Molecular machines stimulate intercellular calcium waves and cause muscle contraction.

Intercellular calcium waves (ICW) are complex signalling phenomena that control many essential biological activities, including smooth muscle contraction, vesicle secretion, gene expression and changes in neuronal excitability. Accordingly, the remote stimulation of ICW could result in versatile biomodulation and therapeutic strategies. Here we demonstrate that light-activated molecular machines (MM)-molecules that perform mechanical work on the molecular scale-can remotely stimulate ICW. MM consist of a polycyclic rotor and stator that rotate around a central alkene when activated with visible light. Live-cell calcium-tracking and pharmacological experiments reveal that MM-induced ICW are driven by the activation of inositol-triphosphate-mediated signalling pathways by unidirectional, fast-rotating MM. Our data suggest that MM-induced ICW can control muscle contraction in vitro in cardiomyocytes and animal behaviour in vivo in Hydra vulgaris. This work demonstrates a strategy for directly controlling cell signalling and downstream biological function using molecular-scale devices.

Real-Time In Vivo Sensing of Nitric Oxide Using Photonic Microring Resonators.

Real-time in vivo detection of biomarkers, particularly nitric oxide (NO), is of utmost importance for critical healthcare monitoring, therapeutic dosing, and fundamental understanding of NO's role in regulating many physiological processes. However, detection of NO in a biological medium is challenging due to its short lifetime and low concentration. Here, we demonstrate for the first time that photonic microring resonators (MRRs) can provide real-time, direct, and in vivo detection of NO in a mouse wound model. The MRR encodes the NO concentration information into its transfer function in the form of a resonance wavelength shift. We show that these functionalized MRRs, fabricated using complementary metal oxide semiconductor (CMOS) compatible processes, can achieve sensitive detection of NO (sub-µM) with excellent specificity and no apparent performance degradation for more than 24 h of operation in biological medium. With alternative functionalizations, this compact lab-on-chip optical sensing platform could support real-time in vivo detection of myriad of biochemical species.

Subsecond multichannel magnetic control of select neural circuits in freely moving flies.

Precisely timed activation of genetically targeted cells is a powerful tool for the study of neural circuits and control of cell-based therapies. Magnetic control of cell activity, or 'magnetogenetics', using magnetic nanoparticle heating of temperature-sensitive ion channels enables remote, non-invasive activation of neurons for deep-tissue applications and freely behaving animal studies. However, the in vivo response time of thermal magnetogenetics is currently tens of seconds, which prevents precise temporal modulation of neural activity. Moreover, magnetogenetics has yet to achieve in vivo multiplexed stimulation of different groups of neurons. Here we produce subsecond behavioural responses in Drosophila melanogaster by combining magnetic nanoparticles with a rate-sensitive thermoreceptor (TRPA1-A). Furthermore, by tuning magnetic nanoparticles to respond to different magnetic field strengths and frequencies, we achieve subsecond, multichannel stimulation. These results bring magnetogenetics closer to the temporal resolution and multiplexed stimulation possible with optogenetics while maintaining the minimal invasiveness and deep-tissue stimulation possible only by magnetic control.

Regio- and Stereoselective Synthesis of Fully Substituted Silyl Enol Ethers of Ketones and Aldehydes in Acyclic Systems.

The regio- and stereoselective preparation of fully substituted and stereodefined silyl enol ethers of ketones and aldehydes through an allyl-Brook rearrangement is reported. This fast and efficient method proceeds from a mixture of E and Z isomers of easily accessible starting materials.

Magnetic Entropy as a Proposed Gating Mechanism for Magnetogenetic Ion Channels.

Magnetically sensitive ion channels would allow researchers to better study how specific brain cells affect behavior in freely moving animals; however, recent reports of "magnetogenetic" ion channels based on biogenic ferritin nanoparticles have been questioned because known biophysical mechanisms cannot explain experimental observations. Here, we reproduce a weak magnetically mediated calcium response in HEK cells expressing a previously published TRPV4-ferritin fusion protein. We find that this magnetic sensitivity is attenuated when we reduce the temperature sensitivity of the channel but not when we reduce the mechanical sensitivity of the channel, suggesting that the magnetic sensitivity of this channel is thermally mediated. As a potential mechanism for this thermally mediated magnetic response, we propose that changes in the magnetic entropy of the ferritin particle can generate heat via the magnetocaloric effect and consequently gate the associated temperature-sensitive ion channel. Unlike other forms of magnetic heating, the magnetocaloric mechanism can cool magnetic particles during demagnetization. To test this prediction, we constructed a magnetogenetic channel based on the cold-sensitive TRPM8 channel. Our observation of a magnetic response in cold-gated channels is consistent with the magnetocaloric hypothesis. Together, these new data and our proposed mechanism of action provide additional resources for understanding how ion channels could be activated by low-frequency magnetic fields.

