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Aberrant activity of the histone methyltransferase polycomb repressive complex 2 (PRC2) has been linked to several cancers, with small-molecule inhibitors of the catalytic subunit of the PRC2 enhancer of zeste homologue 2 (EZH2) being recently approved for the treatment of epithelioid sarcoma (ES) and follicular lymphoma (FL). Compounds binding to the EED subunit of PRC2 have recently emerged as allosteric inhibitors of PRC2 methyltransferase activity. In contrast to orthosteric inhibitors that target EZH2, small molecules that bind to EED retain their efficacy in EZH2 inhibitor-resistant cell lines. In this paper we disclose the discovery of potent and orally bioavailable EED ligands with good solubilities. The solubility of the EED ligands was optimized through a variety of design tactics, with the resulting compounds exhibiting in vivo efficacy in EZH2-driven tumors.

The compartmentalization of cellular function is achieved largely through the existence of membrane-bound organelles. However, recent work suggests a novel mechanism of compartmentalization mediated by membraneless structures that have liquid droplet-like properties and arise through phase separation. Cytoplasmic stress granules (SGs) are the best characterized and are induced by various stressors including arsenite, heat shock, and glucose deprivation. Current models suggest that SGs play an important role in protein homeostasis by mediating reversible translation attenuation. Protein phosphatase-1 (PP1) is a central cellular regulator responsible for most serine/threonine dephosphorylation. Here, we show that upon arsenite stress, PP1's catalytic subunit Glc7 relocalizes to punctate cytoplasmic granules. This altered localization requires PP1's recently described maturation pathway mediated by the multifunctional ATPase Cdc48 and PP1's regulatory subunit Ypi1. Glc7 relocalization is mediated by its regulatory subunit Reg1 and its target Snf1, the AMP-dependent protein kinase. Surprisingly, Glc7 granules are highly specific to arsenite and appear distinct from canonical SGs. Arsenite induces potent translational inhibition, and translational recovery is strongly dependent on Glc7, but independent of Glc7's well-established role in regulating eIF2α. These results suggest a novel form of stress-induced cytoplasmic granule and a new mode of translational control by Glc7.

Organic semiconductors have attracted increasing attention due to their low cost, solution processability, and tunable properties. Of special interest are molecules with enhanced environmental stab...

Tauopathies are a subset of neurodegenerative diseases characterized by abnormal tau inclusions. Specifically, three-repeat tau and four-repeat tau in Alzheimer’s disease, three-repeat tau in Pick’s disease (PiD) and four-repeat tau in progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) form amyloid-like fibrous structures that accumulate in neurons and/or glial cells. Amplification and cell-to-cell transmission of abnormal tau based on the prion hypothesis are believed to explain the onset and progression of tauopathies. Recent studies support not only the self-propagation of abnormal tau, but also the presence of conformationally distinct tau aggregates, namely tau strains. Cryogenic electron microscopy analyses of patient-derived tau filaments have revealed disease-specific ordered tau structures. However, it remains unclear whether the ultrastructural and biochemical properties of tau strains are inherited during the amplification of abnormal tau in the brain. In this study, we investigated template-dependent amplification of tau aggregates using a cellular model of seeded aggregation. Tau strains extracted from human tauopathies caused strain-dependent accumulation of insoluble filamentous tau in SH-SY5Y cells. The seeding activity towards full-length four-repeat tau substrate was highest in CBD-tau seeds, followed by PSP-tau and Alzheimer’s disease (AD)-tau seeds, while AD-tau seeds showed higher seeding activity than PiD-tau seeds towards three-repeat tau substrate. Abnormal tau amplified in cells inherited the ultrastructural and biochemical properties of the original seeds. These results strongly suggest that the structural differences of patient-derived tau strains underlie the diversity of tauopathies, and that seeded aggregation and filament formation mimicking the pathogenesis of sporadic tauopathy can be reproduced in cultured cells. Our results indicate that the disease-specific conformation of tau aggregates determines the tau isoform substrate that is recruited for templated amplification, and also influences the prion-like seeding activity., Tarutani et al. show that pathogenic tau seeds extracted from the brains of patients with tauopathies induce disease-specific seeded tau aggregation and filament formation in cultured cells. The results indicate that template-dependent tau aggregation is key to the development of various types of tauopathy.

Abiotic reduction of nitrite (NO2−) by Fe(II) species (i.e., chemodenitrification) has been demonstrated in a variety of natural environments and laboratory studies, and is a potentially significant source of atmospheric nitrous oxide (N2O) emissions. It is, however, unclear how chemodenitrification rates and N2O yields vary among heterogeneous Fe(II) species under similar conditions and whether abiotic reduction competes with biological NO2− reduction. Here, we measured rates of NO2− reduction and extents of N2O production by several Fe(II) species under consistent, environmentally relevant conditions (i.e., pH 7.0, bicarbonate buffer, and 0.1 mM NO2−). Nitrite reduction rates varied significantly among the heterogeneous Fe(II) species with half-lives (t1/2) ranging from as low as an hour to over two weeks following the trend of goethite/Fe(II) ∼ hematite/Fe(II) ∼ magnetites > maghemite/Fe(II) > sediment/Fe(II). Interestingly, we observed no clear trend of increasing NO2− reduction rates with higher magnetite stoichiometry (x = Fe2+/Fe3+). Nitrogen recovery as N2O also varied significantly among the Fe species ranging from 21% to 100% recovery. We further probed both chemodenitrification and biological denitrification in the absence and presence of added aqueous Fe(II) with a sediment collected from the floodplain of an agricultural watershed. While abiotic NO2− reduction by the sediment + Fe(II) was much slower than the laboratory Fe(II) species, we found near complete mass N balance during chemodenitrification, as well as evidence for both abiotic and biological NO2− reduction potentially occurring in the sediment under anoxic conditions. Our results suggest that in redox active sediments and soils both chemodenitrification and biological denitrification are likely to occur simultaneously, and that agricultural watersheds may be significant sources of N2O emissions.

