Haploid Saccharomyces cerevisiae yeast cells use a prototypic cell signaling system to transmit information about the extracellular concentration of mating pheromone secreted by potential mating partners. The ability for cells to respond distinguishably to different pheromone concentrations depends on how much information about pheromone concentration the system can transmit. Here we show that the MAPK Fus3 mediates fast-acting negative feedback that adjusts the dose-response of downstream system response to match that of receptor-ligand binding. This “dose-response alignment”, defined by a linear relationship between receptor occupancy and downstream response, can improve the fidelity of information transmission by making downstream responses corresponding to different receptor occupancies more distinguishable and reducing amplification of stochastic noise during signal transmission. We also show that one target of the feedback is a novel signal-promoting function of the RGS protein Sst2. Our work suggests that negative feedback is a general mechanism used in signaling systems to align dose-responses and thereby increase the fidelity of information transmission.

As a part of an ongoing investigation of the use of isoelectric focusing (IEF) in microfluidic devices, pH gradients were electrochemically formed and optically quantified in microfluidic channels using acid-base indicators. The microchannels consisted of two parallel 40-mm-long electrodes with an interelectrode gap of 2.54 mm; top and bottom transparent windows were separated by 0.2 mm. Gradients in pH were formed as a result of the electrochemical decomposition of water at an applied potential not higher than 2.5 V to avoid generation of gas bubbles. Solutions contained low concentrations of a single buffer. The stability of the pH gradients and their sensitivity to changes in initial conditions were investigated under static (nonflow) conditions. Isoelectric focusing of sample biological analytes, bovine hemoglobin and bovine serum albumin, was performed to illustrate the potential of "microfluidic transverse IEF" for use in continuous concentration and separation systems.

Mix-and-inject serial crystallography (MISC) is a technique designed to image enzyme catalyzed reactions in which small protein crystals are mixed with a substrate just prior to being probed by an X-ray pulse. This approach offers several advantages over flow cell studies. It provides (i) room temperature structures at near atomic resolution, (ii) time resolution ranging from microseconds to seconds, and (iii) convenient reaction initiation. It outruns radiation damage by using femtosecond X-ray pulses allowing damage and chemistry to be separated. Here, we demonstrate that MISC is feasible at an X-ray free electron laser by studying the reaction of M. tuberculosis ß-lactamase microcrystals with ceftriaxone antibiotic solution. Electron density maps of the apo-ß-lactamase and of the ceftriaxone bound form were obtained at 2.8 Å and 2.4 Å resolution, respectively. These results pave the way to study cyclic and non-cyclic reactions and represent a new field of time-resolved structural dynamics for numerous substrate-triggered biological reactions.

BackgroundEver since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases.ResultsHere, we demonstrate a general method for capturing enzyme catalysis “in action” by mix-and-inject serial crystallography (MISC). Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis β-lactamase with the third-generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation from 30 ms to 2 s.ConclusionsMISC is a versatile and generally applicable method to investigate reactions of biological macromolecules, some of which are of immense biological significance and might be, in addition, important targets for structure-based drug design. With megahertz X-ray pulse rates expected at the Linac Coherent Light Source II and the European X-ray free-electron laser, multiple, finely spaced time delays can be collected rapidly, allowing a comprehensive description of biomolecular reactions in terms of structure and kinetics from the same set of X-ray data.Electronic supplementary materialThe online version of this article (10.1186/s12915-018-0524-5) contains supplementary material, which is available to authorized users.

M icrofluidic phenomena are present in all of nature's exquisitely designed biological systems (1). A complex oxygen transport network consisting of plasma, red blood cells ~6 to 8 gm in diameter, and capillaries -8 gm in diameter carries sustenance to cells throughout the body, including the eyes through which you are reading this article. Without microfluidic channels and diffusion transport systems, life as we know it would not be possible (1).Most microfluidic systems draw their advantages from low thermal mass and efficient mass transport, as well as from large channel surface area-to-channel volume ratios. Manmade microfluidic systems have been made possible by microfabrication methods developed for the electronics industry. Microfluidic systems are of intense interest because of their characteristic controlled mixing that expedites chemical reactions, reduced consumption of expensive materials, high sensitivity, and efficient product yields. Here, we aim to share a summary of the applications and design of microfluidic devices. Welcome to an expansive world of the very small.Microfluidic systems are composed of fluid channels and chambers with critical dimensions of tens to hundreds of micrometers (for reference, a relatively fine human hair is about 80 gm in diameter). Integrated systems of microfluidic channels combined with pumps, valves, and detectors are known as Micro Total Analysis Systems (gTAS) or "lab-on-a-chip" systems (2, 3). Modern microfluidic devices are generally hybrids of various materials and processes integrated with electro-optical components for detection and, importantly, manipulation of molecules of interest (2).Rapid and efficient mixing of components is critical in the control of chemical reactions. In nature, the limit of diffusion has determined, in part, how single-cell organisms evolved and how they behave (4). Microfluidic channels provide specialized environments for controlling mixing. Only laminar flow exists in confined spaces such as microchannels, because fluid viscosity, not inertia, dominates fluid behavior. Therefore, fluid flow in these systems is not turbulent or random. In macrofluidic systems, a much larger fluid mass is in motion and inertial forces can dominate and lead to turbulent conditions (see figure, this page). In laminar flow, two nonreacting fluids that merge will flow smoothly next to each other as diffusion brings the two solutions into a well-mixed equilibrium. This is achieved by layering the fluid or minimizing the channel dimensions (see figure, next page). The equilibration time is proportional to the square of the diffusion distance, in this case the microchannel dimension in the direction of the concentration gradient.The small dimensions of microfluidic devices cause the ratio of surface area-to-volume to be large. Capture of specific molecules and particles is achieved by exploiting the binding affinity of these substances to specialty coatings of the wall surfaces. Captured molecules and particles can be released all at (D.M.); holl@u.washi...

