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Sepsis is an extremely dangerous medical condition that emanates from the bodys response to a pre-existing infection. Early detection of sepsis-inducing bacterial infections can greatly enhance the treatment process and potentially prevent the onset of sepsis. However, current point-of-care (POC) sensors are often complex and costly or lack the ideal sensitivity for effective bacterial detection. Therefore, it is crucial to develop rapid and sensitive biosensors for the on-site detection of sepsis-inducing bacteria. Herein, we developed a graphene oxide CRISPR-Cas12a (GO-CRISPR) biosensor for the detection of sepsis-inducing bacteria in human serum. In this strategy, single-stranded (ssDNA) FAM probes were quenched with single-layer graphene oxide (GO). Target-activated Cas12a trans-cleavage was utilized for the degradation of the ssDNA probes, detaching the short ssDNA probes from GO and recovering the fluorescent signals. Under optimal conditions, we employed our GO-CRISPR system for the detection of Salmonella Typhimurium (S. Typhimurium) with a detection sensitivity of as low as 3 × 103 CFU/mL in human serum, as well as a good detection specificity toward other competing bacteria. In addition, the GO-CRISPR biosensor exhibited excellent sensitivity to the detection of S. Typhimurium in spiked human serum. The GO-CRISPR system offers superior rapidity for the detection of sepsis-inducing bacteria and has the potential to enhance the early detection of bacterial infections in resource-limited settings, expediting the response for patients at risk of sepsis.

We present super-resolution microscopy of isolated functional mitochondria, enabling real-time studies of structure and function (voltages) in response to pharmacological manipulation. Changes in mitochondrial membrane potential as a function of time and position can be imaged in different metabolic states (not possible in whole cells), created by the addition of substrates and inhibitors of the electron transport chain, enabled by the isolation of vital mitochondria. By careful analysis of structure dyes and voltage dyes (lipophilic cations), we demonstrate that most of the fluorescent signal seen from voltage dyes is due to membrane bound dyes, and develop a model for the membrane potential dependence of the fluorescence contrast for the case of super-resolution imaging, and how it relates to membrane potential. This permits direct analysis of mitochondrial structure and function (voltage) of isolated, individual mitochondria as well as submitochondrial structures in the functional, intact state, a major advance in super-resolution studies of living organelles.

Multiphoton microscopy can resolve fluorescent structures and dynamics deep in scattering tissue and has transformed neural imaging, but applying this technique in vivo can be limited by the mechanical and optical constraints of conventional objectives. Short working distance objectives can collide with compact surgical windows or other instrumentation and preclude imaging. Here we present an ultra-long working distance (20 mm) air objective called the Cousa objective. It is optimized for performance across multiphoton imaging wavelengths, offers a more than 4 mm2 field of view with submicrometer lateral resolution and is compatible with commonly used multiphoton imaging systems. A novel mechanical design, wider than typical microscope objectives, enabled this combination of specifications. We share the full optical prescription, and report performance including in vivo two-photon and three-photon imaging in an array of species and preparations, including nonhuman primates. The Cousa objective can enable a range of experiments in neuroscience and beyond.

Protein properties and interactions have been widely investigated by using external labels. However, the micromolar sensitivity of the current dyes limits their applicability due to the high material consumption and assay cost. In response to this challenge, we synthesized a series of cyanine5 (Cy5) dye-based quencher molecules to develop an external dye technique to probe proteins at the nanomolar protein level in a high-throughput one-step assay format. Several families of Cy5 dye-based quenchers with ring and/or side-chain modifications were designed and synthesized by introducing organic small molecules or peptides. Our results showed that steric hindrance and electrostatic interactions are more important than hydrophobicity in the interaction between the luminescent negatively charged europium-chelate-labeled peptide (Eu-probe) and the quencher molecules. The presence of substituents on the quencher indolenine rings reduces their quenching property, whereas the increased positive charge on the indolenine side chain improved the interaction between the quenchers and the luminescent compound. The designed quencher structures entirely altered the dynamics of the Eu-probe (protein-probe) for studying protein stability and interactions, as we were able to reduce the quencher concentration 100-fold. Moreover, the new quencher molecules allowed us to conduct the experiments using neutral buffer conditions, known as the peptide-probe assay. These improvements enabled us to apply the method in a one-step format for nanomolar protein-ligand interaction and protein profiling studies instead of the previously developed two-step protocol. These improvements provide a faster and simpler method with lower material consumption.

Flexible, water-soluble hosts are capable of selective molecular recognition in cellular environments and can detect neurotransmitters such as choline in cells. Both cationic and anionic water-soluble self-folded deep cavitands can recognize suitable styrylpyridinium dyes in cellular interiors. The dyes selectively accumulate in nucleotide-rich regions of the cell nucleus and cytoplasm. The hosts bind the dyes and promote their relocation to the outer cell membrane: the lipophilic cavitands predominantly reside in membrane environments but are still capable of binding suitable targets in other cellular organelles. Incubating the cells with structurally similar biomarkers such as choline, cholamine, betaine, or butyrylcholine illustrates the selective recognition. Choline and butyrylcholine can be bound by the hosts, but minimal binding is seen with betaine or cholamine. Varying the dye allows control of the optical detection method, and both "turn-on" sensing and "turn-off" sensing are possible.

A plaque assay-the gold-standard method for measuring the concentration of replication-competent lytic virions-requires staining and usually more than 48 h of runtime. Here we show that lens-free holographic imaging and deep learning can be combined to expedite and automate the assay. The compact imaging device captures phase information label-free at a rate of approximately 0.32 gigapixels per hour per well, covers an area of about 30 × 30 mm2 and a 10-fold larger dynamic range of virus concentration than standard assays, and quantifies the infected area and the number of plaque-forming units. For the vesicular stomatitis virus, the automated plaque assay detected the first cell-lysing events caused by viral replication as early as 5 h after incubation, and in less than 20 h it detected plaque-forming units at rates higher than 90% at 100% specificity. Furthermore, it reduced the incubation time of the herpes simplex virus type 1 by about 48 h and that of the encephalomyocarditis virus by about 20 h. The stain-free assay should be amenable for use in virology research, vaccine development and clinical diagnosis.