Enhanced Image Sensor Module for Head-Mounted Microscopes.

Several research groups have developed head-mounted fluorescence microscopes as a modality for recording neural activity in freely behaving mice. The current designs have shown exciting results from in vivo imaging of the bright dynamics of genetically encoded calcium indicators (GECI). However, despite their potential, head-mounted microscopes are not in use with genetically encoded voltage indicators (GEVI) or bioluminescence indicators. Due to its ability to match the temporal resolution of neuron spiking, GEVIs offer great benefits to experiments designed to provide feedback after real-time detection of specific neural activity such as the less than 250ms replay events that can occur in the hippocampus. Orthogonally, the emerging bioluminescence activity reporters have the potential to eliminate autofluorescence and photobleaching that can occur in fluorescence imaging. There are two important properties of the head-mounted microscope's image sensor affecting the ability to image GEVIs and bioluminescence indicators. First, the low signal to noise ratio (SNR) characteristics of GEVIs and bioluminescent indicators make signal detection difficult. Second, in order to take advantage of the GEVIs faster fluorescence kinetics, the image sensor must be capable of matching frame rates. Here, we present the design of a new imaging module for head-mounted microscopes incorporating the latest CMOS sensor technology aimed at increasing image sensor sensitivity and frame rates for use in real-time detection experiments. The design builds off an existing open-source project and can integrate into the existing data acquisition hardware and microscope housing.

Molecular machines open cell membranes.

Beyond the more common chemical delivery strategies, several physical techniques are used to open the lipid bilayers of cellular membranes. These include using electric and magnetic fields, temperature, ultrasound or light to introduce compounds into cells, to release molecular species from cells or to selectively induce programmed cell death (apoptosis) or uncontrolled cell death (necrosis). More recently, molecular motors and switches that can change their conformation in a controlled manner in response to external stimuli have been used to produce mechanical actions on tissue for biomedical applications. Here we show that molecular machines can drill through cellular bilayers using their molecular-scale actuation, specifically nanomechanical action. Upon physical adsorption of the molecular motors onto lipid bilayers and subsequent activation of the motors using ultraviolet light, holes are drilled in the cell membranes. We designed molecular motors and complementary experimental protocols that use nanomechanical action to induce the diffusion of chemical species out of synthetic vesicles, to enhance the diffusion of traceable molecular machines into and within live cells, to induce necrosis and to introduce chemical species into live cells. We also show that, by using molecular machines that bear short peptide addends, nanomechanical action can selectively target specific cell-surface recognition sites. Beyond the in vitro applications demonstrated here, we expect that molecular machines could also be used in vivo, especially as their design progresses to allow two-photon, near-infrared and radio-frequency activation.

Diflunisal inhibits prestin by chloride-dependent mechanism.

The motor protein prestin is a member of the SLC26 family of anion antiporters and is essential to the electromotility of cochlear outer hair cells and for hearing. The only direct inhibitor of electromotility and the associated charge transfer is salicylate, possibly through direct interaction with an anion-binding site on prestin. In a screen to identify other inhibitors of prestin activity, we explored the effect of the non-steroid anti-inflammatory drug diflunisal, which is a derivative of salicylate. We recorded prestin activity by whole-cell patch clamping HEK cells transiently expressing prestin and mouse outer hair cells. We monitored the impact of diflunisal on the prestin-dependent non-linear capacitance and electromotility. We found that diflunisal triggers two prestin-associated effects: a chloride independent increase in the surface area and the specific capacitance of the membrane, and a chloride dependent inhibition of the charge transfer and the electromotility in outer hair cells. We conclude that diflunisal affects the cell membrane organization and inhibits prestin-associated charge transfer and electromotility at physiological chloride concentrations. The inhibitory effects on hair cell function are noteworthy given the proposed use of diflunisal to treat neurodegenerative diseases.

