A novel method for automated crystal visualization and quantification in murine folic acid-induced acute kidney injury.

Folic acid (FA)-induced acute kidney injury (FA-AKI) is an increasingly prevalent rodent disease model involving the injection of a high dose of FA that culminates in renal FA crystal deposition and injury. However, the literature characterizing the FA-AKI model is sparse and dated in part due to the absence of a well-described methodology for the visualization and quantification of renal FA crystals. Using widely available materials and tools, we developed a straightforward and crystal-preserving histological protocol that can be coupled with automated imaging for renal FA crystal visualization and generated an automated macro for downstream crystal content quantification. The applicability of the method was demonstrated by characterizing the model in male and female C57BL6/JRj mice after 3 and 30 h of FA treatment. Kidneys from both sexes and timepoints showed a bimodal distribution of FA crystal deposition in the cortical and medullary regions while, compared with males, females exhibited higher renal FA crystal content at the 30-h timepoint accompanied by greater kidney weight and higher plasma urea. Despite comparable plasma phosphate concentrations, FA-AKI resulted in a substantially more elevated plasma intact fibroblast growth factor 23 (FGF23) in females, reflected by a similar pattern in osseous Fgf23 mRNA expression. Therefore, the presented method constitutes a valuable tool for the quantification of renal FA crystals, which can aid the mechanistic characterization of the FA-AKI model and serves as a means to control for confounding changes in FA crystallization when using the model for investigating early and prophylactic AKI therapeutic interventions.NEW & NOTEWORTHY Here, we describe a novel method for the visualization and quantification of renal folic acid (FA) crystals in the rodent FA-induced acute kidney injury (FA-AKI) model. The protocol involves a straightforward histological approach followed by fully automated imaging and quantification steps. Applicability was confirmed by showing that the FA-AKI model is sex-dependent. The method can serve as a tool to aid in characterizing FA-AKI and to control for studies investigating prophylactic therapeutic avenues using FA-AKI.

Hybrid machine-learning framework for volumetric segmentation and quantification of vacuoles in individual yeast cells using holotomography.

The precise, quantitative evaluation of intracellular organelles in three-dimensional (3D) imaging data poses a significant challenge due to the inherent constraints of traditional microscopy techniques, the requirements of the use of exogenous labeling agents, and existing computational methods. To counter these challenges, we present a hybrid machine-learning framework exploiting correlative imaging of 3D quantitative phase imaging with 3D fluorescence imaging of labeled cells. The algorithm, which synergistically integrates a random-forest classifier with a deep neural network, is trained using the correlative imaging data set, and the trained network is then applied to 3D quantitative phase imaging of cell data. We applied this method to live budding yeast cells. The results revealed precise segmentation of vacuoles inside individual yeast cells, and also provided quantitative evaluations of biophysical parameters, including volumes, concentration, and dry masses of automatically segmented vacuoles.

Positive feedback induces switch between distributive and processive phosphorylation of Hog1.

Cellular decision making often builds on ultrasensitive MAPK pathways. The phosphorylation mechanism of MAP kinase has so far been described as either distributive or processive, with distributive mechanisms generating ultrasensitivity in theoretical analyses. However, the in vivo mechanism of MAP kinase phosphorylation and its activation dynamics remain unclear. Here, we characterize the regulation of the MAP kinase Hog1 in Saccharomyces cerevisiae via topologically different ODE models, parameterized on multimodal activation data. Interestingly, our best fitting model switches between distributive and processive phosphorylation behavior regulated via a positive feedback loop composed of an affinity and a catalytic component targeting the MAP kinase-kinase Pbs2. Indeed, we show that Hog1 directly phosphorylates Pbs2 on serine 248 (S248), that cells expressing a non-phosphorylatable (S248A) or phosphomimetic (S248E) mutant show behavior that is consistent with simulations of disrupted or constitutively active affinity feedback and that Pbs2-S248E shows significantly increased affinity to Hog1 in vitro. Simulations further suggest that this mixed Hog1 activation mechanism is required for full sensitivity to stimuli and to ensure robustness to different perturbations.

Simple Assessment of Red Blood Cell Deformability Using Blood Pressure in Capillary Channels for Effective Detection of Subpopulations in Red Blood Cells.

