PAPipe: A Pipeline for Comprehensive Population Genetic Analysis.

Advancements in next-generation sequencing (NGS) technologies have led to a substantial increase in the availability of population genetic variant data, thus prompting the development of various population analysis tools to enhance our understanding of population structure and evolution. The tools that are currently used to analyze population genetic variant data generally require different environments, parameters, and formats of the input data, which can act as a barrier preventing the wide-spread usage of such tools by general researchers who may not be familiar with bioinformatics. To address this problem, we have developed an automated and comprehensive pipeline called PAPipe to perform nine widely used population genetic analyses using population NGS data. PAPipe seamlessly interconnects and serializes multiple steps, such as read trimming and mapping, genetic variant calling, data filtering, and format converting, along with nine population genetic analyses such as principal component analysis, phylogenetic analysis, population tree analysis, population structure analysis, linkage disequilibrium decay analysis, selective sweep analysis, population admixture analysis, sequentially Markovian coalescent analysis, and fixation index analysis. PAPipe also provides an easy-to-use web interface that allows for the parameters to be set and the analysis results to be browsed in intuitive manner. PAPipe can be used to generate extensive results that provide insights that can help enhance user convenience and data usability. PAPipe is freely available at https://github.com/jkimlab/PAPipe.

Identification and characterization of structural variants related to meat quality in pigs using chromosome-level genome assemblies.

BACKGROUND: Many studies have been performed to identify various genomic loci and genes associated with the meat quality in pigs. However, the full genetic architecture of the trait still remains unclear in part because of the lack of accurate identification of related structural variations (SVs) which resulted from the shortage of target breeds, the limitations of sequencing data, and the incompleteness of genome assemblies. The recent generation of a new pig breed with superior meat quality, called Nanchukmacdon, and its chromosome-level genome assembly (the NCMD assembly) has provided new opportunities. RESULTS: By applying assembly-based SV calling approaches to various genome assemblies of pigs including Nanchukmacdon, the impact of SVs on meat quality was investigated. Especially, by checking the commonality of SVs with other pig breeds, a total of 13,819 Nanchukmacdon-specific SVs (NSVs) were identified, which have a potential effect on the unique meat quality of Nanchukmacdon. The regulatory potentials of NSVs for the expression of nearby genes were further examined using transcriptome- and epigenome-based analyses in different tissues. CONCLUSIONS: Whole-genome comparisons based on chromosome-level genome assemblies have led to the discovery of SVs affecting meat quality in pigs, and their regulatory potentials were analyzed. The identified NSVs will provide new insights regarding genetic architectures underlying the meat quality in pigs. Finally, this study confirms the utility of chromosome-level genome assemblies and multi-omics analysis to enhance the understanding of unique phenotypes.

msPIPE: a pipeline for the analysis and visualization of whole-genome bisulfite sequencing data.

BACKGROUND: DNA methylation is an important epigenetic modification that is known to regulate gene expression. Whole-genome bisulfite sequencing (WGBS) is a powerful method for studying cytosine methylation in a whole genome. However, it is difficult to obtain methylation profiles using the WGBS raw reads and is necessary to be proficient in all types of bioinformatic tools for the study of DNA methylation. In addition, recent end-to-end pipelines for DNA methylation analyses are not sufficient for addressing those difficulties. RESULTS: Here we present msPIPE, a pipeline for DNA methylation analyses with WGBS data seamlessly connecting all the required tasks ranging from data pre-processing to multiple downstream DNA methylation analyses. The msPIPE can generate various methylation profiles to analyze methylation patterns in the given sample, including statistical summaries and methylation levels. Also, the methylation levels in the functional regions of a genome are computed with proper annotation. The results of methylation profiles, hypomethylation, and differential methylation analysis are plotted in publication-quality figures. The msPIPE can be easily and conveniently used with a Docker image, which includes all dependent packages and software related to DNA methylation analyses. CONCLUSION: msPIPE is a new end-to-end pipeline designed for methylation calling, profiling, and various types of downstream DNA methylation analyses, leading to the creation of publication-quality figures. msPIPE allows researchers to process and analyze the WGBS data in an easy and convenient way. It is available at https://github.com/jkimlab/msPIPE and https://hub.docker.com/r/jkimlab/mspipe .

Aggregation and gelation in a tunable aqueous colloid-polymer bridging system.

