Immunochemical Characterization of Salix alba (S. alba) Pollen Allergens and Evaluation of the Cross-Reactivity Pattern with Common Allergenic Pollen Grains.

BACKGROUND: Pollens, particularly tree and plant pollens, are one of the major causes of allergic respiratory diseases worldwide. Allergy to pollens of different species of Salix trees has been reported in various regions of the world. The most common type of Salix tree in Iran is white willow (Salix alba). OBJECTIVES: This study aimed to identify and determine the immunochemical characteristics of allergenic proteins in S. alba tree pollen extract using SDS-PAGE and IgE- immunoblotting methods. Moreover, the cross-reaction pattern of the specific IgE antibody of S. alba tree pollen proteins with pollen allergens of common allergenic trees, i.e., Populus nigra (P. nigra), Cupressus sempervirens (C. sempervirens), Pinus brutia (P. brutia) and Platanus orientalis (P. orientalis) in the region was investigated. METHODS: The reaction of allergenic proteins in S. alba pollen extract with specific IgE antibodies in patients' sera was investigated using SDS-PAGE and IgE-immunoblotting methods. The cross-reaction of specific IgE antibodies of the proteins present in S. alba pollen extract with pollen allergens of common allergenic trees in the region was investigated using ELISA and immunoblotting inhibition methods. In silico methods such as phylogenetic tree drawing and alignment of amino acid sequences were used to examine the evolutionary relationship and homology structure of common allergenic proteins (Panallergens) responsible for cross reactions. RESULTS: More than 11 protein bands binding to specific IgE antibodies in patients' sera with a molecular weight between 13 and 95 kDa were identified in the S. alba tree pollen extract. ELISA and immunoblotting inhibition results showed that P. nigra extract could inhibit the binding of IgE antibodies to S. alba pollen extract proteins to a greater extent than C. sempervirens, P. brutia, and P. orientalis tree extracts. In silico methods investigated the results of ELISA and immunoblotting inhibition methods. Moreover, a high structural homology and evolutionary relationship were observed between S. alba and P. nigra tree pollen panallergens. CONCLUSION: In this study, it was found that more than 80 % of the sensitive patients who were examined had specific IgE antibodies reacting with the approximately a 15 kDa-protein present in the S. alba pollen extract. Furthermore, the specific IgE-binding proteins found in the pollens of S. alba and P. nigra trees had relative structural homology, and it is likely that if recombinant forms are produced, they can be used for diagnostic and therapeutic purposes for both of the trees.

Neobavaisoflavone-mediated TH9 cell differentiation ameliorates bowel inflammation.

Neobavaisoflavone (Neo), is the active constituent of the herb Psoralea corylifolial, used in the traditional Chinese medicine, and has anti-inflammatory activity, but whether Neo could regulate colitis remains unclear. T helper 9 (TH9) cells, a subset of CD4+ T helper cells characterized by secretion of IL-9, have been reported to be involved in the pathogenesis of many autoimmune and inflammatory diseases, but whether Neo could control TH9 cell differentiation also remains unclear. Here, we found that Neo could decrease IL-9 production of CD4+ T cells by targeting PU.1 in vitro. Importantly, Neo had therapeutic effects on DSS-induced colitis. Furthermore, we identified TH9 cells as the direct target of Neo for attenuating bowel inflammation. Therefore, Neo could serve as a lead for developing new therapeutics against inflammatory bowel disease.

Schisandrin B promotes TH1 cell differentiation by targeting STAT1.

Schisandrin B (Sch B) is the major active ingredient of the traditional Chinese medicine Schisandra chinensis and has antitumor activity, anti-inflammatory activity. CD4+ Th subsets orchestrate immune responses to plenty of pathogen infections and participate in the pathogenesis of many immune-related diseases. However, little is known about the relationship between Sch B and T cell differentiation. Here, we showed that Sch B might participate in T cell receptor signaling pathway by using the TCMIO database. Importantly, Sch B promoted TH1 cell differentiation. Furthermore, Sch B did not affect TH2 cell and Treg differentiation. Mechanismly, Sch B increased the level of IFN-γ of CD4+ T cells by upregulating the phosphorylation of STAT1 protein. Then, STAT1 promoted T-bet expression in CD4+ T cells. In conclusion, Sch B modulates the differentiation of naïve CD4+ T cells into TH1 subset by STAT1/T-bet signaling, which may have the potential for the treatment of T cell-mediated-immune diseases.