Assessment of red blood cell (RBC) deformability as a biomarker requires expensive equipment to induce and monitor deformation. In this study, we present a simple method for quantifying RBC deformability. We designed a microfluidic channel consisting of a micropillar channel and a coflowing channel connected in series. When blood (loading volume = 100 µL) was injected continuously into the device under constant pressure (1 bar), we monitored the boundary position of the blood and the reference flow in the coflowing channel. A decrease in the deformability of RBCs results in a growing pressure drop in the micropillar channel, which is mirrored by a decrease in blood pressure in the coflowing channel. Analysis of this temporal variation in blood pressure allowed us to define the clogging index (CI) as a new marker of RBC deformability. As a result of the analytical study and numerical simulation, we have demonstrated that the coflowing channel may serve as a pressure sensor that allows the measurement of blood pressure with accuracy. We have shown experimentally that a higher hematocrit level (i.e., more than 40%) does not have a substantial influence on CI. The CI tended to increase to a higher degree in glutaraldehyde-treated hardened RBCs. Furthermore, we were able to resolve the difference in deformability of RBCs between two different RBC density subfractions in human blood. In summary, our approach using CI provides reliable information on the deformability of RBCs, which is comparable to the readouts obtained by ektacytometry. We believe that our microfluidic device would be a useful tool for evaluating the deformability of RBCs, which does not require expensive instruments (e.g., high-speed camera) or time-consuming micro-PIV analysis.

Unveiling host-guest-solvent interactions in solution by identifying highly unstable host-guest configurations in thermal non-equilibrium gas phase.

We propose a novel scheme of examining the host-guest-solvent interactions in solution from their gas phase structures. By adopting the permethylated ß-cyclodextrin (perm ß-CD)-protonated L-Lysine non-covalent complex as a prototypical system, we present the infrared multiple photon dissociation (IRMPD) spectrum of the gas phase complex produced by electrospray ionization technique. In order to elucidate the structure of perm ß-CD)/LysH+ complex in the gas phase, we carry out quantum chemical calculations to assign the two strong peaks at 3,340 and 3,560 cm-1 in the IRMPD spectrum, finding that the carboxyl forms loose hydrogen bonding with the perm ß-CD, whereas the ammonium group of L-Lysine is away from the perm ß-CD unit. By simulating the structures of perm ß-CD/H+/L-Lysine complex in solution using the supramolecule/continuum model, we find that the extremely unstable gas phase structure corresponds to the most stable conformer in solution.

Ingested nano- and microsized polystyrene particles surpass the intestinal barrier and accumulate in the body.

Plastic pollution is a major global challenge of our times, baring a potential threat for the environment and the human health. The increasing abundance of nanoplastic (NP) and microplastic (MP) particles in the human diet might negatively affect human health since they - particularly in patients suffering from inflammatory bowel disease (IBD) - might surpass the intestinal barrier. To investigate whether ingested plastic particles cross the intestinal epithelium and promote bowel inflammation, mice were supplemented with NP or MP polystyrene (PS) particles for 24 or 12 weeks before inducing acute or chronic dextran sodium sulfate (DSS) colitis with continuous plastic administration. Although ingested PS particles accumulated in the small intestine and organs distant from the gastrointestinal tract, PS ingestion did not affect intestinal health nor did it promote colitis severity. Although the lack of colitis-promoting effects of small PS particles might be a relief for IBD patients, potential accumulative effects of ingested plastic particles on the gastrointestinal health cannot be excluded.

Mre11-Rad50 oligomerization promotes DNA double-strand break repair.

The conserved Mre11-Rad50 complex is crucial for the detection, signaling, end tethering and processing of DNA double-strand breaks. While it is known that Mre11-Rad50 foci formation at DNA lesions accompanies repair, the underlying molecular assembly mechanisms and functional implications remained unclear. Combining pathway reconstitution in electron microscopy, biochemical assays and genetic studies, we show that S. cerevisiae Mre11-Rad50 with or without Xrs2 forms higher-order assemblies in solution and on DNA. Rad50 mediates such oligomerization, and mutations in a conserved Rad50 beta-sheet enhance or disrupt oligomerization. We demonstrate that Mre11-Rad50-Xrs2 oligomerization facilitates foci formation, DNA damage signaling, repair, and telomere maintenance in vivo. Mre11-Rad50 oligomerization does not affect its exonuclease activity but drives endonucleolytic cleavage at multiple sites on the 5'-DNA strand near double-strand breaks. Interestingly, mutations in the human RAD50 beta-sheet are linked to hereditary cancer predisposition and our findings might provide insights into their potential role in chemoresistance.

