulation through dynamic variation of its spatial anisotropy. Upon external temperature change the overall effective functionality of the material switches between a "truncated-cloak" and "concentrator" for electric currents. Possible applications may include adaptive matching resistor networks, multifunctional electronic devices, and equivalent artificial materials in the magnetic domain. Additionally, the proposed technology could also be relevant for thermal management of integrated circuits
Combination therapy has become one of the leading approaches for treating complex diseases because it coadministers clinically proven drugs to concurrently target multiple signaling pathways of diseased cells. Identification of synergic drug combinations at their respective effective doses without unwanted accumulative side effects is the key to success for such therapy. In this work, we demonstrate the feasibility of the vortex-assisted microfluidic electroporation system for direct drug cocktail analyses where drug substances were individually delivered into cytosols in a sequential and dosage-controlled manner. Through quantitative analyses, the synergic combinational dosage ratios of the chemotherapeutic drug and the anticancer flavonoid were identified. When integrated with high-throughput label-free rare cell purification techniques, the presented system has the potential for development of personalized medicines as the system would be capable of comprehensively assessing drug combinations directly on patients’ cellular samples.
Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system’s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (>70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.
Schonbrun E, Malka R, Caprio GD, Schaak D, Higgins JM.
Digital cameras would be colorblind if they did not have pixelated color filters integrated into their image sensors. Integration of conventional fixed filters, however, comes at the expense of an inability to modify the camera’s spectral properties. Instead, we demonstrate a micropolarizer-based camera that can reconfigure its spectral response. Color is encoded into a linear polarization state by a chiral dispersive element and then read out in a single exposure. The polarization encoded color camera is capable of capturing three-color images at wavelengths spanning the visible to the near infrared.