Delivery of membrane impermeable molecules to primary mouse T lymphocytes.

The pore-forming toxin streptolysin-O (SLO) enables intracellular delivery of molecules up to 100 kDa and has been used for short-term delivery of membrane-impermeable substances to assess their effects on cellular activities. A limitation of this technique is the loss of intracellular components and the potential unpredicted alterations of cellular metabolism and signaling. This protocol, optimized for primary mouse T lymphocytes, describes steps for SLO-mediated cell membrane permeabilization and substance supplementation, followed by immunoblotting and immunofluorescent microscopy for assessing cellular effects. For complete details on the use and execution of this protocol, please refer to Xu et al., 2021a, Xu et al., 2021b.

Adiponectin receptor agonist AdipoRon ameliorates renal inflammation in diet-induced obese mice and endotoxin-treated human glomeruli ex vivo.

AIMS/HYPOTHESIS: Chronic low-grade inflammation with local upregulation of proinflammatory molecules plays a role in the progression of obesity-related renal injury. Reduced serum concentration of anti-inflammatory adiponectin may promote chronic inflammation. Here, we investigated the potential anti-inflammatory and renoprotective effects and mechanisms of action of AdipoRon, an adiponectin receptor agonist. METHODS: Wild-type DBA/2J mice were fed with high-fat diet (HFD) supplemented or not with AdipoRon to model obesity-induced metabolic endotoxaemia and chronic low-grade inflammation and we assessed changes in the glomerular morphology and expression of proinflammatory markers. We also treated human glomeruli ex vivo and human podocytes in vitro with AdipoRon and bacterial lipopolysaccharide (LPS), an endotoxin upregulated in obesity and diabetes, and analysed the secretion of inflammatory cytokines, activation of inflammatory signal transduction pathways, apoptosis and migration. RESULTS: In HFD-fed mice, AdipoRon attenuated renal inflammation, as demonstrated by reduced expression of glomerular activated NF-κB p65 subunit (NF-κB-p65) (70%, p < 0.001), TNFα (48%, p < 0.01), IL-1ß (51%, p < 0.001) and TGFß (46%, p < 0.001), renal IL-6 and IL-4 (21% and 20%, p < 0.05), and lowered glomerular F4/80-positive macrophage infiltration (31%, p < 0.001). In addition, AdipoRon ameliorated HFD-induced glomerular hypertrophy (12%, p < 0.001), fibronectin accumulation (50%, p < 0.01) and podocyte loss (12%, p < 0.001), and reduced podocyte foot process effacement (15%, p < 0.001) and thickening of the glomerular basement membrane (18%, p < 0.001). In cultured podocytes, AdipoRon attenuated the LPS-induced activation of the central inflammatory signalling pathways NF-κB-p65, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38-MAPK) (30%, 36% and 22%, respectively, p < 0.001), reduced the secretion of TNFα (32%, p < 0.01), and protected against podocyte apoptosis and migration. In human glomeruli ex vivo, AdipoRon reduced the LPS-induced secretion of inflammatory cytokines IL-1ß, IL-18, IL-6 and IL-10. CONCLUSIONS/INTERPRETATION: AdipoRon attenuated the renal expression of proinflammatory cytokines in HFD-fed mice and LPS-stimulated human glomeruli, which apparently contributed to the amelioration of glomerular inflammation and injury. Mechanistically, based on assays on cultured podocytes, AdipoRon reduced LPS-induced activation of the NF-κB-p65, JNK and p38-MAPK pathways, thereby impelling the decrease in apoptosis, migration and secretion of TNFα. We conclude that the activation of the adiponectin receptor by AdipoRon is a potent strategy to attenuate endotoxaemia-associated renal inflammation.

The effects of melatonin daily supplementation to aged rats on the ability to withstand cold, thermoregulation and body weight.