DNA circles promote yeast ageing in part through stimulating the reorganization of nuclear pore complexes.

The nuclear pore complex (NPC) mediates nearly all exchanges between nucleus and cytoplasm, and in many species, it changes composition as the organism ages. However, how these changes arise and whether they contribute themselves to ageing is poorly understood. We show that SAGA-dependent attachment of DNA circles to NPCs in replicatively ageing yeast cells causes NPCs to lose their nuclear basket and cytoplasmic complexes. These NPCs were not recognized as defective by the NPC quality control machinery (SINC) and not targeted by ESCRTs. They interacted normally or more effectively with protein import and export factors but specifically lost mRNA export factors. Acetylation of Nup60 drove the displacement of basket and cytoplasmic complexes from circle-bound NPCs. Mutations preventing this remodeling extended the replicative lifespan of the cells. Thus, our data suggest that the anchorage of accumulating circles locks NPCs in a specialized state and that this process is intrinsically linked to the mechanisms by which ERCs promote ageing.

Ultraviolet-Infrared Mixing in Marginal Fermi Liquids.

When Fermi surfaces (FSs) are subject to long-range interactions that are marginal in the renormalization-group sense, Landau Fermi liquids are destroyed, but only barely. With the interaction further screened by particle-hole excitations through one-loop quantum corrections, it has been believed that these marginal Fermi liquids (MFLs) are described by weakly coupled field theories at low energies. In this Letter, we point out a possibility in which higher-loop processes qualitatively change the picture through UV-IR mixing, in which the size of the FS enters as a relevant scale. The UV-IR mixing effect enhances the coupling at low energies, such that the basin of attraction for the weakly coupled fixed point of a (2+1)-dimensional MFL shrinks to a measure-zero set in the low-energy limit. This UV-IR mixing is caused by gapless virtual Cooper pairs that spread over the entire FS through marginal long-range interactions. Our finding signals a possible breakdown of the patch description for the MFL and questions the validity of using the MFL as the base theory in a controlled scheme for non-Fermi liquids that arise from relevant long-range interactions.

Inter- and Intra-Molecular Organocatalysis of SN2 Fluorination by Crown Ether: Kinetics and Quantum Chemical Analysis.

We present the intra- and inter-molecular organocatalysis of SN2 fluorination using CsF by crown ether to estimate the efficacy of the promoter and to elucidate the reaction mechanism. The yields of intramolecular SN2 fluorination of the veratrole substrates are measured to be very small (<1% in 12 h) in the absence of crown ether promoters, whereas the SN2 fluorination of the substrate possessing a crown ether unit proceeds to near completion (~99%) in 12 h. We also studied the efficacy of intermolecular rate acceleration by an independent promoter 18-crown-6 for comparison. We find that the fluorinating yield of a veratrole substrate (leaving group = -OMs) in the presence of 18-crown-6 follows the almost identical kinetic course as that of intramolecular SN2 fluorination, indicating the mechanistic similarity of intra- and inter-molecular organocatalysis of the crown ether for SN2 fluorination. The calculated relative Gibbs free energies of activation for these reactions, in which the crown ether units act as Lewis base promoters for SN2 fluorination, are in excellent agreement with the experimentally measured yields of fluorination. The role of the metal salt CsF is briefly discussed in terms of whether it reacts as a contact ion pair or as a "free" nucleophile F-.

Mechanistic study of nucleophilic fluorination for the synthesis of fluorine-18 labeled fluoroform with high molar activity from N-difluoromethyltriazolium triflate.

The synthesis of fluorine-18 labeled fluoroform with high molar activity has grown in importance for the development of fluorine-18 labeled aryl-CF3 radiopharmaceuticals that are useful as diagnostic radiotracers for the powerful technique of positron emission tomography (PET). We designed a strategy of synthesizing fluorine-18 labeled fluoroform from N1-difluoromethyl-N3-methyltriazolium triflate (1) via SN2 fluorination without stable fluorine isotope scrambling. Fluoroform was generated at rt in 10 min by fluorination of the triazolium precursor with TBAF (6 equiv.). We propose three routes (a), (b), and (c) for this fluorination. Quantum chemical calculations have been carried out to elucidate the mechanism of experimentally observed nucleophilic attack of fluoride at difluoromethyl group via route (a), not N3-methyl via route (b). 1H and 19F NMR studies using deuterium source have been performed to examine the competition between SN2 fluorination (route (a)) and the formation of difluorocarbene (route (c)). The observed superiority of SN2 pathway to formation of difluorocarbene in the reaction of the precursor using CsF in (CD3CN/(CD3)3COD (17.8 : 1)) gives the possibility of preparing the fluorine-18 labeled fluoroform in high molar activity.