SignificanceThis third biennial intraoperative molecular imaging (IMI) conference shows how optical contrast agents have been applied to develop clinically significant endpoints that improve precision cancer surgery.AimNational and international experts on IMI presented ongoing clinical trials in cancer surgery and preclinical work. Previously known dyes (with broader applications), new dyes, novel nonfluorescence-based imaging techniques, pediatric dyes, and normal tissue dyes were discussed.ApproachPrincipal investigators presenting at the Perelman School of Medicine Abramson Cancer Center's third clinical trials update on IMI were selected to discuss their clinical trials and endpoints.ResultsDyes that are FDA-approved or currently under clinical investigation in phase 1, 2, and 3 trials were discussed. Sections on how to move benchwork research to the bedside were also included. There was also a dedicated section for pediatric dyes and nonfluorescence-based dyes that have been newly developed.ConclusionsIMI is a valuable adjunct in precision cancer surgery and has broad applications in multiple subspecialties. It has been reliably used to alter the surgical course of patients and in clinical decision making. There remain gaps in the utilization of IMI in certain subspecialties and potential for developing newer and improved dyes and imaging techniques.

Visual processing in the retina depends on the collective activity of large ensembles of neurons organized in different layers. Current techniques for measuring activity of layer-specific neural ensembles rely on expensive pulsed infrared lasers to drive 2-photon activation of calcium-dependent fluorescent reporters. We present a 1-photon light-sheet imaging system that can measure the activity in hundreds of neurons in the ex vivo retina over a large field of view while presenting visual stimuli. This allows for a reliable functional classification of different retinal cell types. We also demonstrate that the system has sufficient resolution to image calcium entry at individual synaptic release sites across the axon terminals of dozens of simultaneously imaged bipolar cells. The simple design, large field of view, and fast image acquisition make this a powerful system for high-throughput and high-resolution measurements of retinal processing at a fraction of the cost of alternative approaches.

Fluorescence lifetime imaging captures the spatial distribution of chemical species across cellular environments employing pulsed illumination confocal setups. However, quantitative interpretation of lifetime data continues to face critical challenges. For instance, fluorescent species with known in vitro excited-state lifetimes may split into multiple species with unique lifetimes when introduced into complex living environments. What is more, mixtures of species, which may be both endogenous and introduced into the sample, may exhibit 1) very similar lifetimes as well as 2) wide ranges of lifetimes including lifetimes shorter than the instrumental response function or whose duration may be long enough to be comparable to the interpulse window. By contrast, existing methods of analysis are optimized for well-separated and intermediate lifetimes. Here, we broaden the applicability of fluorescence lifetime analysis by simultaneously treating unknown mixtures of arbitrary lifetimes-outside the intermediate, Goldilocks, zone-for data drawn from a single confocal spot leveraging the tools of Bayesian nonparametrics (BNP). We benchmark our algorithm, termed BNP lifetime analysis, using a range of synthetic and experimental data. Moreover, we show that the BNP lifetime analysis method can distinguish and deduce lifetimes using photon counts as small as 500.

Integrated optofluidic devices have become subjects of high interest for rapid biosensor devices due to their unique ability to combine the fluidic processing of small volumes of microfluidics with the analysis capabilities of photonic structures. By integrating dynamically reconfigurable optofluidic lasers on-chip, complex coupling can be eliminated while further increasing the capabilities of sensors to detect an increasing number of target biomarkers. Here, we report a polydimethylsiloxane-based device with two on-chip fluidic distributed feedback (DFB) laser cavities that are integrated with an orthogonal analyte channel for multiplexed fluorescence excitation. One DFB grating is filled with 4-(dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran dissolved in dimethyl sulfoxide. The second grating is filled with rhodamine 6G dissolved in a diluted ethylene glycol solution. We present characterization of both lasers through analysis of the lasing spectra for spectral narrowing along with a power series to observe threshold behavior. We then demonstrate simultaneous detection of two different fluorescent microbeads as a proof of concept for scalable, single biomarker analysis using on-chip optofluidic lasers.

The development of genetically encoded dopamine sensors such as dLight has provided a new approach to measuring slow and fast dopamine dynamics both in brain slices and in vivo, possibly enabling dopamine measurements in areas like the dorsolateral striatum (DLS) where previously such recordings with fast-scan cyclic voltammetry (FSCV) were difficult. To test this, we first evaluated dLight photometry in mouse brain slices with simultaneous FSCV and found that both techniques yielded comparable results, but notable differences in responses to dopamine transporter inhibitors, including cocaine. We then used in vivo fiber photometry with dLight in mice to examine responses to cocaine in DLS. We also compared dopamine responses during Pavlovian conditioning across the striatum. We show that dopamine increases were readily detectable in DLS and describe transient dopamine kinetics, as well as slowly developing signals during conditioning. Overall, our findings indicate that dLight photometry is well suited to measuring dopamine dynamics in DLS.

The small Ultra-Red Fluorescent Protein (smURFP) represents a new class of fluorescent protein with exceptional photostability and brightness derived from allophycocyanin in a previous directed evolution. Here, we report the smURFP crystal structure to better understand properties and enable further engineering of improved variants. We compare this structure to the structures of allophycocyanin and smURFP mutants to identify the structural origins of the molecular brightness. We then use a structure-guided approach to develop monomeric smURFP variants that fluoresce with phycocyanobilin but not biliverdin. Furthermore, we measure smURFP photophysical properties necessary for advanced imaging modalities, such as those relevant for two-photon, fluorescence lifetime, and single-molecule imaging. We observe that smURFP has the largest two-photon cross-section measured for a fluorescent protein, and that it produces more photons than organic dyes. Altogether, this study expands our understanding of the smURFP, which will inform future engineering toward optimal FPs compatible with whole organism studies.