The sigma 1 receptor (S1R) is widely expressed in the CNS and is mainly located on the endoplasmic reticulum. The S1R is involved in the regulation of many neurotransmission systems and, indirectly, in neurodegenerative diseases. The S1R may therefore represent an interesting neuronal biomarker in neurodegenerative diseases such as Parkinson's (PD) or Alzheimer's diseases (AD). Here we present the characterisation of the S1R-specific 18F-labelled tracer 18F-IAM6067 in two animal models and in human brain tissue. Methods: Wistar rats were used for PET-CT imaging (60 min dynamic acquisition) and metabolite analysis (1, 2, 5, 10, 20, 60 min post-injection). To verify in vivo selectivity, haloperidol, BD1047 (S1R ligand), CM398 (S2R ligand) and SB206553 (5HT2B/C antagonist) were administrated for pre-saturation studies. Excitotoxic lesions induced by intra-striatal injection of AMPA were also imaged by 18F-IAM6067 PET-CT to test the sensitivity of the methods in a well-established model of neuronal loss. Tracer brain uptake was also verified by autoradiography in rats and in a mouse model of PD (intrastriatal 6-hydroxydopamine (6-OHDA) unilateral lesion). Finally, human cortical binding was investigated by autoradiography in three groups of subjects (control subjects with Braak ≤2, and AD patients, Braak >2 & ≤4 and Braak >4 stages). Results: We demonstrate that despite rapid peripheral metabolism of 18F-IAM6067, radiolabelled metabolites were hardly detected in brain samples. Brain uptake of 18F-IAM6067 showed differences in S1R anatomical distribution, namely from high to low uptake: pons-raphe, thalamus medio-dorsal, substantia nigra, hypothalamus, cerebellum, cortical areas and striatum. Pre-saturation studies showed 79-90% blockade of the binding in all areas of the brain indicated above except with the 5HT2B/C antagonist SB206553 and S2R ligand CM398 which induced no significant blockade, indicating good specificity of 18F-IAM6067 for S1Rs. No difference between ipsi- and contralateral sides of the brain in the mouse model of PD was detected. AMPA lesion induced a significant 69% decrease in 18F-IAM6067 uptake in the globus pallidus matching the neuronal loss as measured by NeuN, but only a trend to decrease (-16%) in the caudate putamen despite a significant 91% decrease in neuronal count. Moreover, no difference in the human cortical binding was shown between AD groups and controls. Conclusion: This work shows that 18F-IAM6067 is a specific and selective S1R radiotracer. The absence or small changes in S1R detected here in animal models and human tissue warrants further investigations and suggests that S1R might not be the anticipated ideal biomarker for neuronal loss in neurodegenerative diseases such as AD and PD.

The metalloenzyme protein phosphatase 1 (PP1), which is responsible for ≥50% of all dephosphorylation reactions, is regulated by scores of regulatory proteins, including the highly conserved SDS22 protein. SDS22 has numerous diverse functions, surprisingly acting as both a PP1 inhibitor and as an activator. Here, we integrate cellular, biophysical, and crystallographic studies to address this conundrum. We discovered that SDS22 selectively binds a unique conformation of PP1 that contains a single metal (M2) at its active site, i.e., SDS22 traps metal-deficient inactive PP1. Furthermore, we showed that SDS22 dissociation is accompanied by a second metal (M1) being loaded into PP1, as free metal cannot dissociate the complex and M1-deficient mutants remain constitutively trapped by SDS22. Together, our findings reveal that M1 metal loading and loss are essential for PP1 regulation in cells, which has broad implications for PP1 maturation, activity, and holoenzyme subunit exchange.

Organic semiconductors are of interest for (opto)electronic applications due to their low cost, solution processability, and tunable properties. Recently, natural product-derived organic pigments attracted attention due to their extraordinary environmental stability and unexpectedly good optoelectronic performance, in spite of only partially conjugated molecular structure. Fungi-derived pigments are a naturally sourced, sustainable class of materials that are largely unexplored as organic semiconductor materials. We present a study of the optical and electronic properties of a fungi-derived pigment xylindein, which is secreted by the wood-staining fungi Chlorociboria aeruginosa, and its blends with poly(methyl methacrylate) (PMMA) and crystalline nanocellulose (CNC). Optical absorption spectra of xylindein revealed the presence of two tautomers whose structures and properties were established using density functional theory. Pronounced pigment aggregation in polar solvents and in films, driven by intermolecular hydrogen bonding, was also observed. The pigment exhibited high photostability, electron mobility up to 0.4 cm2/(V s) in amorphous films, and thermally activated charge transport and photoresponse with activation energies of ∼0.3 and 0.2 eV, respectively. The dark and photocurrents in xylindein:PMMA blends were comparable to those in pristine xylindein film, whereas blends with CNC exhibited lower currents due to inhomogeneous distribution of xylindein in the CNC.

Disruption of interleukin-13 (IL-13) signaling with large molecule antibody therapies has shown promise in diseases of allergic inflammation. Given that IL-13 recruits several members of the Janus Kinase family (JAK1, JAK2, and TYK2) to its receptor complex, JAK inhibition may offer an alternate small molecule approach to disrupting IL-13 signaling. Herein we demonstrate that JAK1 is likely the isoform most important to IL-13 signaling. Structure-based design was then used to improve the JAK1 potency of a series of previously reported JAK2 inhibitors. The ability to impede IL-13 signaling was thereby significantly improved, with the best compounds exhibiting single digit nM IC50's in cell-based assays dependent upon IL-13 signaling. Appropriate substitution was further found to influence inhibition of a key off-target, LRRK2. Finally, the most potent compounds were found to be metabolically labile, which makes them ideal scaffolds for further development as topical agents for IL-13 mediated diseases of the lungs and skin (for example asthma and atopic dermatitis, respectively).

We present a study of optical and electronic properties of solutions and films based on the fungi-derived pigment xylindein, extracted from decaying wood and processed without and with a simple purification step (“ethanol wash”). The “post-wash” xylindein solutions exhibited considerably lower absorption in the ultraviolet spectral range and dramatically reduced photoluminescence below 600 nm, due to removal of contaminants most likely to be fungal secondary metabolites. The “post-wash” xylindein-based films were characterized by two orders of magnitude higher charge carrier mobilities as compared to “pre-wash” samples. This underlines the importance of minimizing contaminants that disrupt the conductive xylindein network in xylindein-based electronic devices.