A series of new naphthalimide derivatives were synthesized and studied. Three of the materials (SM1, SM2, and SM3) possess methacrylate(s) moieties as pH sensor monomers, enabling these compounds to be polymerized with other monomers for thin film preparation for extracellular pH sensing. Herein, poly(2-hydroxyethyl methacrylate)-co-poly(acrylamide) (PHEMA-co-PAM) was chosen as the polymer matrix. Structure influences on pH responses and pKa values were studied. The film P3 composed of the sensing moiety SM3 has a pKa close to the usual biological environmental pH of ~7. It was used as an extracellular pH sensor to monitor pH change during the metabolism of prokaryotic Escherichia coli (E. coil). On the other hand, the three sensor monomers are new intracellular biomarkers to sense lysosomes of eukaryotic cells since (1) their pKa values are in a range of 5.9 to 6.8; (2) their emission intensities at acidic conditions (such as at pH 5) are much stronger than those at a neutral condition of pH 7; (3) lysosomes range in size from 0.1 to 1.2 μm in diameter with pH ranging from 4.5 to 5.0, which is much more acidic than the pH value of the cytoplasm (usually with a pH value of ~7.2); and (4) the acidity of lysosomes enables a protonation of the amino groups of the pH probes making the sensors emit brightly in acidic organelles by inhibiting the photo-induced electron transfer from the amino groups to the fluorophores. Lysosome sensing was demonstrated using live human brain glioblastoma U87MG cell line, human cervical cancer HeLa cell line, and human esophagus premalignant CP-A and CP-D cell lines by observations of small acidic spherical organelles (lysosomes) and significant colocalizations (82 ~ 95%) of the sensors with a commercially available lysosome-selective staining probe LysoTracker Red® under confocal fluorescence microscopy.

The development of a high-throughput single-cell metabolic rate monitoring system relies on the use of transparent substrate material for a single cell-trapping platform. The high optical transparency, high chemical resistance, improved surface quality and compatibility with the silicon micromachining process of fused silica make it very attractive and desirable for this application. In this paper, we report the results from the development and characterization of a hydrofluoric acid (HF) based deep wet-etch process on fused silica. The pin holes and notching defects of various single-coated masking layers during the etching are characterized and the most suitable masking materials are identified for different etch depths. The dependence of the average etch rate and surface roughness on the etch depth, impurity concentration and HF composition are also examined. The resulting undercut from the deep HF etch using various masking materials is also investigated. The developed and characterized process techniques have been successfully implemented in the fabrication of micro-well arrays for single cell trapping and sensor deposition. Up to 60 μm deep micro-wells have been etched in a fused silica substrate with over 90% process yield and repeatability. To our knowledge, such etch depth has never been achieved in a fused silica substrate by using a non-diluted HF etchant and a single-coated masking layer at room temperature.

A novel system that has enabled the measurement of single-cell oxygen consumption rates is presented. The experimental apparatus includes a temperature controlled environmental chamber, an array of microwells etched in glass, and a lid actuator used to seal cells in the microwells. Each microwell contains an oxygen sensitive platinum phosphor sensor used to monitor the cellular metabolic rates. Custom automation software controls the digital image data collection for oxygen sensor measurements, which are analyzed using an image-processing program to yield the oxygen concentration within each microwell versus time. Two proof-of-concept experiments produced oxygen consumption rate measurements for A549 human epithelial lung cancer cells of 5.39 and 5.27 fmol/min/cell, closely matching published oxygen consumption rates for bulk A549 populations.