This paper presents a method for time-lapse 3D cell analysis. Specifically, we consider the problem of accurately localizing and quantitatively analyzing sub-cellular features, and for tracking individual cells from time-lapse 3D confocal cell image stacks. The heterogeneity of cells and the volume of multi-dimensional images presents a major challenge for fully automated analysis of morphogenesis and development of cells. This paper is motivated by the pavement cell growth process, and building a quantitative morphogenesis model. We propose a deep feature based segmentation method to accurately detect and label each cell region. An adjacency graph based method is used to extract sub-cellular features of the segmented cells. Finally, the robust graph based tracking algorithm using multiple cell features is proposed for associating cells at different time instances. We also demonstrate the generality of our tracking method on C. elegans fluorescent nuclei imagery. Extensive experiment results are provided and demonstrate the robustness of the proposed method. The code is available on GitHub and the method is available as a service through the BisQue portal.

Concern that synthetic food dyes may impact behavior in children prompted a review by the California Office of Environmental Health Hazard Assessment (OEHHA). OEHHA conducted a systematic review of the epidemiologic research on synthetic food dyes and neurobehavioral outcomes in children with or without identified behavioral disorders (particularly attention and activity). We also conducted a search of the animal toxicology literature to identify studies of neurobehavioral effects in laboratory animals exposed to synthetic food dyes. Finally, we conducted a hazard characterization of the potential neurobehavioral impacts of food dye consumption. We identified 27 clinical trials of children exposed to synthetic food dyes in this review, of which 25 were challenge studies. All studies used a cross-over design and most were double blinded and the cross-over design was randomized. Sixteen (64%) out of 25 challenge studies identified some evidence of a positive association, and in 13 (52%) the association was statistically significant. These studies support a relationship between food dye exposure and adverse behavioral outcomes in children. Animal toxicology literature provides additional support for effects on behavior. Together, the human clinical trials and animal toxicology literature support an association between synthetic food dyes and behavioral impacts in children. The current Food and Drug Administration (FDA) acceptable daily intakes are based on older studies that were not designed to assess the types of behavioral effects observed in children. For four dyes where adequate dose-response data from animal and human studies were available, comparisons of the effective doses in studies that measured behavioral or brain effects following exposure to synthetic food dyes indicate that the basis of the ADIs may not be adequate to protect neurobehavior in susceptible children. There is a need to re-evaluate exposure in children and for additional research to provide a more complete database for establishing ADIs protective of neurobehavioral effects.

Photoacoustic (PA) imaging is a powerful biomedical imaging modality. We designed KeTMR and KeJuR, two xanthene-based dyes that were readily obtained through a 2-step synthetic route. KeJuR has low molecular weight, good aqueous solubility, and superior chemical stability compared to KeTMR. KeJuR shows a robust PA signal under 860 nm excitation and can be paired with traditional PA dyes for multiplex imaging in blood samples under a tissue-mimicking environment.

Background: The widespread presence of organic dyes in the various industries’ effluent such as paper, textile, and clothing has led to significant environmental pollution. Titanium oxide (TiO2) nanoparticles immobilized on zeolite (ZSM-5) were investigated under UV light for activation of peroxymonosulfate (PMS) for photocatalytic degradation of Orange II (OII) dye in aqueous solutions. Methods: In this study, the photocatalyst used was prepared by immobilizing different amounts of TiO2 nanoparticles on ZSM-5. Characterization analyses including X-ray diffractometer (XRD), Fourier transform infrared (FTIR), BET, scanning electron microscope (SEM), and energy dispersive X-ray spectroscopy (EDS) were performed on the synthesized samples. Then, the effect of various parameters, such as TiO2 nanoparticles loading onto the ZSM-5, pH, contact time, dye concentration, and TiO2/ ZSM-5 dosage, is investigated for the removal of OII as a model molecule under the UV irradiation with 15 W power. Results: The highest removal percentage of OII dye (97.44%) was obtained in the optimal operating conditions of pH = 3, the initial dye concentration = 5 ppm, amount of TiO2/ZSM-5 = 10 mg/L, amount of PMS = 50 mg/L, and reaction time = 120 minutes. The Langmuir–Hinshelwood kinetics model fitted with the experimental data. Conclusion: The obtained results of this research showed that PMS can be used as a suitable oxidant activated with ZSM-5/TiO2 nanocomposite in OII degradation in different water environments, by optimizing the effective operating factors.

BackgroundEarly experience with indocyanine green-based fluorescent cholangiography during laparoscopic cholecystectomy suggests the potential to improve outcomes. However, the cost-effectiveness of routine use has not been studied. Our objective was to evaluate the cost-effectiveness of fluorescent cholangiography versus standard bright light laparoscopic cholecystectomy for noncancerous gallbladder disease.MethodsA Markov model decision analysis was performed comparing fluorescent cholangiography versus standard bright light laparoscopic cholecystectomy alone. Probabilities of outcomes, survival, toxicities, quality-adjusted life-years, and associated costs were determined from literature review and pooled analysis of currently available studies on fluorescent cholangiography (n = 37). Uncertainty in the model parameters was evaluated with 1-way and probabilistic sensitivity analyses, varying parameters up to 40% of their means. Cost-effectiveness was measured with an incremental cost-effectiveness ratio expressed as the dollar amount per quality-adjusted life-year.ResultsThe model predicted that fluorescent cholangiography reduces lifetime costs by $1,235 per patient and improves effectiveness by 0.09 quality-adjusted life-years compared to standard bright light laparoscopic cholecystectomy. Reduced costs were due to a decreased operative duration (21.20 minutes, P < .0001) and rate of conversion to open (1.62% vs 6.70%, P < .0001) associated with fluorescent cholangiography. The model was not influenced by the rate of bile duct injury. Probabilistic sensitivity analysis found that fluorescent cholangiography was both more effective and less costly in 98.83% of model iterations at a willingness-to-pay threshold of $100,000/quality-adjusted life year.ConclusionThe current evidence favors routine use of fluorescent cholangiography during laparoscopic cholecystectomy as a cost-effective surgical strategy. Our model predicts that fluorescent cholangiography reduces costs while improving health outcomes, suggesting fluorescence imaging may be considered standard surgical management for noncancerous gallbladder disease. Further study with prospective trials should be considered to verify findings of this predictive model.