Free milk oligosaccharides are bioactive molecules that function as prebiotics and prevent infections that commonly afflict developing infants. To date, few publications have examined the factors affecting bovine milk oligosaccharide production among cattle in the dairy industry. Here we have applied a high-throughput isobaric labeling technique to measure oligosaccharide abundances in milk collected from Danish Holstein-Friesian and Jersey dairy cattle by liquid chromatography-mass spectrometry. With a total of 634 milk samples, this collection represents the largest sample set used for milk oligosaccharide profiling in the current literature. This study is also the first to use isobaric labeling for the purpose of measuring free oligosaccharides in a real sample set. We have identified 13 oligosaccharides that vary significantly by breed, with most structures being more abundant in the milk of Jersey cattle. The abundances of several oligosaccharides were increased in second-parity cows, and correlations between the abundances of oligosaccharide pairs were identified, potentially indicating similarities in their synthetic pathways. Fucosylated oligosaccharide structures were widely identified among both breeds. Improving our understanding of oligosaccharide production will aid in developing strategies to recover these compounds from processing streams and may enable their use as a functional ingredient in foods for infants and adults.

Peptidomics and glycomics are recently established disciplines enabling researchers to characterize functional characteristics of foods at a molecular level. Milk-derived bioactive peptides and oligosaccharides have garnered both scientific and commercial interest because they possess unique functional properties, such as anti-hypertensive, immunomodulatory and prebiotic activities; therefore, the objective of this work was to employ peptidomic and glycomic tools to identify and measure relative and absolute quantities of peptides and oligosaccharides in widely consumed dairy products. Specifically, we identified up to 2117 unique peptides in 10 commercial dairy products, which together represent the most comprehensive peptidomic profiling of dairy milk in the literature to date. The quantity of peptides, measured by ion-exchange chromatography, varied between 60 and 130 mg/L among the same set of dairy products, which the majority originated from caseins, and the remaining from whey proteins. A recently published bioactive peptide database was used to identify 66 unique bioactive peptides in the dataset. In addition, 24 unique oligosaccharide compositions were identified in all the samples by nano LC Chip QTOF. Neutral oligosaccharides were the most abundant class in all samples (66–91.3%), followed by acidic (8.6–33.7%), and fucosylated oligosaccharides (0–4.6%). Variation of total oligosaccharide concentration ranged from a high of 65.78 to a low of 24.82 mg/L. Importantly, characterizing bioactive peptides and oligosaccharides in a wider number of dairy products may lead to innovations that go beyond the traditional vision of dairy components used for nutritional purposes but that will rather focus on improving human health.

Little is known about the extent of variation and activity of naturally occurring milk glycosidases and their potential to degrade milk glycans. A multi-omics approach was used to investigate the relationship between glycosidases and important bioactive compounds such as free oligosaccharides and O-linked glycans in bovine milk. Using 4-methylumbelliferone (4-MU) assays activities of eight indigenous glycosidases were determined, and by mass spectrometry and 1H NMR spectroscopy various substrates and metabolite products were quantified in a subset of milk samples from eight native North European cattle breeds. The results showed a clear variation in glycosidase activities among the native breeds. Interestingly, negative correlations between some glycosidases including β-galactosidase, N-acetyl-β-d-glucosaminidase, certain oligosaccharide isomers as well as O-linked glycans of κ-casein were revealed. Further, a positive correlation was found for free fucose content and α-fucosidase activity (r = 0.37, p-value &lt, 0.001) indicating cleavage of fucosylated glycans in milk at room temperature. The results obtained suggest that milk glycosidases might partially degrade valuable glycans, which would result in lower recovery of glycans and thus represent a loss for the dairy ingredients industry if these activities are pronounced.

Insulin and muscle contractions mediate glucose transporter 4 (GLUT4) translocation and insertion into the plasma membrane (PM) for glucose uptake in skeletal muscles. Muscle contraction results in AMPK activation, which promotes GLUT4 translocation and PM insertion. However, little is known regarding AMPK effectors that directly regulate GLUT4 translocation. We aim to identify novel AMPK effectors in the regulation of GLUT4 translocation. We performed biochemical, molecular biology and fluorescent microscopy imaging experiments using gain- and loss-of-function mutants of tropomodulin 3 (Tmod3). Here we report Tmod3, an actin filament capping protein, as a novel AMPK substrate and an essential mediator of AMPK-dependent GLUT4 translocation and glucose uptake in myoblasts. Furthermore, Tmod3 plays a key role in AMPK-induced F-actin remodeling and GLUT4 insertion into the PM. Our study defines Tmod3 as a key AMPK effector in the regulation of GLUT4 insertion into the PM and glucose uptake in muscle cells, and offers new mechanistic insights into the regulation of glucose homeostasis.

Organic semiconductor materials have recently gained momentum due to their non-toxicity, low cost, and sustainability. Xylindein is a remarkably photostable pigment secreted by fungi that grow on decaying wood, and its relatively strong electronic performance is enabled by π-π stacking and hydrogen-bonding network that promote charge transport. Herein, femtosecond transient absorption spectroscopy with a near-IR probe was used to unveil a rapid excited-state intramolecular proton transfer reaction. Conformational motions potentially lead to a conical intersection that quenches fluorescence in the monomeric state. In concentrated solutions, nascent aggregates exhibit a faster excited state lifetime due to excimer formation, confirmed by the excimer→charge-transfer excited-state absorption band of the xylindein thin film, thus limiting its optoelectronic performance. Therefore, extending the xylindein sidechains with branched alkyl groups may hinder the excimer formation and improve optoelectronic properties of naturally derived materials.

Aside from their amino acid content, dairy proteins are valuable for their ability to carry encrypted bioactive peptides whose activities are latent until released by digestive enzymes or endogenous enzymes within the food. Peptides can possess a wide variety of functionalities, such as antibacterial, antihypertensive, and antioxidative properties, as demonstrated by in vitro and in vivo studies. This phenomenon raises the question as to what impact various traditional cheese-making processes have on the formation of bioactive peptides in the resulting products. In this study, we have profiled the naturally-occurring peptides in two hard and two soft traditional cheeses and have identified their known bioactive sequences. While past studies have typically identified fewer than 100 peptide sequences in a single cheese, we have used modern instrumentation to identify between 2900 and 4700 sequences per cheese, an increase by a factor of about 50. We demonstrated substantial variations in proteolysis and peptide formation between the interior and rind of each cheese, which we ascribed to the differences in microbial composition between these regions. We identified a total of 111 bioactive sequences among the four cheeses, with the greatest number of sequences, 89, originating from Mimolette. The most common bioactivities identified were antimicrobial and inhibition of the angiotensin-converting enzyme. This work revealed that cheese proteolysis and the resulting peptidomes are more complex than originally thought in terms of the number of peptides released, variation in peptidome across sites within a single cheese, and variation in bioactive peptides among cheese-making techniques.