We report a colloid-polymer model system with tunable bridging interactions for microscopic studies of structure and dynamics using confocal imaging. The interactions between trifluoroethyl methacrylate-co-tert-butyl methacrylate copolymer particles and poly(acrylic acid) (PAA) polymers were controllable via polymer concentration and pH. The strength of adsorption of PAA on the particles, driven by pH-dependent interactions with polymer brush stabilizers on the particle surfaces, was tuned via solution pH. Particle-polymer suspensions formulated at low pH, where polymers strongly adsorbed to the particles, contained clusters or weak gels at particle volume fractions of ϕ = 0.15 and ϕ = 0.40. At high pH, where the PAA only weakly adsorbed to the particle surface, particles largely remained dispersed, and the suspensions behaved as a dense fluid. The ability to visualize the suspension structure is likely to provide insight into the role of polymer-driven bridging interactions in the behavior of colloidal suspensions.

Lobeglitazone attenuates fibrosis in corneal fibroblasts by interrupting TGF-beta-mediated Smad signaling.

PURPOSE: Transforming growth factor beta 1 (TGF-ß1) is an important cytokine released after ocular surface injury to promote wound healing. However, its persistence at the injury site triggers a fibrotic response that leads to corneal scarring and opacity. Thiazolidinediones (TZDs) are synthetic peroxisome proliferator-activated receptor gamma (PPAR-γ) ligands used to regulate glucose and lipid metabolism in the management of type 2 diabetes. Studies have also showed TZDs have antifibrotic effect. In this study, we investigated the antifibrotic effect of the TZD lobeglitazone on TGF-ß1-induced fibrosis in corneal fibroblasts. METHODS: Human primary corneal fibroblasts were cultivated and treated with TGF-ß1 (5 ng/mL) to induce fibrosis, with or without pre-treatments with different concentrations of lobeglitazone. Myofibroblast differentiation and extracellular matrix (ECM) protein expression was evaluated by western blotting, immunofluorescence, real-time PCR, and collagen gel contraction assay. The effect of lobeglitazone on TGF-ß1-induced reactive oxygen species (ROS) generation was evaluated by DCFDA-cellular ROS detection assay kit. Signaling proteins were evaluated by western blotting to determine the mechanism underlying the antifibrotic effect. RESULTS: Our results showed lobeglitazone attenuated TGF-ß1-induced ECM synthesis and myofibroblast differentiation of corneal fibroblasts. This antifibrotic effect appeared to be independent of PPAR signaling and rather due to the inhibition of the TGF-ß1-induced Smad signaling. Lobeglitazone also blocked TGF-ß1-induced ROS generation and nicotinamide adenine dinucleotide phosphate oxidase (Nox) 4 transcription. CONCLUSION: These findings indicate that lobeglitazone may be a promising therapeutic agent for corneal scarring. KEY MESSAGES.

Farnesol Ameliorates Demyelinating Phenotype in a Cellular and Animal Model of Charcot-Marie-Tooth Disease Type 1A.

Charcot-Marie-Tooth disease (CMT) is a genetically heterogeneous disease affecting the peripheral nervous system that is caused by either the demyelination of Schwann cells or degeneration of the peripheral axon. Currently, there are no treatment options to improve the degeneration of peripheral nerves in CMT patients. In this research, we assessed the potency of farnesol for improving the demyelinating phenotype using an animal model of CMT type 1A. In vitro treatment with farnesol facilitated myelin gene expression and ameliorated the myelination defect caused by PMP22 overexpression, the major causative gene in CMT. In vivo administration of farnesol enhanced the peripheral neuropathic phenotype, as shown by rotarod performance in a mouse model of CMT1A. Electrophysiologically, farnesol-administered CMT1A mice exhibited increased motor nerve conduction velocity and compound muscle action potential compared with control mice. The number and diameter of myelinated axons were also increased by farnesol treatment. The expression level of myelin protein zero (MPZ) was increased, while that of the demyelination marker, neural cell adhesion molecule (NCAM), was reduced by farnesol administration. These data imply that farnesol is efficacious in ameliorating the demyelinating phenotype of CMT, and further elucidation of the underlying mechanisms of farnesol's effect on myelination might provide a potent therapeutic strategy for the demyelinating type of CMT.

Tissue-specific roles of GCN2 in aging and autosomal dominant retinitis pigmentosa.