Intramolecular Inverse Electron-Demand [4 + 2] Cycloadditions of Ynamides with Pyrimidines: Scope and Density Functional Theory Insights.

4-Aminopyridines are valuable scaffolds for the chemical industry in general, from life sciences to catalysis. We report herein a collection of structurally diverse polycyclic fused and spiro-4-aminopyridines that are prepared in only three steps from commercially available pyrimidines. The key step of this short sequence is a [4 + 2]/retro-[4 + 2] cycloaddition between a pyrimidine and an ynamide, which constitutes the first examples of ynamides behaving as electron-rich dienophiles in [4 + 2] cycloaddition reactions. In addition, running the ihDA/rDA reaction in continuous mode in superheated toluene, to overcome the limited scalability of MW reactions, results in a notable production increase compared to batch mode. Finally, density functional theory investigations shed light on the energetic and geometric requirements of the different steps of the ihDA/rDA sequence.

Membrane prestin expression correlates with the magnitude of prestin-associated charge movement.

Full expression of electromotility, generation of non-linear capacitance (NLC), and high-acuity mammalian hearing require prestin function in the lateral wall of cochlear outer hair cells (OHCs). Estimates of the number of prestin molecules in the OHC membrane vary, and a consensus has not emerged about the correlation between prestin expression and prestin-associated charge movement in the OHC. Using an inducible prestin-expressing cell line, we demonstrate that the charge density, but not the voltage at peak capacitance, directly correlates with the amount of prestin in the plasma membrane. This correlation is evident in studies involving a controlled increase of prestin expression with time after induction and inducer dose-response. Conversely, membrane prestin levels and charge density gradually decline together following the reduction of prestin levels from a steady state by removal of the inducer. Thus, charge density directly correlates with the level of membrane prestin expression, whereas changing membrane levels of prestin have no effect on the voltage at peak capacitance in this inducible prestin-expressing cell line.

Inverse Electron-Demand [4 + 2]-Cycloadditions of Ynamides: Access to Novel Pyridine Scaffolds.

Functionalized polycyclic aminopyridines are central to the chemical sciences, but their syntheses are still hampered by a number of shortcomings. These nitrogenated heterocycles can be efficiently prepared by an intramolecular inverse electron demand hetero Diels-Alder (ihDA) cycloaddition of ynamides to pyrimidines. This ihDA/rDA sequence is general in scope and affords expedient access to novel types of aminopyridinyl scaffolds that hold great promise in terms of exit vector patterns.

Boron chemistry in a new light.

Photocatalysis has recently opened up new avenues for the generation of radical species under visible light irradiation conditions. A particularly fascinating class of photocatalyzed transformations relies on the activation of stable boron species with visible-light since it allows the creation of boryl and/or carbon radicals through single electron transfer or energy transfer without the need for specific and costly equipment. This new paradigm has found numerous applications in synthetic organic chemistry, catalysis, and macromolecular chemistry. In this minireview, the concepts underlying photoactivation of boron-species as well as applications to the creation of C-H, C-C, C-O, B-C and B-S bond are discussed.

Functional prokaryotic-eukaryotic chimera from the pentameric ligand-gated ion channel family.

Pentameric ligand-gated ion channels (pLGICs), which mediate chemo-electric signal transduction in animals, have been recently found in bacteria. Despite clear sequence and 3D structure homology, the phylogenetic distance between prokaryotic and eukaryotic homologs suggests significant structural divergences, especially at the interface between the extracellular (ECD) and the transmembrane (TMD) domains. To challenge this possibility, we constructed a chimera in which the ECD of the bacterial protein GLIC is fused to the TMD of the human α1 glycine receptor (α1GlyR). Electrophysiology in Xenopus oocytes shows that it functions as a proton-gated ion channel, thereby locating the proton activation site(s) of GLIC in its ECD. Patch-clamp experiments in BHK cells show that the ion channel displays an anionic selectivity with a unitary conductance identical to that of the α1GlyR. In addition, pharmacological investigations result in transmembrane allosteric modulation similar to the one observed on α1GlyR. Indeed, the clinically active drugs propofol, four volatile general anesthetics, alcohols, and ivermectin all potentiate the chimera while they inhibit GLIC. Collectively, this work shows the compatibility between GLIC and α1GlyR domains and points to conservation of the ion channel and transmembrane allosteric regulatory sites in the chimera. This provides evidence that GLIC and α1GlyR share a highly homologous 3D structure. GLIC is thus a relevant model of eukaryotic pLGICs, at least from the anionic type. In addition, the chimera is a good candidate for mass production in Escherichia coli, opening the way for investigations of "druggable" eukaryotic allosteric sites by X-ray crystallography.