Spalting fungal pigments have shown potential in technologies ranging from green energy generation to natural colorants. However, their unknown toxicity has been a barrier to industrial adoption. In order to gain an understanding of the safety of the pigments, zebrafish embryos were exposed to multiple forms of liquid media and solvent-extracted pigments with concentrations of purified pigment ranging from 0 to 50 mM from Chlorociboria aeruginosa, Chlorociboria aeruginascens, and Scytalidium cuboideum. Purified xylindein from Chlorociboria sp. did not show toxicity at any tested concentration, while the red pigment dramada from S. cuboideum was only associated with significant toxicity above 23.2 uM. However, liquid cultures and pigment extracted into dichloromethane (DCM) showed toxicity, suggesting the co-production of bioactive secondary metabolites. Future research on purification and the bioavailability of the red dramada pigment will be important to identify appropriate use, however, purified forms of the blue-green pigment xylindein are likely safe for use across industries. This opens the door to the adoption of green technologies based on these pigments, with potential to replace synthetic colorants and less stable natural pigments.

Xylindein, a stable quinonic blue-green fungal pigment, has shown potential for use not only as a colorant but also as an (opto)electronic material. As no method presently exists to synthesize the pigment, organic production by slow-growing fungi from the genus Chlorociboria is the only method to obtain it. This has resulted in limited quantities of impure xylindein, hampering research. In order to improve quantity and quality of pigment for optoelectronic applications, speed of xylindein production by Chlorociboria aeruginosa and its relative purity were compared across liquid and solid-state fermentation conditions on selected nutrient sources. Liquid 2% malt shaking cultures produced the same amount of pigment in 5 weeks that previous testing produced in 2 months. Xylindein generation speed, purity, and conductive properties of produced pigment for (opto)electronics was then compared between two Chlorociboria species native to North America, Chlorociboria aeruginosa and Chlorociboria aeruginascens. Differences were seen in the conductivity of extracted pigment between species and strains, with xylindein from C. aeruginascens strain UAMH 7614 producing films with the highest effective electron mobility. The identification of the most effective growth conditions and the strain with highest purity xylindein production should support further development of sustainable organic (opto)electronics. Future work identifying new strains with reduced production of interfering metabolites and new extraction methodologies will help to produce very low cost xylindein, supporting sustainable technologies based on the pigment.

The use of post-mortem human tissue is indispensable in studies investigating alterations in metabolite levels in neurodegenerative conditions such as Alzheimer&rsquo, s disease (AD). However, variability between samples may have unknown effects on metabolite concentrations. The aim of this study was to characterize the impact of such variables. Cingulate gyrus was obtained from AD cases and controls, from three brain banks. Gas chromatography-mass spectrometry (GC-MS) was used to measure and compare the levels of 66 identifiable metabolites in these tissues to determine effects of tissue-collection variables. The effect of PMD was further investigated by analysis of rat brain cortex and cerebellum collected following post-mortem delays (PMDs) of zero to 72 h. Metabolite levels between cases and controls were not replicable across cohorts with variable age- and gender-matching, PMD, and control Braak staging. Analysis of rat tissues found significant effects of PMD on 31 of 63 identified metabolites over periods up to 72 h. PMD must be kept under 24 h for metabolomics analyses on brain tissues to yield replicable results. Tissues should also be well age- and gender-matched, and Braak stage in controls should be kept to a minimum in order to minimize the impact of these variables in influencing metabolite variability.

The risk of coronavirus disease 2019 (COVID-19) and disease progression among patients using immunomodulatory therapy is unclear. Accordingly, we implemented an active surveillance project with USA/Canada Infectious Disease specialists via the Emerging Infections Network (EIN) to identify COVID-19 cases occurring in patients who use immunomodulatory therapy and to describe their clinical outcomes. EIN listserv members include 2396 infectious disease physicians in the USA/Canada linked via a moderated listserv. On 8 April via listserv, we requested reports of COVID-19 cases among patients receiving immunomodulatory therapy. Two weekly reminders were later sent and case reports were collected until 22 May. We collected information regarding patient demographics, COVID-19 test results, symptoms, hospitalisation details, complications, treatment, pre-existing conditions, concomitant therapies, and patient outcomes. We conducted descriptive analyses of these patient factors and compared differences between survivors and non-survivors. We grouped immunosuppressive therapies by class (table 1). View this table: Table 1 COVID-19 outcomes among autoimmune patients receiving immunomodulatory therapy Thirty-eight physicians screened over 2500 COVID-19 cases from which 77 (3%) were identified using immunomodulatory drugs. Of these, 52% were female, median age of 60 years (range, 16–84) and 83.1% had autoimmune disease (rheumatoid arthritis (19, 24.7%), ulcerative colitis (5, 6.5%) and sarcoidosis (5, 6.5%) were most common). Comorbidities included hypertension (26, 33.8%), diabetes (19, 24.7%), underlying chronic kidney disease (11, 14.3%) and others. All patients had PCR-confirmed COVID-19. Symptoms included dyspnoea (70.1%), fever (68.8%) and cough (64.9%). At time of COVID-19 diagnosis, 31 (40%) were using biologic therapies including anti tumor necrosis factor (anti-TNF) therapies (n=16), rituximab (n=6), abatacept (n=2), tocilizumab (n=2) and other (n=5). Among those using non-biologics at baseline (46, 60%), the following therapies were in use: janus kinase (JAK) inhibitors (3, 6.5%), non-biologic disease-modifying antirheumatic drugs (DMARDs) (11, 24%), prednisone alone (5, 11%) or other (27, 59%). Among …