Viscoelastic particle focusing in human biofluids.

Saliva and blood plasma are non-Newtonian viscoelastic fluids that play essential roles in the transport of particulate matters (e.g., food and blood cells). However, whether the viscoelasticity of such biofluids alters the dynamics of suspended particles is still unknown. In this study, we report that under pressure-driven microflows of both human saliva and blood plasma, spherical particles laterally migrate and form a focused stream along the channel centerline by their viscoelastic properties. We observed that the particle focusing varied among samples on the basis of sampling times/donors, thereby demonstrating that the viscoelasticity of the human biofluids can be affected by their compositions. We showed that the particle focusing, observed in bovine submaxillary mucin solutions, intensified with the increase in mucin concentration. We expect that the findings from this study will contribute to the understanding of the physiological roles of viscoelasticity of human biofluids.

Gear-shaped micromixer for synthesis of silica particles utilizing inertio-elastic flow instability.

Mixing in microscale flows, where turbulence is inherently difficult to generate, has been a challenging issue owing to its laminar flow characteristics. Either the diffusion-based mixing process, or the convective mixing based on the cross-stream secondary flow, has been exploited as a passive mixing scheme that does not require any external force field. However, these techniques suffer from insufficient mixing or complicated channel design step. In this study, we propose an efficient mixing scheme by combining inertio-elastic flow instability in a viscoelastic dilute polymer solution and a modified serpentine channel, termed a gear-shape channel, which has side wells along the serpentine channel. We achieved highly efficient mixing in the gear-shaped channel for a significantly wider range of flow rates than in a conventional serpentine channel. Further, we applied our novel mixing scheme to the continuous synthesis of silica nanoparticles, which demonstrated the synthesis of nanoparticles with more uniform size distribution and regular shape, than those in a Newtonian fluid. In addition, the adsorption of inorganic materials on the channel walls was significantly suppressed by the flow instability of the viscoelastic dilute polymer solution in the gear-shaped channel.

Noncovalent Complexes of Cyclodextrin with Small Organic Molecules: Applications and Insights into Host-Guest Interactions in the Gas Phase and Condensed Phase.

Cyclodextrins (CDs) have drawn a lot of attention from the scientific communities as a model system for host-guest chemistry and also due to its variety of applications in the pharmaceutical, cosmetic, food, textile, separation science, and essential oil industries. The formation of the inclusion complexes enables these applications in the condensed phases, which have been confirmed by nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography, and other methodologies. The advent of soft ionization techniques that can transfer the solution-phase noncovalent complexes to the gas phase has allowed for extensive examination of these complexes and provides valuable insight into the principles governing the formation of gaseous noncovalent complexes. As for the CDs' host-guest chemistry in the gas phase, there has been a controversial issue as to whether noncovalent complexes are inclusion conformers reflecting the solution-phase structure of the complex or not. In this review, the basic principles governing CD's host-guest complex formation will be described. Applications and structures of CDs in the condensed phases will also be presented. More importantly, the experimental and theoretical evidence supporting the two opposing views for the CD-guest structures in the gas phase will be intensively reviewed. These include data obtained via mass spectrometry, ion mobility measurements, infrared multiphoton dissociation (IRMPD) spectroscopy, and density functional theory (DFT) calculations.

Modular microfluidics enables kinetic insight from time-resolved cryo-EM.

Mechanistic understanding of biochemical reactions requires structural and kinetic characterization of the underlying chemical processes. However, no single experimental technique can provide this information in a broadly applicable manner and thus structural studies of static macromolecules are often complemented by biophysical analysis. Moreover, the common strategy of utilizing mutants or crosslinking probes to stabilize intermediates is prone to trapping off-pathway artefacts and precludes determining the order of molecular events. Here we report a time-resolved sample preparation method for cryo-electron microscopy (trEM) using a modular microfluidic device, featuring a 3D-mixing unit and variable delay lines that enables automated, fast, and blot-free sample vitrification. This approach not only preserves high-resolution structural detail but also substantially improves sample integrity and protein distribution across the vitreous ice. We validate the method by visualising reaction intermediates of early RecA filament growth across three orders of magnitude on sub-second timescales. The trEM method reported here is versatile, reproducible, and readily adaptable to a broad spectrum of fundamental questions in biology.