Size and dynamics of the Vibrio cholerae porins OmpU and OmpT probed by polymer exclusion.

The trimeric OmpU and OmpT porins form large, triple-barrel hydrophilic channels in the outer membrane of the pathogen Vibrio cholerae. They have distinct pore properties, such as conductance, block by deoxycholic acid, and sensitivity to acidic pH. Their three-dimensional structures are unknown, but they share significant sequence homologies. To gain insight into the molecular basis for the distinct functional properties of these two similar porins, we carried out polymer exclusion experiments using planar lipid bilayer and patch-clamp electrophysiology. By studying the partitioning of polyethylene glycols (PEGs) of different molecular weights into each porin, we determined an effective radius of 0.55 nm and 0.43 nm for OmpU and OmpT respectively, and found an increased OmpU effective radius at acidic pH. PEGs or high buffer ionic strength promotes the appearance of single step closures in OmpU similar to the acidic-pH induced closures we documented previously. In addition, these closing events can be triggered by nonpenetrating PEGs applied asymmetrically. We believe our results support a model whereby acidic pH, high ionic strength, or exposure to PEGs stabilizes a less conductive state that corresponds to the appearance of an additional resistive element on one side of the OmpU protein and common to the three monomers.

Modulating effects of the plug, helix, and N- and C-terminal domains on channel properties of the PapC usher.

The chaperone/usher system is one of the best characterized pathways for protein secretion and assembly of cell surface appendages in Gram-negative bacteria. In particular, this pathway is used for biogenesis of the P pilus, a key virulence factor used by uropathogenic Escherichia coli to adhere to the host urinary tract. The P pilus individual subunits bound to the periplasmic chaperone PapD are delivered to the outer membrane PapC usher, which serves as an assembly platform for subunit incorporation into the pilus and secretion of the pilus fiber to the cell surface. PapC forms a dimeric, twin pore complex, with each monomer composed of a 24-stranded transmembrane beta-barrel channel, an internal plug domain that occludes the channel, and globular N- and C-terminal domains that are located in the periplasm. Here we have used planar lipid bilayer electrophysiology to characterize the pore properties of wild type PapC and domain deletion mutants for the first time. The wild type pore is closed most of the time but displays frequent short-lived transitions to various open states. In comparison, PapC mutants containing deletions of the plug domain, an alpha-helix that caps the plug domain, or the N- and C-terminal domains form channels with higher open probability but still exhibiting dynamic behavior. Removal of the plug domain results in a channel with extremely large conductance. These observations suggest that the plug gates the usher channel closed and that the periplasmic domains and alpha-helix function to modulate the gating activity of the PapC twin pore.

Folding and trimerization of signal sequence-less mature TolC in the cytoplasm of Escherichia coli.

TolC is a multifunctional outer-membrane protein (OMP) of Escherichia coli that folds into a unique alpha/beta-barrel structure. Previous studies have shown that unlike the biogenesis of beta-barrel OMPs, such as porins, TolC assembles independently from known periplasmic folding factors. Yet, the assembly of TolC, like that of beta-barrel OMPs, is dependent on BamA and BamD, two essential components of the beta-barrel OMP assembly machinery. We have investigated the folding properties and cellular trafficking of a TolC derivative that lacks the entire signal sequence (TolCDelta2-22). A significant amount of TolCDelta2-22 was found to be soluble in the cytoplasm, and a fraction of it folded and trimerized into a conformation similar to that of the normal outer membrane-localized TolC protein. Some TolCDelta2-22 was found to associate with membranes, but failed to assume a wild-type-like folded conformation. The null phenotype of TolCDelta2-22 was exploited to isolate suppressor mutations, the majority of which mapped in secY. In the secY suppressor background, TolCDelta2-22 resumed normal function and folded like wild-type TolC. Proper membrane insertion could not be achieved upon in vitro incubation of cytoplasmically folded TolCDelta2-22 with purified outer membrane vesicles, showing that even though TolC is intrinsically capable of folding and trimerization, for successful integration into the outer membrane these events need to be tightly coupled to the insertion process, which is mediated by the Bam machinery. Genetic and biochemical data attribute the unique folding and assembly pathways of TolC to its large soluble alpha-helical domain.