AIMS: Investigate the role of melatonin on the regulation of body temperature in aged animals that have impaired melatonin production. MATERIAL AND METHODS: Aged Male Wistar rats were randomly assigned to the following groups: 1) control (vehicle added to the water bottles during the dark phase) and 2) melatonin-treated (10 mg/kg melatonin added to the water bottles during the dark phase). Before and after 16 weeks of vehicle or melatonin treatment, control group and melatonin-treated animals were acutely exposed to 18 °C for 2 h for an acute cold challenge and thermal images were obtained using an infrared camera. After 16 weeks, animals were euthanized and brown and beige adipocytes were collected for analysis of genes involved in the thermogenesis process by real-time PCR, and the uncoupling protein expression was evaluated by immunoblotting. Browning intensity of beige adipocytes were quantified by staining with hematoxylin-eosin. KEY FINDINGS: Chronic melatonin supplementation induced a minor increase in body mass and increased the animal's thermogenic potential in the cold acute challenge. Brown and beige adipocytes acted in a coordinated and complementary way to ensure adequate heat production. SIGNIFICANCE: Melatonin plays an important role in the thermoregulatory mechanisms, ensuring greater capacity to withstand cold and, also, participating in the regulation of energy balance.

An immunochemistry-based screen for chemical inhibitors of DNA-protein interactions and its application to human CGGBP1.

BACKGROUND: Inhibition of DNA-binding of proteins by small-molecule chemicals holds immense potential in manipulating the activities of DNA-binding proteins. Such a chemical inhibition of DNA-binding of proteins can be used to modulate processes such as replication, transcription, DNA repair and maintenance of epigenetic states. This prospect is currently challenged with the absence of robust and generic protocols to identify DNA-protein interactions. Additionally, much of the current approaches to designing inhibitors requires structural information of the target proteins. METHODS: We have developed a simple dot blot and immunodetection-based assay to screen chemical libraries for inhibitors of DNA-protein interactions. The assay has been applied to a library of 1685 FDA-approved chemicals to discover inhibitors of CGGBP1, a multifunctional DNA-binding protein with no known structure. Additional in vitro and in cellulo assays have been performed to verify and supplement the findings of the screen. RESULTS: Our primary screen has identified multiple inhibitors of direct or indirect interactions between CGGBP1 and genomic DNA. Of these, one inhibitor, Givinostat, was found to inhibit direct DNA-binding of CGGBP1 in the secondary screen using purified recombinant protein as the target. DNA and chromatin immunoprecipitation assays reinforced the findings of the screen that Givinostat inhibits CGGBP1-DNA binding. CONCLUSIONS: The assay we have described successfully identifies verifiable inhibitors of DNA-binding of protein; in this example, the human CGGBP1. This assay is customizable for a wide range of targets for which primary antibodies are available. It works with different sources of the target protein, cell lysates or purified recombinant preparations and does not require special equipment, DNA modifications or protein structural data. This assay is scalable and highly adaptable with the potential to discover inhibitors of transcription factors with implications in cancer biology.

NF-κB inhibitor with Temozolomide results in significant apoptosis in glioblastoma via the NF-κB(p65) and actin cytoskeleton regulatory pathways.

Glioblastoma (GBM) is the most malignant brain tumor characterized by intrinsic or acquired resistance to chemotherapy. GBM tumors show nuclear factor-κB (NF-κB) activity that has been associated with tumor formation, growth, and increased resistance to therapy. We investigated the effect of NF-κB inhibitor BAY 11-7082 with Temozolomide (TMZ) on the signaling pathways in GBM pathogenesis. GBM cells and patient-derived GBM cells cultured in 3D microwells were co-treated with BAY 11-7082 and TMZ or BAY 11-7082 and TMZ alone, and combined experiments of cell proliferation, apoptosis, wound healing assay, as well as reverse-phase protein arrays, western blot and immunofluorescence staining were used to evaluate the effects of drugs on GBM cells. The results revealed that the co-treatment significantly altered cell proliferation by decreasing GBM viability, suppressed NF-κB pathway and enhanced apoptosis. Moreover, it was found that the co-treatment of BAY 11-7082 and TMZ significantly contributed to a decrease in the migration pattern of patient-derived GBM cells by modulating actin cytoskeleton pathway. These findings suggest that in addition to TMZ treatment, NF-κB can be used as a potential target to increase the treatment's outcomes. The drug combination strategy, which is significantly improved by NF-κB inhibitor could be used to better understand the underlying mechanism of GBM pathways in vivo and as a potential therapeutic tool for GBM treatment.