Background. Droplet simulation often requires expensive and inaccessible equipment. Herein, we develop and assess a low-cost droplet simulation model using easily accessible materials, open-source software, and a smartphone-based cobalt blue light. Methods. The simulation model was developed using commercial-grade materials and fluorescein dye. A clear face shield was assessed ten times following a simulated cough using fluorescein dye. A conventional ultraviolet Woods lamp was compared to a smartphone-based cobalt blue light to detect fluorescein illumination. Results. The simulation platform and smartphone-based cobalt blue light cost $20.18. A Wilcoxon signed rank test revealed that the median droplet area of fluorescence under the UV Wood's lamp was not significantly different than that of the smartphone-based cobalt blue light (2.89 vs 2.94, P = .386). Conclusions. This simulation model is inexpensive and easily reproducible. The smartphone application may be a convenient alternative to standard ultraviolet lights. This model has great potential for use in financially restricted academic centers during the COVID-19 pandemic and beyond.

A new fluorescent ribonucleoside alphabet (mth N) consisting of pyrimidine and purine analogues, all derived from methylthieno[3,4-d]pyrimidine as the heterocyclic core, is described. Large bathochromic shifts and high microenvironmental susceptibility of their emission relative to previous alphabets derived from thieno[3,4-d]pyrimidine (th N) and isothiazole[4,3-d]pyrimidine (tz N) scaffolds are observed. Subjecting the purine analogues to adenosine deaminase, guanine deaminase and T7 RNA polymerase indicate that, while varying, all but one enzyme tolerate the corresponding mth N/mth NTP substrates. The robust emission quantum yields, high photophysical responsiveness and enzymatic accommodation suggest that the mth N alphabet is a biophysically viable tool and can be used to probe the tolerance of nucleoside/tide-processing enzymes to structural perturbations of their substrates.

Mapping the localization of multiple proteins in their native three-dimensional (3D) context would be useful across many areas of biomedicine, but multiplexed fluorescence imaging has limited intrinsic multiplexing capability, and most methods for increasing multiplexity can only be applied to thin samples (

The aim of this study is to evaluate the color stability, translucency and hardness of eight different composite resins, after thermal cycling and immersion in beverages. Six light-cured dimethacrylate-based composite (Clearfil Majesty Esthetic, Clearfil Majesty Posterior, Grandio, Grandio Flow, Arabesk Top, Valux Plus), an ormocer (Admira) and a silorane-based composite (Filtek Silorane) were tested in this study. Fifteen samples (8x2 mm) from each composite were prepared. All samples were subjected to thermal cycling [(5.0 ± 0.5)~(55.0 ± 1.0) °C, 1000 cycles]. After thermal cycling, fifteen specimens in each group were divided into three subgroups (n = 5): distilled water (control), coffee, and red wine at 37°C for 30 days. With a spectrophotometer, the △E and Translucency Parameter (TP) were calculated against white and black backgrounds. Color, translucency and hardness of all samples were remeasured baseline, after thermal cycles and after immersion in beverages. The data were analyzed using repeated measured analysis of variance and Bonferroni multiple-comparison test (P3.3). The effect of thermal cycles on color and transluceny was not statistically significant (P>0.05). TP values generally decreased especially after immersion in red wine (P

Applying natural food additives extracted from plants is a sustainable development trend in the food industry. Compared with synthetic food additives, plant-based food additives have garnered considerable attention owing to their advantages such as green safety, health, and environmental protection. Therefore, the development and utilization of plant-based food additives is becoming the most active field and future direction for the development of food industry. However, there are several problems associated with large-scale production and application, and more studies are required to evaluate their potential side effects and safety level. In addition, it is necessary to adopt multiple technologies to overcome the shortcomings of natural additives and develop plant-based composite food additives and functional food additives to industrialize natural resources and maximize benefits. This review focuses on the grouping of food additives according to the scope of Food and Drug Administration, including preservation, flavoring agents, coloring agents, texturing agents, nutritional additives, and miscellaneous agents. The application, safety, and major challenges of typical natural food additives extracted from plants are discussed. In addition, it provides a theoretical basis for the development of safer and effective plant-based food additives in the future. [ABSTRACT FROM AUTHOR]

PurposeTo characterize the association of reticular pseudodrusen (RPD) with late-onset retinal degeneration (L-ORD) using multimodal imaging.DesignProspective, 2-center, longitudinal case series.ParticipantsTwenty-nine patients with L-ORD.MethodsAll patients were evaluated within a 3-year interval with near-infrared reflectance, fundus autofluorescence, and spectral-domain OCT. In addition, a subset of patients also underwent indocyanine green angiography, fundus fluorescein angiography, mesopic microperimetry, and multifocal electroretinography.Main outcome measuresPrevalence, topographic distribution, and temporal phenotypic changes of RPD in L-ORD.ResultsA total of 29 patients with molecularly confirmed L-ORD were included in this prospective study. Reticular pseudodrusen was detected in 18 patients (62%) at baseline, 10 of whom were men. The prevalence of RPD varied with age. The mean age of RPD patients was 57.3 ± 7.2 years. Reticular pseudodrusen was not seen in patients younger than the fifth decade of life (n = 3 patients) or in the eighth decade of life (n = 5 patients). Reticular pseudodrusen were found commonly in the macula with relative sparing of the fovea and also were identified in the peripheral retina. The morphologic features of RPD changed with follow-up. Two patients (3 eyes) demonstrated RPD regression.ConclusionsReticular pseudodrusen is found frequently in patients with L-ORD and at a younger age than in individuals with age-related macular degeneration (AMD). Reticular pseudodrusen exhibits quick formation and collapse, change in type and morphologic features with time, and relative foveal sparing and also has a peripheral retinal location in L-ORD.