Crosstalk and spatiotemporal regulation between stress-induced MAP kinase pathways and pheromone signaling in budding yeast.

Budding yeast, Saccharomyces cerevisiae, has been widely used as a model system to study cellular signaling in response to internal and external cues. Yeast was among the first organisms in which the architecture, feedback mechanisms and physiological responses of various MAP kinase signaling cascades were studied in detail. Although these MAP kinase pathways are activated by different signals and elicit diverse cellular responses, such as adaptation to stress and mating, they function as an interconnected signaling network, as they influence each other and, in some cases, even share components. Indeed, various stress signaling pathways interfere with pheromone signaling that triggers a distinct cellular differentiation program. However, the molecular mechanisms responsible for this crosstalk are still poorly understood. Here, we review the general topology of the yeast MAP kinase signaling network and highlight recent and new data revealing how conflicting intrinsic and extrinsic signals are interpreted to orchestrate appropriate cellular responses.

Quantitative analysis of yeast MAPK signaling networks and crosstalk using a microfluidic device.

Eukaryotic cells developed complex mitogen-activated protein kinase (MAPK) signaling networks to sense their intra- and extracellular environment and respond to various stress conditions. For example, S. cerevisiae uses five distinct MAP kinase pathways to orchestrate meiosis or respond to mating pheromones, osmolarity changes and cell wall stress. Although each MAPK module has been studied individually, the mechanisms underlying crosstalk between signaling pathways remain poorly understood, in part because suitable experimental systems to monitor cellular outputs when applying different signals are lacking. Here, we investigate the yeast MAPK signaling pathways and their crosstalk, taking advantage of a new microfluidic device coupled to quantitative microscopy. We designed specific micropads to trap yeast cells in a single focal plane, and modulate the magnitude of a given stress signal by microfluidic serial dilution while keeping other signaling inputs constant. This approach enabled us to quantify in single cells nuclear relocation of effectors responding to MAPK activation, like Yap1 for oxidative stress, and expression of stress-specific reporter expression, like pSTL1-qV and pFIG1-qV for high-osmolarity or mating pheromone signaling, respectively. Using this quantitative single-cell analysis, we confirmed bimodal behavior of gene expression in response to Hog1 activation, and quantified crosstalk between the pheromone- and cell wall integrity (CWI) signaling pathways. Importantly, we further observed that oxidative stress inhibits pheromone signaling. Mechanistically, this crosstalk is mediated by Pkc1-dependent phosphorylation of the scaffold protein Ste5 on serine 185, which prevents Ste5 recruitment to the plasma membrane.

Nanoadhesive layer to prevent protein absorption in a poly(dimethylsiloxane) microfluidic device.

Poly(dimethylsiloxane) (PDMS) is widely used as a microfluidics platform material; however, it absorbs various molecules, perturbing specific chemical concentrations in microfluidic channels. We present a simple solution to prevent adsorption into a PDMS microfluidic device. We used a vapor-phase-deposited nanoadhesive layer to seal PDMS microfluidic channels. Absorption of fluorescent molecules into PDMS was efficiently prevented in the nanolayer-treated PDMS device. Importantly, when cultured in a nanolayer-treated PDMS device, yeast cells exhibited the expected concentration-dependent response to a mating pheromone, including mating-specific morphological and gene expression changes, while yeast cultured in an untreated PDMS device did not properly respond to the pheromone. Our method greatly expands microfluidic applications that require precise control of molecule concentrations.

Stable Flatbands, Topology, and Superconductivity of Magic Honeycomb Networks.

We propose a new principle to realize flatbands which are robust in real materials, based on a network superstructure of one-dimensional segments. This mechanism is naturally realized in the nearly commensurate charge-density wave of 1T-TaS_{2} with the honeycomb network of conducting domain walls, and the resulting flatband can naturally explain the enhanced superconductivity. We also show that corner states, which are a hallmark of the higher-order topological insulators, appear in the network superstructure.