Nonalcoholic fatty liver disease (NAFLD), is associated with metabolic disease and estimated to affect 25% of adults. NAFLD can progress to nonalcoholic steatohepatitis (NASH) which may lead to cirrhosis and hepatocellular carcinoma. There is no current treatment or cure for NASH. AMPK (5’AMP-activated protein kinase) activators have shown potential for treating NAFLD/NASH due to their effects on fatty acid inhibition and cholesterol synthesis. To evaluate this potential, an AMPK activator (herein known as C455) was selected for a preclinical NASH study. C455 demonstrated potent and selective activation of AMPK (cellular EC50= 85 nM). X-ray crystallography showed the compound binds at the ADaM site, and pharmacokinetics determined a bioavailability of 45% and half-life of 3h. Mice (male ob/ob on Amylin-NASH diet for 14-weeks or low-fat control) were dosed orally with vehicle or C455 at 3 or 30mg/kg daily. After 6 weeks of treatment, plasma ALT and terminal liver weight were decreased 32% and 22% respectively in mice treated with 30mg/kg C455 vs. vehicle. This dose also decreased liver lipid from 33% to 23%. Fibrosis via collagen staining did not improve with treatment, but significant decreases in mRNA transcripts for Col1a1(62%), Col1a2(59%), Col4a1(56%), and Timp1(55%) were seen with the high dose. Histology showed dose dependent increases in heart weight relative to body weight, and increased expression of genes associated with cardiac hypertrophy. Mice given 30mg/kg C455 also had increased cardiac glycogen storage compared to vehicle treated or LFD control mice. Our study results show that systemic activation of AMPK in a diet-induced mouse model of NASH reduces plasma ALT, decreases liver lipid, and suppressed hepatic collagen gene expression but shows no histological improvement in fibrosis. Coupled with increased heart weight and glycogen storage, the data suggests that current small molecule activators of AMPK may not be viable therapies for the treatment of NASH-related fibrotic disease. Disclosure K.M. Mather: None. M.L. Boland: None. E. Rivers: None. A. Srivastava: None. M. Schimpl: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. J. Robinson: None. P.T.C. Wan: None. J.L. Hansen: None. J.A. Read: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. J.L. Trevaskis: Employee; Self; Gilead Sciences, Inc. D.M. Smith: Employee; Self; AstraZeneca.

Human milk is a dynamic protein-protease system that delivers bioactive peptides to infants. The pH of milk changes from the mother’s mammary gland to the infant’s digestive tract. Although the release of human milk peptides has been studied during in vivo or in vitro digestion, these models did not explicitly vary nor observe the effect of pH. The objective of this research was to determine the effect of pH on the proteolysis of human milk. Using high-resolution accurate-mass Orbitrap mass spectrometry, profiles of endogenous human milk peptides before and after incubation at various pH levels have been mapped. Over 5000 peptides were identified. Comparative analyses classified 74 peptides that were consistently found independent of pH alterations, and 8 peptides that were released only at pH 4 or 5 (typical infant gastric pH). Results documented that the proteolysis of milk proteins, particularly β-casein, polymeric immunoglobulin receptor, and α-lactalbumin, is pH-dependent.

Aim: First, evaluate if patients carrying putatively diminished activity CYP2C8 genotype have longer paclitaxel exposure (e.g., time above threshold concentration of 0.05 μM [Tc >0.05]). Second, screen additional pharmacogenes for associations with Tc >0.05. Methods: Pharmacogene panel genotypes were translated into genetic phenotypes for associations with Tc >0.05 (n = 58). Results: Patients with predicted low-activity CYP2C8 had shorter Tc >0.05 after adjustment for age, body surface area and race (9.65 vs 11.03 hrs, β = 5.47, p = 0.02). This association was attributed to CYP2C8*3 (p = 0.006), not CYP2C8*4 (p = 0.58). Patients with predicted low-activity SLCO1B1 had longer Tc >0.05 (12.12 vs 10.15 hrs, β = 0.85, p = 0.012). Conclusion: Contrary to previous publications, CYP2C8*3 may confer increased paclitaxel metabolic activity. SLCO1B1 and CYP2C8 genotype may explain some paclitaxel pharmacokinetic variability.

Sickle cell disease (SCD) is caused by a single nucleotide polymorphism on chromosome 11 in the β-globin gene. The resulting mutant hemoglobin S (HbS) is a poor oxygen transporter and causes a variety of vascular symptoms and organ failures. At birth, the DRED epigenetic complex forms and silences the γ-globin gene, and fetal hemoglobin (HbF, 2 α-, and 2 γ-subunits) is replaced by adult HbA (α2β2) or HbS (α2βs2) in SCD patients. HbF is a potent inhibitor of HbS polymerization, thus alleviating the symptoms of SCD. The current therapy, hydroxyurea (HU), increases γ-globin and the HbF content in sickle cells but is highly underutilized due to concern for adverse effects and other complications. The DRED complex contains the epigenetic eraser lysine-specific demethylase 1 (LSD1), which appears to serve as a scaffolding protein. Our recently discovered 1,2,4-triazole derivatives and cyclic peptide LSD1 inhibitors promote the upregulation of γ-globin production in vitro without significant toxicity. Herein, we demonstrate that these LSD1 inhibitors can be used to disrupt the DRED complex and increase the cellular HbF content in vitro and in vivo. This approach could lead to an innovative and effective treatment for SCD.

Identification of effective natural dyes with the potential for low environmental impact has been a recent focus of the textile industry. Pigments derived from spalting fungi have previously shown promise as textile dyes, however, their use has required numerous organic solvents with human health implications. This research explored the possibility of using linseed oil as a carrier for the pigment from Scytalidium cuboideum as a textile dye. Colored linseed oil effectively dyed a range of fabrics, with natural fibers showing better coloration. Scanning electron microscopy (SEM) revealed a pigment film over the fabric surface. While mechanical testing showed no strength loss in treated fabric, colorfastness tests showed significant changes in color in response to laundering and bleach exposure with variable effects across fabric varieties. SEM investigation confirmed differences in pigmented oil layer loss and showed variation in pigment crystal formation between fabric varieties. Heating of the pigmented oil layer was found to result in a bright, shiny fabric surface, which may have potential for naturally weatherproof garments.

Oxidative stress alters cell viability, from microorganism irradiation sensitivity to human aging and neurodegeneration. Deleterious effects of protein carbonylation by reactive oxygen species (ROS) make understanding molecular properties determining ROS susceptibility essential. The radiation‐resistant bacterium Deinococcus radiodurans accumulates less carbonylation than sensitive organisms, making it a key model for deciphering properties governing oxidative stress resistance. We integrated shotgun redox proteomics, structural systems biology, and machine learning to resolve properties determining protein damage by γ‐irradiation in Escherichia coli and D. radiodurans at multiple scales. Local accessibility, charge, and lysine enrichment accurately predict ROS susceptibility. Lysine, methionine, and cysteine usage also contribute to ROS resistance of the D. radiodurans proteome. Our model predicts proteome maintenance machinery, and proteins protecting against ROS are more resistant in D. radiodurans. Our findings substantiate that protein‐intrinsic protection impacts oxidative stress resistance, identifying causal molecular properties., An integrated approach combining redox proteomics, structural systems biology and machine learning resolves molecular properties underlying superior proteome protection in radiation‐resistant Deinococcus radiodurans.