The worldwide coronavirus disease 2019 pandemic has had devastating effects on health, healthcare infrastructure, social structure, and economics. One of the limiting factors in containing the spread of this virus has been the lack of widespread availability of fast, inexpensive, and reliable methods for testing of individuals. Frequent screening for infected and often asymptomatic people is a cornerstone of pandemic management plans. Here, we introduce 2 pH-sensitive "LAMPshade" dyes as novel readouts in an isothermal Reverse Transcriptase Loop-mediated isothermal AMPlification amplification assay for severe acute respiratory syndrome coronavirus 2 RNA. The resulting JaneliaLAMP assay is robust, simple, inexpensive, and has low technical requirements, and we describe its use and performance in direct testing of contrived and clinical samples without RNA extraction.

Pathology is practiced by visual inspection of histochemically stained tissue slides. While the hematoxylin and eosin (H&E) stain is most commonly used, special stains can provide additional contrast to different tissue components. Here, we demonstrate the utility of supervised learning-based computational stain transformation from H&E to special stains (Masson's Trichrome, periodic acid-Schiff and Jones silver stain) using kidney needle core biopsy tissue sections. Based on the evaluation by three renal pathologists, followed by adjudication by a fourth pathologist, we show that the generation of virtual special stains from existing H&E images improves the diagnosis of several non-neoplastic kidney diseases, sampled from 58 unique subjects (P = 0.0095). A second study found that the quality of the computationally generated special stains was statistically equivalent to those which were histochemically stained. This stain-to-stain transformation framework can improve preliminary diagnoses when additional special stains are needed, also providing significant savings in time and cost.

Growing evidence suggests that many essential intracellular signaling events are compartmentalized within kinetically distinct microdomains in cells. Genetically encoded fluorescent biosensors are powerful tools to dissect compartmentalized signaling, but current approaches to probe these microdomains typically rely on biosensor fusion and overexpression of critical regulatory elements. Here, we present a novel class of biosensors named FluoSTEPs (fluorescent sensors targeted to endogenous proteins) that combine self-complementing split green fluorescent protein, CRISPR-mediated knock-in, and fluorescence resonance energy transfer biosensor technology to probe compartmentalized signaling dynamics in situ. We designed FluoSTEPs for simultaneously highlighting endogenous microdomains and reporting domain-specific, real-time signaling events including kinase activities, guanosine triphosphatase activation, and second messenger dynamics in live cells. A FluoSTEP for 3,5-cyclic adenosine monophosphate (cAMP) revealed distinct cAMP dynamics within clathrin microdomains in response to stimulation of G protein-coupled receptors, showcasing the utility of FluoSTEPs in probing spatiotemporal regulation within endogenous signaling architectures.

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Introduction: Denture cleansers effectively reduce plaque formation; nonetheless, there is a dearth of studies on their efficiency in stain removal and the reduction of multispecies biofilms. The present study aimed to determine the ability of different denture cleansers to remove different stains. Methods: A total of 90 central incisors with A1 color were randomly assigned to three groups. Specimens were immersed into three staining solutions (black tea, coffee, and wine). After staining, colorimetry measurements were made, and specimens were immersed in denture cleansers (Protefix Active Cleanser and Aktident Cleansing Tablet) or tap water (control). Color difference (ΔE*ab) was determined after two, four, and six months (n=10). Data were statistically analyzed using the Kruskal-Wallis analysis and Dunn's multiple comparison test at the %95 confidence level. Results: There was no significant difference between the stain removal efficacy of denture cleansers and the control group for all staining solutions. When the ΔE*ab values were compared according to different time periods, specimens exposed to three different staining solutions and cleaned by denture cleaners and tap water showed no significant difference (P>0.05). In all time periods, teeth exposed to tea and immersed in the Protefix demonstrated higher color difference values (ΔE*ab>5.5). Color differences of all groups were perceptible to the eye (ΔE*ab>1.8). There were no significant differences among denture cleansers in different time periods (P>0.05). Conclusion: There was no significant difference among the denture cleansers regarding stain removal capacity. The soaking time of stained artificial teeth in denture cleansers did not change their color.

The trinuclear ruthenium amine ruthenium red (RuR) inhibits diverse ion channels, including K2P potassium channels, TRPs, the calcium uniporter, CALHMs, ryanodine receptors, and Piezos. Despite this extraordinary array, there is limited information for how RuR engages targets. Here, using X-ray crystallographic and electrophysiological studies of an RuR-sensitive K2P, K2P2.1 (TREK-1) I110D, we show that RuR acts by binding an acidic residue pair comprising the Keystone inhibitor site under the K2P CAP domain archway above the channel pore. We further establish that Ru360, a dinuclear ruthenium amine not known to affect K2Ps, inhibits RuR-sensitive K2Ps using the same mechanism. Structural knowledge enabled a generalizable design strategy for creating K2P RuR super-responders having nanomolar sensitivity. Together, the data define a finger in the dam inhibition mechanism acting at a novel K2P inhibitor binding site. These findings highlight the polysite nature of K2P pharmacology and provide a new framework for K2P inhibitor development.

Excited state proton transfer (ESPT) is thought to be responsible for the photostability of biological molecules, including DNA and proteins, and natural dyes such as indigo. However, the mechanistic role of the solvent interaction in driving ESPT is not well understood. Here, the electronic excited state deactivation dynamics of indigo carmine (InC) is mapped by visible pump-infrared probe and two-dimensional electronic-vibrational (2DEV) spectroscopy and complemented by electronic structure calculations. The observed dynamics reveal notable differences between InC in a protic solvent, D2O, and an aprotic solvent, deuterated dimethyl sulfoxide (dDMSO). Notably, an acceleration in the excited state decay is observed in D2O (

We present an autocatalytic system for the detection and amplification of thiols termed the Methionase Chain Reaction (MCR). MCR is based on the reversible modification of the thiol producing enzyme Methionine Gamma-Lyase (MGL). MCR was able to amplify the concentration of thiols by a factor of 560 and was able to visually detect thiols at concentrations as low as 50 nM.