The TpsB translocator HMW1B of haemophilus influenzae forms a large conductance channel.

The Haemophilus influenzae HMW1 adhesin is secreted via the two-partner secretion pathway and requires HMW1B for translocation across the outer membrane. HMW1B belongs to the Omp85-TpsB superfamily of transporters and consists of two structural domains, a C-terminal transmembrane beta-barrel and an N-terminal periplasmic domain. We investigated the electrophysiological properties of the purified full-length HMW1B and the C-terminal domain using planar lipid bilayers. Both the full-length and the truncated proteins formed conductive pores with a low open probability, two well defined conductance states, and other substates. The kinetic patterns of the two conductance states were distinct, with rapid and frequent transitions to the small conductance state and occasional and more prolonged openings to the large conductance state. The channel formed by the full-length HMW1B showed selectivity for cations, which decreased when measured at pH 5.2, suggesting the presence of acidic residues in the pore. The C-terminal domain of HMW1B was less stable and required reconstitution into liposomes prior to insertion in the bilayer. It formed a channel of smaller conductance but a similar gating pattern as the full-length protein, demonstrating the ability of the last 312 C-terminal amino acids to form a pore and suggesting that the periplasmic domain is not involved in occluding the pore, nor in controlling the inherent basal kinetics of the channel. The HMW1 pro-piece containing the secretion domain, although binding to the channel with high affinity, did not induce channel opening.

Modulation of Vibrio cholerae porin function by acidic pH.

The outer membrane of Gram-negative bacteria contains porins, large pore-forming proteins which allow the traffic of hydrophilic compounds between the external medium and the periplasm. The oral mode of infection of Vibrio cholerae, the agent of cholera, implies that the bacteria must adapt to severe changes in the environment, such as acidic pH and the presence of bile. Because of their localization and the regulation of their expression in response to these external factors, the OmpU and OmpT porins of V. cholerae are thought to be involved in the adaptation of the bacteria to the host environment. Using patch clamp and planar lipid bilayer electrophysiology, we assessed the effect of pH on the channel properties of OmpU and OmpT. OmpT does not show any major modification in its activity between pH 4 and pH 7.2. In the case of OmpU, the effect of acidic pH is manifested by promoting single-step closures, whose duration, frequency and current size increase as pH is lowered, thereby producing a pH-dependent decrease in the channel open probability. Surprisingly, the increase in current size of this single-step closure is not coupled with an increase of the total current through the porin, indicating that the trimeric conductance remains unchanged. This observation suggests that coordinated events take place at the level of the trimer, and various explanations for this peculiar effect of acidic pH on porin gating and conductance are provided.

Deoxycholic acid blocks vibrio cholerae OmpT but not OmpU porin.

OmpT and OmpU are general diffusion porins of the human intestinal pathogen Vibrio cholerae. The sole presence of OmpT in the outer membrane sensitizes cells to the bile component deoxycholic acid, and the repression of OmpT in the intestine may play an important role in the adaptation of cells to the host environment. Here we report a novel important functional difference between the two porins, namely the sensitivity to deoxycholic acid. Single channel recordings show that submicellar concentrations of sodium deoxycholate induce time-resolved blocking events of OmpT but are devoid of any effect on OmpU. The effects are dose-, voltage-, and pH-dependent. They are elicited by deoxycholate applied to either side of the membrane, with some asymmetry in the sensitivity. The voltage dependence remains even when deoxycholate is applied symmetrically, indicating that it is intrinsic to the binding site. The pH dependence suggests that the active form is the neutral deoxycholic acid and not the negatively charged species. The results are interpreted as deoxycholic acid acting as an open-channel blocker, which may relate to deoxycholic acid permeation.