The organisms have the capacity to sense and adapt to their surroundings for their life in a dynamic environment. In response to amino acid starvation, cells activate a rectifying physiological program, termed the integrated stress response (ISR), to restore cellular homeostasis. General controlled non-repressed (GCN2) kinase is a master regulator of the ISR and modulates protein synthesis in response to amino acid starvation. We previously established the GCN2/ATF4/4E-BP pathway in development and aging. Here, we investigated the tissue-specific roles of GCN2 upon dietary restriction of amino acid in a Drosophila model. The knockdown of GCN2 in the gut and fat body, an energy sensing organ in Drosophila, abolished the beneficial effect of GCN2 in lifespan extension upon dietary restriction of amino acids. Proteome analysis in an autosomal dominant retinitis pigmentosa (ADRP) model showed that dietary restriction of amino acids regulates the synthesis of proteins in several pathways, including mitochondrial translation, mitochondrial gene expression, and regulation of biological quality, and that gcn2-mutant flies have reduced levels of these mitochondria-associated proteins, which may contribute to retinal degeneration in ADRP. These results indicate that the tissue-specific regulation of GCN2 contributes to normal physiology and ADRP progression.

Cyclo(Phe-Pro) produced by Vibrio species passes through biological membranes by simple diffusion.

Cyclo(Phe-Pro) (cFP), produced by the Vibrio species, plays the dual roles of being a signaling molecule and a virulence factor. Acting modes of this compound have recently been characterized at the molecular level. Nevertheless, the method by which this compound passes across biological membranes remains obscure. Using radiolabeled cFP, we examined the kinetics of transport for this compound across membranes using V. vulnificus, Escherichia coli, and sheep red blood cells. We observed that cFP was taken up by these cells in a concentration-dependent manner and was not affected by the addition of the proton ionophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP), suggesting that cFP is taken up by passive transport. The kinetics of uptake of cFP by the above three types of cells revealed no significant differences, indicating that no specific protein is involved in this process. When the intracellular accumulation of cFP in the tested cells was measured, the concentrations did not exhibit significant differences between the 1-min and 10-min time points after cFP was added to the culture. In contrast, the intracellular concentration of fumarate, which is well known to be taken up by cells via active transport, was significantly higher at the 10-min than at the 1-min time point after addition. Taken together, this study shows that cFP is a diffusible molecule that does not require energy for transportation across biological membranes, and that cFP does not need membrane machinery in order to cross membranes and consequently act as a virulence factor or signal. KEY POINTS: • Kinetics of cFP uptake into cells of V. vulnificus, E. coli, or RBS was studied. • The uptake was not saturated and required no energy, indicating passive transport. • The lack of cell specificity in cFP uptake means no specific protein is needed. • Therefore, the cFP moves across the biological membrane by simple diffusion.

Contact Networks Enhance Shear Thickening in Attractive Colloid-Polymer Mixtures.

Increased shear thinning arising due to strong attractive interactions between colloidal particles is thought to obscure shear thickening. Here, we demonstrate how moderate attractions, induced by adding a nonadsorbing polymer, can instead enhance shear thickening. We measure the rheology of colloidal suspensions at a constant particle volume fraction of ϕ=0.40 with dilute to weakly semidilute concentrations of three polyacrylamide depletants of different molecular weights. Suspensions containing large polymer exhibit increased shear thickening and positive first normal stress differences at high shear stress, and increased heterogeneous fluctuations in the boundary stress. These results are consistent with a friction-based model for shear thickening, suggesting that the presence of large, extended polymers induces the formation of near-spanning networks of interparticle contacts.

Cyclo-(l-Phe-l-Pro), a Quorum-Sensing Signal of Vibrio vulnificus, Induces Expression of Hydroperoxidase through a ToxR-LeuO-HU-RpoS Signaling Pathway To Confer Resistance against Oxidative Stress.

Vibrio vulnificus, an opportunistic human pathogen, produces cyclo-(l-Phe-l-Pro) (cFP), which serves as a signaling molecule controlling the ToxR-dependent expression of innate bacterial genes, and also as a virulence factor eliciting pathogenic effects on human cells by enhancing intracellular reactive oxygen species levels. We found that cFP facilitated the protection of V. vulnificus against hydrogen peroxide. At a concentration of 1 mM, cFP enhanced the level of the transcriptional regulator RpoS, which in turn induced expression of katG, encoding hydroperoxidase I, an enzyme that detoxifies H2O2 to overcome oxidative stress. We found that cFP upregulated the transcription of the histone-like proteins vHUα and vHUß through the cFP-dependent regulator LeuO. LeuO binds directly to upstream regions of vhuA and vhuB to enhance transcription. vHUα and vHUß then enhance the level of RpoS posttranscriptionally by stabilizing the mRNA. This cFP-mediated ToxR-LeuO-vHUαß-RpoS pathway also upregulates genes known to be members of the RpoS regulon, suggesting that cFP acts as a cue for the signaling pathway responsible for both the RpoS and the LeuO regulons. Taken together, this study shows that cFP plays an important role as a virulence factor, as well as a signal for the protection of the cognate pathogen.