Mechanistic-understanding-based selection of excipients may improve formulation development strategies for generic drug products and potentially accelerate their approval. Our study aimed at investigating the effects of molecular excipients present in orally administered FDA-approved drug products on the intestinal efflux transporter, BCRP (ABCG2), which plays a critical role in drug absorption with potential implications on drug safety and efficacy. We determined the interactions of 136 oral molecular excipients with BCRP in isolated membrane vesicles and identified 26 excipients as BCRP inhibitors with IC50 values less than 5 μM using 3H-cholecystokinin octapeptide (3H-CCK8). These BCRP inhibitors belonged to three functional categories of excipients: dyes, surfactants, and flavoring agents. Compared with noninhibitors, BCRP inhibitors had significantly higher molecular weights and SLogP values. The inhibitory effects of excipients identified in membrane vesicles were also evaluated in BCRP-overexpressing HEK293 cells at similar concentrations. Only 1 of the 26 inhibitors of BCRP identified in vesicles inhibited BCRP-mediated 3H-oxypurinol uptake by more than 50%, consistent with the notion that BCRP inhibition depends on transmembrane or intracellular availability of the inhibitors. Collectively, the results of this study provide new information on excipient selection during the development of drug products with active pharmaceutical ingredients that are BCRP substrates.

The ability to predict when an individual will die can be extremely useful for many research problems in aging. A technique for predicting death in the model organism, Drosophila melanogaster, has been proposed which relies on an increase in the permeability of the fly intestinal system, allowing dyes from the diet to permeate the body of the fly shortly before death. In this study we sought to verify this claim in a large cohort study using different populations of D. melanogaster and different dyes. We found that only about 50% of the individuals showed a visible distribution of dye before death. This number did not vary substantially with the dye used. Most flies that did turn a blue color before death did so within 24 hours of death. There was also a measurable effect of the dye on the fly mean longevity. These results would tend to limit the utility of this method depending on the application the method was intended for.

PurposeTo perform a comprehensive clinical, diagnostic, and imaging characterization of the ocular surface in West Highland White Terriers (WHWTs) diagnosed with aqueous deficient dry eye (ADDE) disease.MethodsSix ADDE-affected and 13 ADDE-unaffected WHWT dogs were enrolled and underwent clinical assessment and disease scoring, tear osmolarity, phenol red thread test, Schirmer tear test, tear film breakup time, fluorescein staining, Rose bengal and lissamine green vital dye staining, meibometry, corneal esthesiometry, ultrasound pachymetry, optical coherence tomography, in vivo confocal microscopy, and conjunctival biopsy. Subjective assessment of their condition was provided by owner-reported surveys.ResultsADDE-affected WHWT dogs had higher median clinical disease (conjunctiva: 5.75 vs. 0.00; cornea: 14.00 vs. 5.00; total: 17.50 vs. 5.00), vital staining (Rose bengal: 2.25 vs. 1.50; lissamine green: 2.00 vs. 1.00), and histologic disease (conjunctiva: 2 vs. 0) scores when compared with the controls. In addition, ADDE-affected WHWTs had significantly lower phenol red thread test (5.0 vs. 17.5, mm/15 s), Schirmer tear test (3 vs. 20, mm/min), tear film breakup time (3.6 vs. 13.9, s) values and higher area under the curve values for meibometry (394 vs. 245, meibometry units [MU]). There were no significant differences in other tear film tests performed. Advanced imaging revealed decreased tear meniscus height (optical coherence tomography) and variable pigment deposition within corneal epithelial cells (in vivo confocal microscopy).ConclusionsThis comprehensive assessment of ADDE-affected WHWTs depicts the ocular surface changes associated with quantitative lacrimal gland dysfunction. Importantly, ADDE-affected WHWTs may prove a valuable naturally occurring ADDE model for investigating underlying pathophysiological mechanisms and the development of novel therapeutics.

We introduce a method for quantitative hyperspectral optical imaging in the spatial frequency domain (hs-SFDI) to image tissue absorption (μa) and reduced scattering (μs') parameters over a broad spectral range. The hs-SFDI utilizes principles of spatial scanning of the spectrally dispersed output of a supercontinuum laser that is sinusoidally projected onto the tissue using a digital micromirror device. A scientific complementary metal-oxide-semiconductor camera is used for capturing images that are demodulated and analyzed using SFDI computational models. The hs-SFDI performance is validated using tissue-simulating phantoms over a range of μa and μs' values. Quantitative hs-SFDI images are obtained from an ex-vivo beef sample to spatially resolve concentrations of oxy-, deoxy-, and met-hemoglobin, as well as water and fat fractions. Our results demonstrate that the hs-SFDI can quantitatively image tissue optical properties with 1000 spectral bins in the 580- to 950-nm range over a wide, scalable field of view. With an average accuracy of 6.7% and 12.3% in μa and μs', respectively, compared to conventional methods, hs-SFDI offers a promising approach for quantitative hyperspectral tissue optical imaging.

Systemic amyloidosis is a devastating group of disorders for which there is no current cure. The treatment goal is to reduce the burden of amyloidogenic protein precursors. The treatment is only effective if applied early in the disease process before significant and irreversible end organ damage has taken place. Congo red is still the standard stain used in most histopathology laboratories to identify amyloid material in tissues. The identification of Congophilic amyloid material is challenging because of multiple interfering factors. Here we describe improved sensitivity of identifying Congophilic materials in histologic sections using a metallurgical polarized microscope specifically constructed for polarized microscopy. The microscope is equipped with strain-free optics, matching polarizers, dis-integrated compensators, and a circular mobile stage. Compared to a standard clinical microscope, this setup significantly improves sensitivity of identifying amyloid material in Congo red-stained slides. We also describe the deleterious effect of plastic coverslip which can interfere with the ability to examine the slides under polarized light. We present a series of 10 different patients who had cardiac, brain, and salivary gland biopsies that were either equivocal or deemed negative using a standard clinical microscope but were positive using the equipment described above. These samples were confirmed to be positive by other methods including electron microscopy. We conclude that use of the correct equipment is needed before ruling out amyloidosis in tissue sections.