Oxidative stress alters cell viability, from microorganism irradiation sensitivity to human aging and neurodegeneration. Deleterious effects of protein carbonylation by reactive oxygen species (ROS) make understanding molecular properties determining ROS-susceptibility essential. The radiation-resistant bacterium Deinococcus radiodurans accumulates less carbonylation than sensitive organisms, making it a key model for deciphering properties governing oxidative stress resistance. We integrated shotgun redox proteomics, structural systems biology, and machine learning to resolve properties determining protein damage by γ-irradiation in Escherichia coli and D. radiodurans at multiple scales. Local accessibility, charge, and lysine enrichment accurately predict ROS-susceptibility. Lysine, methionine, and cysteine usage also contribute to ROS-resistance of the D. radiodurans proteome. Our model predicts proteome maintenance machinery and proteins protecting against ROS are more resistant in D. radiodurans. Our findings substantiate that protein-intrinsic protection impacts oxidative stress resistance, identifying causal molecular properties.One Sentence SummaryProteins differ in intrinsic susceptibility to oxidation, a mode of evolutionary adaptation for stress tolerance in bacteria.

Mitochondrial DNA (mtDNA) double-strand break (DSB) repair is essential for maintaining mtDNA integrity, but little is known about the proteins involved in mtDNA DSB repair. Here, we utilize Saccharomyces cerevisiae as a eukaryotic model to identify proteins involved in mtDNA DSB repair. We show that Mhr1, a protein known to possess homologous DNA pairing activity in vitro, binds to mtDNA DSBs in vivo, indicating its involvement in mtDNA DSB repair. Our data also indicate that Yku80, a protein previously implicated in mtDNA DSB repair, does not compete with Mhr1 for binding to mtDNA DSBs. In fact, C-terminally tagged Yku80 could not be detected in yeast mitochondrial extracts. Therefore, we conclude that Mhr1, but not Yku80, is a potential mtDNA DSB repair factor in yeast.

A failure of protein degradation may underpin Lewy body disease (LBD) where α-synuclein is assimilated into the pathognomic Lewy bodies and Lewy neurites. We investigated histological alterations in lysosomes and autophagosomes in the substantia nigra (SN) and cingulate gyrus (CG) in 34 patients with LBD employing antibodies against phosphorylated α-synuclein and lysosomal (lysosomal associated membrane proteins 1 and 2 (LAMP-1 and LAMP-2), cathepsin D (CTSD)) and autophagosomal (microtubule-associated protein light chain 3α (LC3A)) proteins. Immunostained sections were qualitatively and semi-quantitatively assessed for the appearance, distribution and intensity of staining. Four LBD patients had mutations in GBA1. There was significantly less LAMP-1, LAMP-2 and CTSD immunostaining in neurons of the SN in LBD cases compared to control cases and marginally less LAMP-1 in patients with GBA1 mutations compared to those without. Loss of LAMP-1 and CTSD immunoreactivity correlated with cell loss from the SN. There were no changes in LC3A immunoreactivity in the SN, nor any major changes in the CG, or glial cell activity in the SN and CG, for any of the markers. A proportion of amyloid plaques in both the LBD and control cases was immunoreactive for LAMP-1 and LAMP-2, but not CTSD or LC3A proteins. These immunohisochemical features were seen in glial cells, which were negative for amyloid-β. Alterations in lysosomal structure or function, but not macroautophagy, may underpin the pathogenesis of LBD.

Small noncoding RNA molecules (miRNA) regulate protein levels in a post-transcriptional manner by partial base pairing to the 3'-UTR of target genes thus mediating degradation or translational repression. Previous studies indicate that numerous miRNA regulate the biosynthesis of intraovarian hormones, and emerging evidence indicates that one of these, miRNA-221 (MIR221), may be a modulator of ovarian function. However, the hormonal control of ovarian MIR221 is not known. The objectives of this study were to investigate the developmental and hormonal regulation of MIR221 expression in granulosa (GC) and theca cell (TC) and its possible role in regulating follicular function. Bovine ovaries were collected from a local abattoir and GC and TC were obtained from small ( 0.35) on MIR221 expression, whereas treatment with fibroblast growth factor 9 (FGF9) and FGF2 increased (P < 0.001) TC MIR221 abundance 1.7- to 2.5-fold. In Exp. 5, FGF9 increased (P < 0.05) GC MIR221 abundance by 1.7- and 2.0-fold in small and large follicles, respectively. The role of MIR221 in GC steroidogenesis was investigated in Exp. 6 and it was found that transfection with a MIR221 mimic reduced (P < 0.01) GC estradiol and progesterone production induced by FSH and IGF1, whereas transfection with MIR221 inhibitor had little or no effect. We conclude that thecal MIR221 expression is increased by FGF9 and increased MIR221 may act to inhibit GC steroidogenesis in cattle.

Key points Neurons in the hypothalamus of the brain which secrete the peptide kisspeptin are important regulators of reproduction, and normal reproductive development. Electrical activity, in the form of action potentials, or spikes, leads to secretion of peptides and neurotransmitters, influencing the activity of downstream neurons; in kisspeptin neurons, this activity is highly irregular, but the mechanism of this is not known. In this study, we show that irregularity depends on the presence of a particular type of potassium ion channel in the membrane, which opens transiently in response to electrical excitation. The results contribute to understanding how kisspeptin neurons generate and time their membrane potential spikes, and how reliable this process is. Improved understanding of the activity of kisspeptin neurons, and how it shapes their secretion of peptides, is expected to lead to better treatment for reproductive dysfunction and disorders of reproductive development. Abstract Kisspeptin neurons in the hypothalamus are critically involved in reproductive function, via their effect on GnRH neuron activity and consequent gonadotropin release. Kisspeptin neurons show an intrinsic irregularity of firing, but the mechanism of this remains unclear. To address this, we carried out targeted whole-cell patch-clamp recordings of kisspeptin neurons in the arcuate nucleus (Kiss1Arc ), in brain slices isolated from adult male Kiss-Cre:tdTomato mice. Cells fired irregularly in response to constant current stimuli, with a wide range of spike time variability, and prominent subthreshold voltage fluctuations. In voltage clamp, both a persistent sodium (NaP) current and a fast transient (A-type) potassium current were apparent, activating at potentials just below the threshold for spiking. These currents have also previously been described in irregular-spiking cortical interneurons, in which the A-type current, mediated by Kv4 channels, interacts with NaP current to generate complex dynamics of the membrane potential, and irregular firing. In Kiss1Arc neurons, A-type current was blocked by phrixotoxin, a specific blocker of Kv4.2/4.3 channels, and consistent expression of Kv4.2 transcripts was detected by single-cell RT-PCR. In addition, firing irregularity was correlated to the density of A-type current in the membrane. Using conductance injection, we demonstrated that adding Kv4-like potassium conductance (gKv4 ) to a cell produces a striking increase in firing irregularity, and excitability is reduced, while subtracting gKv4 has the opposite effects. Thus, we propose that Kv4 interacting dynamically with NaP is a key determinant of the irregular firing behaviour of Kiss1Arc neurons, shaping their physiological function in gonadotropin release.