Vaccinia-related kinase 3 (VRK3) sets the circadian period and amplitude by affecting the subcellular localization of clock proteins in mammalian cells.

In the eukaryotic circadian clock machinery, negative feedback repression of CLOCK (CLK) and BMAL1 transcriptional activity by PERIOD (PER) and CRYPTOCHROME (CRY) underlies the basis for 24 h rhythmic gene expression. Thus, precise regulation of the time-dependent nuclear entry of circadian repressors is crucial to generating normal circadian rhythms. Here, we sought to identify novel kinase(s) that regulate nuclear entry of mammalian CRY1 (mCRY1) with an unbiased screening using red fluorescent protein (RFP)-tagged human kinome expression plasmids in mammalian cells. Transient expression of human vaccinia-related kinase 3 (hVRK3) reduced the nuclear presence of mCRY1. hVRK3 expression also induced alterations in the subcellular localization of other core clock proteins, including mCRY2, mPER2, and BMAL1. In contrast, the subcellular localization of mCLK was not changed. Given that singly expressed mCLK mostly resides in the cytoplasm and that nuclear localization sequence (NLS) mutation of hVRK3 attenuated the effect of hVRK3 co-expression on subcellular localization, ectopically expressed hVRK3 presumably reduces the retention of proteins in the nucleus. Finally, downregulation of hvrk3 using siRNA reduced the amplitude and lengthened the period of the cellular bioluminescence rhythm. Taken together, these data suggest that VRK3 plays a role in setting the amplitude and period length of circadian rhythms in mammalian cells.

Phase behavior of colloid-polymer depletion mixtures with unary or binary depletants.

Adding depletants to a colloidal suspension induces an attractive interparticle interaction that can be tuned to obtain desired structures or to probe phase behavior. When the depletant is not uniform in size, however, both the range and strength of the attraction become difficult to predict and hence control. We investigated the effects of depletant bidispersity on the non-equilibrium phase behavior of colloid-polymer mixtures. We added unary or binary mixtures of polystyrene as the depletant to suspensions of charged poly(methyl methacrylate) particles. The structure and dynamics of the particles were compared over three sets of samples with various mixtures of two different polystyrenes whose size varied by an order of magnitude. The structure and dynamics were nearly independent of depletant dispersity if the polymer concentration was represented as a sum of normalized concentrations of each species. Near the transition region between a fluid of clusters and an interconnected gel at intermediate volume fractions, partitioning of polymers in a binary mixture into colloid-rich and polymer-rich phase leads to a slightly different gelation pathway.

Correction: Phase behavior of colloid-polymer depletion mixtures with unary or binary depletants.

Correction for 'Phase behavior of colloid-polymer depletion mixtures with unary or binary depletants' by Nayoung Park et al., Soft Matter, 2017, 13, 2781-2792.

Vitamin E γ-Tocotrienol Inhibits Cytokine-Stimulated NF-κB Activation by Induction of Anti-Inflammatory A20 via Stress Adaptive Response Due to Modulation of Sphingolipids.

NF-κB plays a central role in pathogenesis of inflammation and cancer. Many phytochemicals, including γ-tocotrienol (γTE), a natural form of vitamin E, have been shown to inhibit NF-κB activation, but the underlying mechanism has not been identified. In this study, we show that γTE inhibited cytokine-triggered activation of NF-κB and its upstream regulator TGF-ß-activated kinase-1 in murine RAW 264.7 macrophages and primary bone marrow-derived macrophages. In these cells, γTE induced upregulation of A20, an inhibitor of NF-κB. Knockout of A20 partially diminished γTE's anti-NF-κB effect, but γTE increased another NF-κB inhibitor, Cezanne, in A20(-/-) cells. In search of the reason for A20 upregulation, we found that γTE treatment increased phosphorylation of translation initiation factor 2, IκBα, and JNK, indicating induction of endoplasmic reticulum stress. Liquid chromatography-tandem mass spectrometry analyses revealed that γTE modulated sphingolipids, including enhancement of intracellular dihydroceramides, sphingoid bases in de novo synthesis of the sphingolipid pathway. Chemical inhibition of de novo sphingolipid synthesis partially reversed γTE's induction of A20 and the anti-NF-κB effect. The importance of dihydroceramide increase is further supported by the observation that C8-dihydroceramide mimicked γTE in upregulating A20, enhancing endoplasmic reticulum stress, and attenuating TNF-triggered NF-κB activation. Our study identifies a novel anti-NF-κB mechanism where A20 is induced by stress-induced adaptive response as a result of modulation of sphingolipids, and it demonstrates an immunomodulatory role of dihydrocermides.