Normalization of Western blotting data is a critical step that is needed to reduce errors caused by unequal sample loading across lanes in a gel, inconsistent sample preparation, and variations due to experimental errors. Several papers have suggested that total protein normalization may be better than housekeeping protein normalization for Western blotting normalization. Ponceau S is the most commonly used stain for total protein normalization. A review of the literature and commercial websites suggest a multitude of Ponceau S staining protocols for total protein staining of blots. In this study, we explored which Ponceau S staining protocol would result in the highest sensitivity of protein band detection. Unexpectedly, we found that irrespective of the Ponceau S concentration (between 0.001 and 2% (w/v)), acid concentration, and acid type (acetic acid, trichloroacetic acid and/or sulfosalicylic acid), the sensitivity of protein detection remained constant. The most commonly used concentration of Ponceau S is 0.1%, while 0.001% (100-fold less) Ponceau S resulted in the same sensitivity of protein band detection. We suggest the use of the relatively inexpensive 0.01% Ponceau S in 1% acetic acid stain for total protein normalization as it is as effective as all the expensive formulations that are currently used.

Keratopigmentation was first recorded many centuries ago by the philosopher Galeno, as a strategy used for the aesthetic treatment of patients with leukomas. Corneas with leucoma are pathological and often intolerant of cosmetic contact lenses or ocular prostheses, with complaints of excessive discomfort provided by the irregular corneal surface being common. Therefore, keratopigmentation is an alternative for the aesthetic improvement of patients with corneal opacities. We describe the case of a 39-year old male patient, who presented early depigmentation in a case of keratopigmentation associated with necrotizing herpetic keratitis. The patient was treated with Acyclovir 2g/day and Doxycycline 200mg/day, evolving with clinical improvement, despite poor medication adherence. [ABSTRACT FROM AUTHOR]

Objectives: The study aimed to compare the color stability of two different light-cured composites after immersion in three liquids and the effectiveness of 16% carbamide peroxide (CP) in removing the discoloration. Material and methods: Color stability of a microhybrid (Z250, 3M ESPE) and nanocomposite (Z550, 3M ESPE) was evaluated after immersion in instant coffee, tea, Coca-Cola, and deionized water as a control group (n=5). Samples were kept in liquids for four hours daily at 37°C for 30 days. Furthermore, 16% CP was applied for the following 14 days, simulating night whitening. A digital spectrophotometer was used for color measurement based on the CIEL*a*b* color coordinates. The color changes (∆E) were measured at baseline, after immersion in the beverages, and also after the teeth whitening procedure. Mixed and factorial ANOVA followed by Bonferroni’s posthoc test were used for statistical evaluation (p≤0.05). Results: Tested resin composites showed a color change over the acceptability threshold (ΔE*> 3.48) after immersion in coffee and tea. Nanocomposite reported a significant increase in discoloration in coffee after 30 days (p

BackgroundA low adenoma detection rate (ADR) increases risks of interval cancers (ICs). Proximal colon flat polyps, e.g. serrated lesions (SLs), are difficult to find. Missed proximal colon flat lesions likely contribute to IC.AimsWe compared chromoendoscopy with water exchange (CWE), water exchange (WE) and air insufflation (AI) in detecting adenomas in screening colonoscopy.MethodsAfter split-dose preparation, 480 veterans were randomized to AI, WE and CWE.ResultsPrimary outcome of proximal ADR (55.6% vs 53.4% vs 52.2%, respectively) were similar in all groups. Adenoma per colonoscopy (APC) and adenoma per positive colonoscopy (APPC) were comparable. Detection rate of proximal colon SLs was significantly higher for CWE and WE than AI (26.3%, 23.6% and 11.3%, respectively, p = 0.002). Limitations: single operator; SLs only surrogate markers of but not IC.ConclusionsWhen an endoscopist achieves high-quality AI examinations with overall ADR twice (61.6%) the recommended standard (30%), use of WE and CWE does not produce further improvement in proximal or overall ADR. Comparable APC and APPC confirm equivalent withdrawal inspection techniques. WE alone is sufficient to significantly improve detection of proximal SLs. The impact of increased detection of proximal SLs by WE on prevention of IC deserves to be studied. This study is registered at ClinicalTrial.gov (NCT#01607255).

We introduce a simple new approach for time-resolved multiplexed analysis of complex systems using near-infrared (NIR) dyes, applicable to in vitro and in vivo studies. We show that fast and precise in vitro quantification of NIR fluorophores' short (subnanosecond) lifetime and stoichiometry can be done using phasor analysis, a computationally efficient and user-friendly representation of complex fluorescence intensity decays obtained with pulsed laser excitation and time-gated camera imaging. We apply this approach to the study of binding equilibria by Förster resonant energy transfer using two different model systems: primary/secondary antibody binding in vitro and ligand/receptor binding in cell cultures. We then extend it to dynamic imaging of the pharmacokinetics of transferrin engagement with the transferrin receptor in live mice, elucidating the kinetics of differential transferrin accumulation in specific organs, straightforwardly differentiating specific from nonspecific binding. Our method, implemented in a freely-available software, has the advantage of time-resolved NIR imaging, including better tissue penetration and background-free imaging, but simplifies and considerably speeds up data processing and interpretation, while remaining quantitative. These advances make this method attractive and of broad applicability for in vitro and in vivo molecular imaging and could be extended to applications as diverse as image-guided surgery or optical tomography.

Soluble oligomers of the β-amyloid peptide, Aβ, are associated with the progression of Alzheimers disease. Although many small molecules bind to these assemblies, the details of how these molecules interact with Aβ oligomers remain unknown. This paper reports that crystal violet, and other C3 symmetric triphenylmethane dyes, bind to C3 symmetric trimers derived from Aβ17-36. Binding changes the color of the dyes from purple to blue, and causes them to fluoresce red when irradiated with green light. Job plot and analytical ultracentrifugation experiments reveal that two trimers complex with one dye molecule. Studies with several triphenylmethane dyes reveal that three N, N-dialkylamino substituents are required for complexation. Several mutant trimers, in which Phe19, Phe20, and Ile31 were mutated to cyclohexylalanine, valine, and cyclohexylglycine, were prepared to probe the triphenylmethane dye binding site. Size exclusion chromatography, SDS-PAGE, and X-ray crystallographic studies demonstrate that these mutations do not impact the structure or assembly of the triangular trimer. Fluorescence spectroscopy and analytical ultracentrifugation experiments reveal that the dye packs against an aromatic surface formed by the Phe20 side chains and is clasped by the Ile31 side chains. Docking and molecular modeling provide a working model of the complex in which the triphenylmethane dye is sandwiched between two triangular trimers. Collectively, these findings demonstrate that the X-ray crystallographic structures of triangular trimers derived from Aβ can be used to guide the discovery of ligands that bind to soluble oligomers derived from Aβ.