Ionizable residues buried in hydrophobic environments in proteins are essential for many fundamental biochemical processes. These residues titrate with anomalous pKa values that are challenging to reproduce with structure-based calculations owing to the conformational reorganization coupled to their ionization. Detailed characterization of this conformational reorganization is of interest; unfortunately, the properties of buried Lys residues are difficult to study experimentally. Here we demonstrate the utility of 15N NMR spectroscopy to gain insight into the protonation state, state of hydration and conformational dynamics of the Nζ amino group of buried Lys residues. The experiments were applied to five variants of staphylococcal nuclease, with internal Lys residues that titrate with pKa values ranging from 6.2 to 8.1. Direct detection of buried Lys residues with these NMR spectroscopy methods will enable correlation between thermodynamic and structural data as well as unprecedented examination of how c...

Fluctuation analysis is a method that allows measurement of the single-channel current of ion channels even when it is too small to be resolved directly with the patch-clamp technique. This is the case for voltage-gated calcium channels. They are present in all mammalian central neurons, controlling presynaptic release of transmitter, postsynaptic signaling, and synaptic integration. The amplitudes of their single-channel currents in a physiological concentration of extracellular calcium, however, are small and not well determined. But measurement of this quantity is essential for estimating numbers of functional voltage-gated calcium channels in the membrane and the size of channel-associated calcium signaling domains, and for understanding the stochastic nature of calcium signaling. Here, we recorded the voltage-gated calcium channel current in nucleated patches from layer 5 pyramidal neurons in rat neocortex, in physiological external calcium (1-2 mM). The ensemble-averaging of current responses required for conventional fluctuation analysis proved impractical because of the rapid rundown of calcium channel currents. We therefore developed a more robust method, using mean current fitting of individual current responses and band-pass filtering. Furthermore, voltage-ramp stimulation proved useful. We validated the accuracy of the method by analyzing simulated data. At an external calcium concentration of 1 mM, and a membrane potential of -20 mV, we found that the average single-channel current amplitude was ∼0.04 pA, increasing to 0.065 pA at 2 mM external calcium, and 0.12 pA at 5 mM. The relaxation time constant of the fluctuations was in the range 0.2-0.8 ms. The results are relevant to understanding the stochastic properties of dendritic Ca2+ spikes in neocortical layer 5 pyramidal neurons. With the reported method, single-channel current amplitude of native voltage-gated calcium channels can be resolved accurately despite conditions of unstable rundown.

Ionizable groups buried in the hydrophobic interior of proteins are essential for energy transduction. These groups can have highly anomalous pKa values that reflect the incompatibility between charges and dehydrated environments. A systematic study of pKa values of buried ionizable groups in staphylococcal nuclease (SNase) suggests that these pKa values are determined in part by conformational reorganization of the protein. Lys-66 is one of the most deeply buried residues in SNase. We show that its apparent pKa of 5.7 reflects the average of the pKa values of Lys-66 in different conformational states of the protein. In the fully folded state, Lys-66 is deeply buried in the hydrophobic core of SNase and must titrate with a pKa of ≪5.7. In other states, the side chain of Lys-66 is fully solvent-exposed and has a normal pKa of ≈10.4. We show that the pKa of Lys-66 can be shifted from 5.7 toward a more normal value of 7.1 via the insertion of flanking Gly residues at positions 64 and 67 to promote an “open” ...

Wood colored internally by fungi has long been used by woodcrafters and artisans as a substitute for synthetic dyes. Recent advances in the field of spalted wood have led to methods by which the fungal dyes can be extracted from either a fungal solution or colonized wood and then reapplied to clear wood. This takes the “guess work” out of spalting, as well as the time necessary for fungal colonization; however, it requires organic solvents like dichloromethane, which are toxic and not readily available to consumers. Herein, the authors show that the dyes can be successfully carried and blended together (to increase the range of colors) in a range of natural oils. The blue–green dye of Chlorociboria species, called xylindein, carried best in raw linseed oil, the red dye of Scytalidium cuboideum performed best in Danish oil, although more dye could be carried in raw linseed oil, and the yellow dye of Scytalidium ganodermophthorum performed best in walnut oil. The ability to carry and mix these dyes in easily purchased, nontoxic oils opens up their use to woodworkers who seek to follow the traditions of spalted wood, but do not have the skills or time to work with live fungal cultures or fungal dyes suspended in toxic organic solvents.

Brain lesions composed of pathological tau help to drive neurodegeneration in Alzheimer’s disease (AD) and related tauopathies. Here, we identified the mammalian suppressor of tauopathy 2 (MSUT2) gene as a modifier of susceptibility to tau toxicity in two mouse models of tauopathy. Transgenic PS19 mice overexpressing tau, a model of AD, and lacking the Msut2 gene exhibited decreased learning and memory deficits, reduced neurodegeneration, and reduced accumulation of pathological tau compared to PS19 tau transgenic mice expressing Msut2. Conversely, Msut2 overexpression in 4RTauTg2652 tau transgenic mice increased pathological tau deposition and promoted the neuroinflammatory response to pathological tau. MSUT2 is a poly(A) RNA binding protein that antagonizes the canonical nuclear poly(A) binding protein PABPN1. In individuals with AD, MSUT2 abundance in postmortem brain tissue predicted an earlier age of disease onset. Postmortem AD brain tissue samples with normal amounts of MSUT2 showed elevated neuroinflammation associated with tau pathology. We observed co-depletion of MSUT2 and PABPN1 in postmortem brain samples from a subset of AD cases with higher tau burden and increased neuronal loss. This suggested that MSUT2 and PABPN1 may act together in a macromolecular complex bound to poly(A) RNA. Although MSUT2 and PABPN1 had opposing effects on both tau aggregation and poly(A) RNA tail length, we found that increased poly(A) tail length did not ameliorate tauopathy, implicating other functions of the MSUT2/PABPN1 complex in tau proteostasis. Our findings implicate poly(A) RNA binding proteins both as modulators of pathological tau toxicity in AD and as potential molecular targets for interventions to slow neurodegeneration in tauopathies.