Protein-protein interaction of the putative magnetoreceptor cryptochrome 4 expressed in the avian retina.

Migratory birds can sense the Earth's magnetic field and use it for orientation over thousands of kilometres. A light-dependent radical-pair mechanism associated with the visual system is currently discussed as the underlying mechanism of the magnetic compass sense. The blue light receptor cryptochrome 4 (Cry4) is considered as the most likely primary sensory protein that detects the geomagnetic field. Since the protein interaction partners of Cry4 are completely unknown at present, here, we aim to identify potential candidate interaction partners of Cry4 in the avian retina. We used the yeast-two-hybrid system to screen avian cDNA libraries for possible interaction partners of Cry4 in the European robin. The UAS-GAL yeast two hybrid system was applied to confirm a group of candidate Cry4 interaction partners. Six proteins were found to be particularly promising candidates for interacting with European robin Cry4. The identified genes code for guanine nucleotide-binding protein G(t) subunit alpha-2 (GNAT2), long-wavelength-sensitive opsin (LWS, also called iodopsin), guanine nucleotide-binding protein subunit gamma 10 (GNG10), potassium voltage-gated channel subfamily V member 2 (KCNV2), retinol binding protein 1 (RBP1) and retinal G protein-coupled receptor (RGR). All genes are known to be expressed in vertebrate retinae of different species. We conclude by discussing putative signalling pathways that could connect cryptochrome 4 to one or more of these 6 candidates.

Immunochemical Detection of Protein Modification Derived from Metabolic Activation of 8-Epidiosbulbin E Acetate.

Furanoid 8-epidiosbulbin E acetate (EEA) is one of the most abundant diterpenoid lactones in herbal medicine Dioscorea bulbifera L. (DB). Our early work proved that EEA could be metabolized to EEA-derived cis-enedial (EDE), a reactive intermediate, which is required for the hepatotoxicity observed in experimental animals exposed to EEA. Also, we found that EDE could modify hepatic protein by reaction with thiol groups and/or primary amines of protein. The present study was inclined to develop polyclonal antibodies to detect protein modified by EDE. An immunogen was prepared by reaction of EDE with keyhole limpet hemocyanin (KLH), and polyclonal antibodies were raised in rabbits immunized with the immunogen. Antisera collected from the immunized rabbits demonstrated high titers evaluated by enzyme-linked immunosorbent assays (ELISAs). Immunoblot analysis showed that the polyclonal antibodies recognized EDE-modified bovine serum albumin (BSA) in a hapten load-dependent manner but did not cross-react with native BSA. Competitive inhibition experiments elicited high selectivity of the antibodies toward EDE-modified BSA. The antibodies allowed us to detect and enrich EDE-modified protein in liver homogenates obtained from EEA-treated mice. The developed immunoprecipitation technique, along with mass spectrometry, enabled us to succeed in identifying multiple hepatic proteins of animals given EEA. We have successfully developed polyclonal antibodies with the ability to recognize EDE-derived protein adducts, which is a unique tool for us to define the mechanisms of toxic action of EEA.

Stability of extracts from pollens of allergenic importance in Korea.

BACKGROUND/AIMS: Accurate diagnosis and the effects of allergen-specific immunotherapy for pollinosis are greatly dependent on the potency and stability of the extract. This study aimed to examine factors, such as temperature and storage buffer composition, that affect the stability of allergen extracts from pollens of allergenic importance in Korea. METHODS: We prepared four pollen allergen extracts from ragweed, mugwort, Japanese hop, and sawtooth oak, which are the most important causes of seasonal rhinitis in Korea. Changes of protein and major allergen concentration were measured over 1 year by Bradford assay, two-site enzyme-linked immunosorbent assay, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reconstitution of the lyophilized allergen extract in various buffers and stored at room temperature (RT, 18°C to 26°C) or refrigerated (4°C). RESULTS: More than 90% of the original protein concentration in all four extracts examined was detected over 1 year when 50% glycerol was added and refrigerated, whereas 57.9% to 94.5% remained in the extracts at RT. The addition of 50% glycerol to the storage buffer was found to prevent protein degradation at RT. Amb a 1, a major allergen of ragweed, was almost completely degraded in 9 weeks at RT when reconstituted in a buffer without 50% glycerol. However, 55.6% to 92.8% of Amb a 1 content was detected after 1 year of incubation at 4°C in all buffer conditions except 0.3% phenol. CONCLUSION: Addition of 50% glycerol as well as refrigeration was found to be important in increasing the shelf-life of allergen extracts from pollens of allergenic importance.