Vibrio vulnificus Secretes an Insulin-degrading Enzyme That Promotes Bacterial Proliferation in Vivo.

We describe a novel insulin-degrading enzyme, SidC, that contributes to the proliferation of the human bacterial pathogen Vibrio vulnificus in a mouse model. SidC is phylogenetically distinct from other known insulin-degrading enzymes and is expressed and secreted specifically during host infection. Purified SidC causes a significant decrease in serum insulin levels and an increase in blood glucose levels in mice. A comparison of mice infected with wild type V. vulnificus or an isogenic sidC-deletion strain showed that wild type bacteria proliferated to higher levels. Additionally, hyperglycemia leads to increased proliferation of V. vulnificus in diabetic mice. Consistent with these observations, the sid operon was up-regulated in response to low glucose levels through binding of the cAMP-receptor protein (CRP) complex to a region upstream of the operon. We conclude that glucose levels are important for the survival of V. vulnificus in the host, and that this pathogen uses SidC to actively manipulate host endocrine signals, making the host environment more favorable for bacterial survival and growth.

KOTMIN13, a Korean herbal medicine alleviates allergic inflammation in vivo and in vitro.

BACKGROUND: The ethanol extract of KOTMIN13, composed of Inula japonica Flowers, Trichosanthes kirilowii Semen, Peucedanum praeruptorum Radix, and Allium macrostemon Bulbs, was investigated for its anti-asthmatic and anti-allergic activities. METHODS: The anti-asthmatic effects of KOTMIN13 were evaluated on ovalbumin (OVA)-induced murine asthma model. Anti-allergic properties of KOTMIN13 in bone-marrow derived mast cells (BMMC) and passive cutaneous anaphylaxis (PCA) in vivo were also examined. RESULTS: In asthma model, KOTMIN13 effectively suppressed airway hyperresponsiveness induced by aerosolized methacholine when compared to the levels of OVA-induced mice. KOTMIN13 treatment reduced the total leukocytes, eosinophil percentage, and Th2 cytokines in the bronchoalveolar lavage fluids in OVA-induced mice. The increased levels of eotaxin and Th2 cytokines in the lung as well as serum IgE were decreased by KOTMIN13. The histological analysis shows that the increased inflammatory cell infiltration and mucus secretion were also reduced. In addition, the degranulation and leukotriene C4 production were inhibited in BMMC with IC50 values of 3.9 µg/ml and 1.7 µg/ml, respectively. Furthermore, KOTMIN13 treatment attenuated mast-mediated PCA reaction. CONCLUSIONS: These results demonstrate that KOTMIN13 has anti-asthmatic and anti-allergic effects in vivo and in vitro models.

Anti-inflammatory effects of Juncus effusus extract (JEE) on LPS-stimulated RAW 264.7 cells and edema models.

CONTEXT: Juncus effusus L. var. decipiens BUCHEN. f. leschenaultii GAY has been used in traditional medicine for the treatment of anxiety and insomnia. OBJECTIVE: The objective of this study was to evaluate the effects of ethanol extract from the pith of Juncus effusus (JEE) on anti-inflammatory activities in RAW 264.7 cells. MATERIALS AND METHODS: The production of inflammatory mediators and the underlying mechanisms using 3.1, 6.3, and 12.5 µg/mL concentrations of JEE were investigated. In addition, the topical anti-inflammatory effects of JEE (0.5, 1, and 2 mg/mL) on 12-O-tetradecanoylphorobol-13 acetate (TPA)-induced ear edema and oral administration of JEE (50, 100, and 200 mg/kg) on carrageenan-induced paw-edema were studied in mice. RESULTS: JEE reduced the release of nitric oxide (NO, IC50 value = 1.98 µg/mL), prostaglandin E2 (IC50 value = 5.5 µg/mL), and pro-inflammatory cytokines, IL-1ß (IC50 value = 4.74 µg/mL) and IL-6 (IC50 value = 20.48 µg/mL). JEE also suppressed the protein expression of inducible NO synthase and cyclooxygenase-2 in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Mechanism studies showed attenuation of LPS-induced activation of NF-κB by JEE via abrogation of IκBα degradation and a subsequent decrease in nuclear p65 level. Phosphorylation of all three MAP kinases (ERK, JNK, and p38) in LPS-stimulated RAW 264.7 cells was also suppressed in a dose-dependent manner. In acute inflammation models of mice, topical application (1 and 2 mg) and oral administration (50, 100, and 200 mg/kg) of JEE ameliorated TPA-induced ear edema and carrageenan-induced paw edema, respectively, in dose-dependent manners. DISCUSSION AND CONCLUSION: These results indicate that JEE exhibited anti-inflammatory activities by suppressing the production of inflammatory mediators in LPS-stimulated RAW 264.7 cells and by attenuating edema in mice.