Background: Recent evidence suggests that fluorescein-guided microsurgery facilitates resection of intrinsic and metastatic lesions in the brain. Use of sodium fluorescein for angiography has previously been reported without non-specific diffusion into the adjacent spinal parenchyma. Diagnostic biopsy of spinal cord lesion is often challenging and the localization of such lesions intraoperatively is paramount. We present the first known report of use of sodium fluorescein for localization and diagnostic biopsy of intrinsic spinal cord lesions. Methods: Three patients with spinal cord lesions received fluorescein sodium 10% (Alcon Laboratories INC, Fort Worth, TX, USA) at 3 mg/kg prior to surgical resection. Intraoperative visualization of fluorescence was performed using a Zeiss Pentero (Carl Zeiss AG, Oberkochen, Germany) microscope equipped with a Yellow560 filter or a Leica OH6 (Leice Microsystems, Wetzlar, Germany) equipped with a FL560 filter. Results: Administration of sodium fluorescein resulted in lesional fluorescent contrast extravasation and facilitated surgical resection. The addition of sodium fluorescein allowed for identification and facilitated biopsy in three patients. In patients undergoing biopsy, tissue samples were positive for a diagnostic pathology. Conclusion: Fluorescein may be a helpful microsurgical tool in guiding resection and localization for tissue biopsy of intramedullary spinal lesions. Further research is necessary to explore fluorescein sodium applications in the resection of spinal cord lesions. Keywords: Fluorescein sodium, Fluorescence-guided spinal surgery, Spinal tumor resection, Spine tumor biopsy

Introduction Necrotizing enterocolitis (NEC) is a devastating intestinal illness in premature infants characterized by severe intestinal inflammation. Despite medical interventions, NEC mortality remains alarmingly high, which necessitates improved therapies. Lactoferrin is among the most abundant proteins in human milk and has important immunomodulatory functions. While previous studies have indicated protective effects of lactoferrin against neonatal sepsis and NEC, the underlying mechanism remains unclear. We hypothesize that lactoferrin downregulates inflammation and upregulates proliferation in intestinal epithelium during NEC injury. Materials and Methods NEC was induced by hypoxia, gavage feeding of hyperosmolar formula and lipopolysaccharide between postnatal day P5 and P9 (n = 8). Breastfed mice were used as control (n = 7). Lactoferrin (0.3 g/kg/day) was administered once daily by gavage from P6 to P8 in both NEC (NEC + Lac; n = 9) and control mice (Cont + Lac; n = 5). Distal ileum was harvested on P9 and analyzed for disease severity, inflammation, and proliferation. Groups were compared using one-way ANOVA and t-test appropriately; p Results Compared to NEC group, lactoferrin-treated NEC mice had reduced disease severity, reduced inflammation markers IL-6 and TNF-α expression and increased intestinal stem cell marker Lgr5 + expression. Lactoferrin-treated NEC mice exhibited increased nuclear β-catenin, indicating upregulated Wnt pathway, and increased Ki67 positivity, suggesting enhanced proliferation. Furthermore, lactoferrin administration to control mice did not affect intestinal inflammation as well as Lgr5 + stem cell expression and epithelial proliferation. This supports the safety of lactoferrin administration and indicates that the beneficial effects of lactoferrin are present when intestinal injury such as NEC is present. Conclusion Lactoferrin administration reduces the intestinal injury in experimental NEC by downregulating inflammation and upregulating cell proliferation. This beneficial effect of lactoferrin in stimulating cell proliferation is mediated by the Wnt pathway. This experimental study provides insights on the mechanism of action of lactoferrin in NEC and the role of lactoferrin in enteral feeding.

Textile inkjet printing is an increasingly popular process in the textile industry, as it allows for the incorporation of complex and detailed patterns onto fabrics, as well as the production of small and medium volumes of printed text. Unfortunately, most of the dyes used by the textile industry come from synthetic and/or non-renewable sources. There has been some research to date in using fungal pigments from wood rotting fungi (&lsquo, spalting&rsquo, fungi) as textile dyes, however these have never been tested in inkjet printing. Of particular interest is the red crystallizing pigment from Scytalidium cuboideum, which has previously shown exceptional stability on textiles. To test this pigment in an inkjet setting, cotton and polyester fabrics were printed with three different ink formulations involving the red pigment: hexadecyltrimethylammonium bromide (CTAB), ethanol, and acetone. The CTAB and ethanol-based ink formulations formed a &lsquo, mesh-like&rsquo, structure on the surface of the cotton and polyester fibers, and turned the fabric purple. Acetone formulas formed crystal structures on the surface and turned the fabric red. These results show promise for turning the red pigment of S. cuboideum into an environmentally friendly, inkjet colorant, however further research is required to evaluate the crocking and explain the crystallization differences between inks.

Extensive membrane blebbing is one of the earliest observable changes in HeLa cells stimulated with apoptosis inducers. Blebbing caused by actinomycin D or camptothecin, but not by anti-Fas antibody, is accompanied by an almost 10% volume increase as measured by transmission-through-dye microscopy. When the experiment is carried out in DMEM medium, the swelling appears to result from activation of amiloride-sensitive channels. Low-sodium choline-, but not N-methyl(-)D-glucamine-based, medium, also supports swelling during the blebbing phase of apoptosis; this indicates that the membrane becomes permeable to choline as well. Because choline can enter the cells through organic cation transporters (OCT), we tested three fluorescent dyes (2-[4-(dimethylamino)styryl]-1-methylpyridinium iodide, rhodamine 123 and ethidium bromide) that have been reported to utilize OCT for cell entry. Intact HeLa cells are poorly permeable for these fluorophores, and initially they accumulate on the plasma membranes. Blebbing results in an enhanced penetration of these dyes into the cell interior, as was demonstrated both by direct observation and by FRET. The increased membrane permeability is specific for OCT substrates; the other tested cationic dyes apparently cross the membrane by other routes and exhibit a markedly different behavior. Our results reveal a previously unknown feature of apoptosis and the utility of cationic dyes for studying membrane transport.