ABSTRACT Introduction: Composite resins are an extensively used esthetic material that has excellent adaptation, reproduces the characteristics of the tooth structure and is suitable for the conditions of the oral environment. However, this material is also very susceptible to staining due to pigmentation coming from the diet and habits of the patient. Objectives: To evaluate the staining of two brands of composite resin, one nanoparticle and a nanohybrid type (Filtek Z350XT® and IPS Empress Direct®), in direct contact with the acid pH of beverages. Methods: In total 40 specimens were made, 20 of each type of resin studied. After being inserted into a Teflon matrix and light activated, the samples were removed, stored in containers with 2 ml of artificial saliva and kept in the refrigerator. Every day 20 of these samples were placed in direct contact with 2 ml Gatorade, and the other 20 samples, in 2 ml of wine, each set for 30 minutes. After 30 minutes, the acid pH substances were removed, and 2 ml of artificial saliva was again placed in the container. This process was repeated every day for 10 days. Results: The results showed that the nanoparticle resin (Filtek Z350XT) was more susceptible to staining than the nanohybrid type (Empress Direct®) when in contact with the wine. When wine and Gatorade were compared, the solution with higher staining capacity was shown to be wine. Conclusion: The two resins tested were susceptible to staining in a 10-day period. Mottling and more staining was visibly detected after samples were immersed in the solution with the higher pH, and nanoparticulate resin was more susceptible to staining.

Purpose: To describe the first reported case of outer retinal damage following the use of Membrane Blue Dual for epiretinal membrane (ERM) surgery. Observations: A 74-year-old female underwent pars plana vitrectomy and ERM peeling assisted with Membrane Blue Dual for an idiopathic ERM. Postoperatively, the patient reported a decline in visual acuity with a central scotoma. Fundus examination revealed a well-defined retinal whitening in the peeling area which evolved into pigmentary changes as confirmed by fundus autofluorescence. Optical coherence tomography (OCT) showed loss of outer retinal layers and irregular mottling of the retinal pigment epithelium. Fundus and OCT appearance remained unchanged after 4 months and the central scotoma also persisted. Conclusions and Importance: ERM surgery assisted with Membrane Blue Dual can induce major changes in retinal pigment epithelium and outer retinal layers. This adverse event which probably results from combined light and dye toxicity should be considered by all surgeons even though its occurrence is rare.

Previously, we demonstrated that temperature-modulated fluorescence tomography (TM-FT) could provide fluorescence images with high quantitative accuracy and the spatial resolution of focused ultrasound. TM-FT is based on scanning the focused ultrasound across the medium to activate temperature-reversible fluorescent nanoprobes (ThermoDots). This technique can resolve small fluorescent targets located several centimeters deep in turbid media with millimeter resolution. Our past studies with this multimodality technique used agar phantoms, which could not represent the true heterogeneous nature of the acoustic and optical properties of biological tissue. In this work, we report the results of the first TM-FT study performed on ex vivo chicken breast tissue. In order to improve the spatial resolution of this technique, diffuse optical tomography is also used to better estimate the optical property maps of the tissue, which is utilized as functional a priori for the TM-FT reconstruction algorithm. These ex vivo results show that TM-FT can accurately recover the concentration and position of a 1.5  mm×5  mm inclusion filled with ThermoDots. Since the inclusion is embedded 2 cm deep in the chicken breast sample, these results demonstrate the great potential of TM-FT for future in vivo small animal imaging.

PURPOSE: To evaluate the feasibility of safely performing aqueous angiography in intact eyes of living nonhuman primates (NHPs) for evaluating aqueous humor outflow and segmental patterns. DESIGN: Cross-sectional, observational study. SUBJECTS: Six nonhuman primates. METHODS: Aqueous angiography was performed in 6 nonhuman primates. After anesthesia, an anterior chamber (AC) maintainer was placed through a temporal 1-mm side-port wound. Indocyanine green (ICG; 0.4%) or 2.5% fluorescein was introduced (individually or in sequence) into the eye with a gravity-driven constant-pressure system. Aqueous angiography images were obtained with a Spectralis HRA+OCT (Heidelberg Engineering GmbH, Heidelberg, Germany) suspended over the NHP eye using a custom-designed surgical boom arm. Concurrent anterior segment optical coherence tomography (OCT) was performed on distally angiographically positive and negative regions. MAIN OUTCOME MEASURES: Angiographic patterns described by location, time-course, choice of tracer, and anterior-segment OCT. RESULTS: Aqueous angiography in the living NHP eye demonstrated mostly stable angiographic patterns. With multimodal imaging, angiographically positive signal co-localized with episcleral veins as identified by infrared imaging and intrascleral lumens, as demonstrated by anterior segment OCT. Sequential aqueous angiography in individual eyes with ICG followed by fluorescein showed similar angiographic patterns. A pulsatile nature of aqueous angiographic outflow was sometimes observed. Aqueous angiographic patterns could also dynamically change. In some instances, positive angiographic flow suddenly arose in regions previously without an angiographic signal. Alternatively, an angiographic signal could suddenly disappear from regions in which an angiographic signal was initially documented. CONCLUSIONS: Aqueous angiography in living NHPs demonstrated segmental and pulsatile patterns with a newly described ability to dynamically shift. These characteristics further the understanding of live aqueous humor outflow biology and may be useful in improving glaucoma surgeries aimed at trabecular meshwork bypass.