Brain MRI in neuropsychiatric lupus: associations with the 1999 ACR case definitions.

The purpose of this study was to identify the characteristic magnetic resonance imaging (MRI) findings in neuropsychiatric systemic lupus erythematosus (NPSLE) and to investigate the association between MRI findings and neuropsychiatric manifestations in SLE. Brain MRIs with a diagnosis of SLE from 2002 to 2013 from three tertiary university hospitals were screened. All clinical manifestations evaluated by brain MRI were retrospectively reviewed. If the clinical manifestations were compatible with the 1999 NPSLE American College of Rheumatology (ACR) nomenclature and case definitions, the brain MRIs were assessed for the presence of white matter hyperintensities, gray matter hyperintensities, parenchymal defects, atrophy, enhancement, and abnormalities in diffusion-weighted images (DWI). The number, size, and location of each lesion were evaluated. The neuropsychiatric manifestation of each brain MRI was classified according to the 1999 ACR NPSLE case definitions. The associations between MRI findings and NPSLE manifestations were examined. In total, 219 brain MRIs with a diagnosis of SLE were screened, and 133 brain MRIs met the inclusion criteria for NPSLE. The most common MRI abnormality was white matter hyperintensities, which were observed in 76 MRIs (57.1 %). Gray matter hyperintensities were observed in 41 MRIs (30.8 %). Parenchymal defects were found in 31 MRIs (23.3 %), and atrophy was detected in 20 MRIs (15.0 %). Patients who had seizures were more associated with gray matter hyperintensities than patients with other neuropsychiatric manifestations. Patients with cerebrovascular disease were more associated with gray matter hyperintensity, parenchymal defects, and abnormal DWI than patients with other neuropsychiatric manifestations. In addition to white matter hyperintensities, which were previously known as SLE findings, we also noted the presence of gray matter hyperintensities, parenchymal defects, and abnormal DWI in a substantial portion of SLE patients, particularly in those with cerebrovascular disease or seizures.

Toward a chemical marker for inflammatory disease: a fluorescent probe for membrane-localized thioredoxin.

Thioredoxin (Trx) is a redox-active protein that plays a key role in mitigating the effects of oxidative stress. The secretion of Trx on the plasma membrane has been suggested as a distinctive feature of inflammation. However, selective monitoring of membrane-associated Trx activity has proved challenging because of the ubiquity of Trx action in cells. Here, we report a Trx-specific probe that allows visualization of Trx activity associated with the membranes via fluorescence microscopy. The ability of this probe to act as a possible screening tool for agents that modulate Trx secretion was demonstrated in HeLa cells under oxidative stress conditions and in a cellular hepatosteatosis model. Control experiments serve to confirm that the response seen for the present probe is due to Trx and that it is selective over various potentially competing metabolites, including thiol-containing small molecules and test proteins.

Mitochondria-immobilized pH-sensitive off-on fluorescent probe.

We report here a mitochondria-targetable pH-sensitive probe that allows for a quantitative measurement of mitochondrial pH changes, as well as the real-time monitoring of pH-related physiological effects in live cells. This system consists of a piperazine-linked naphthalimide as a fluorescence off-on signaling unit, a cationic triphenylphosphonium group for mitochondrial targeting, and a reactive benzyl chloride subunit for mitochondrial fixation. It operates well in a mitochondrial environment within whole cells and displays a desirable off-on fluorescence response to mitochondrial acidification. Moreover, this probe allows for the monitoring of impaired mitochondria undergoing mitophagic elimination as the result of nutrient starvation. It thus allows for the monitoring of the organelle-specific dynamics associated with the conversion between physiological and pathological states.