Evaluation of the Putative Efficacy of a Methanolic Extract of Ocimum Basilicum in Preventing Disruption of Structural Proteins in an in Vitro System of Selenite-induced Cataractogenesis.

Purpose: To evaluate whether a methanolic extract of Ocimum basilicum (OB) leaves prevented lenticular protein alterations in an in-vitro model of selenite-induced cataractogenesis.Materials and Methods: Transparent lenses extirpated from Wistar rats were divided into three groups: control; selenite only; treated. Control lenses were cultured in Dulbecco's modified Eagle's medium (DMEM) alone, selenite only lenses were cultured in DMEM containing sodium selenite only (100 µM selenite/ml DMEM) and treated lenses were cultured in DMEM containing sodium selenite and the methanolic extract of OB leaves (200 µg of extract/ml DMEM); all lenses were cultured for 24 h and then processed. The parameters assessed in lenticular homogenates were lenticular protein sulfhydryl and carbonyl content, calcium level, insoluble to soluble protein ratio, sodium dodecyl sulphate-polyacrylamide gel electrophoretic (SDS-PAGE) patterns of lenticular proteins, and mRNA transcript and protein levels of αA-crystallin and ßB1-crystallins.Results: Selenite only lenses exhibited alterations in all parameters assessed. Treated lenses exhibited values for these parameters that were comparable to those noted in normal control lenses.Conclusions: The methanolic extract of OB leaves prevented alterations in lenticular protein sulfhydryl and carbonyl content, calcium level, insoluble to soluble protein ratio, SDS-PAGE patterns of lenticular proteins, and expression of αA-crystallin and ßB1-crystallin gene and proteins in cultured selenite-challenged lenses. OB may be further evaluated as a promising agent for the prevention of cataract.

Testing the Specificity of Compounds Designed to Inhibit CPT1A in T Cells.

In response to antigen and costimulation, T cells undergo a series of metabolic transitions that fulfill the biosynthetic demands of clonal expansion, differentiation, and effector function. Following antigen clearance, the oxidation of long-chain fatty acids (LCFAO) has been implicated in the transition from effector to central memory T cells. However, studies demonstrating a role for LCFAO in memory T-cell development have largely relied on the use of etomoxir (ETO), a small molecule inhibitor of the long-chain fatty acid transporter CPT1A. Understanding how the depletion of nutrients including LCFA that might occur in tumor microenvironments affects T-cell proliferation, differentiation, and function has important implications for tumor immunotherapy. Here, we combine the analysis of posttranscriptional gene silencing with extracellular flux assays to determine if etomoxir exerts nonspecific effects on oxidative metabolism. The off-target effects of ETO that we describe highlight the challenges of using pharmacologic inhibitors in loss-of-function approaches in T cells.

Adiponectin Reverses the Hypothalamic Microglial Inflammation during Short-Term Exposure to Fat-Rich Diet.

Adiponectin, an adipokine derived from the adipose tissue, manifests anti-inflammatory effects in the metabolically active organs and is, therefore, beneficial in various metabolic diseases associated with inflammation. However, the role of adiponectin in alleviating the hypothalamic inflammation connected to the pathogenesis of obesity has not yet been clearly interrogated. Here, we identified that the systemic administration of adiponectin suppresses the activation of microglia and thereby reverses the hypothalamic inflammation during short-term exposure to a high-fat diet. Additionally, we show that adiponectin induces anti-inflammatory effects in the microglial cell line subjected to an exogenous treatment with a saturated free fatty acid. In conclusion, the current study suggests that adiponectin suppresses the saturated free fatty acid-triggered the hypothalamic inflammation by modulating the microglial activation and thus maintains energy homeostasis.

Differential requirements for the EF-hand domains of human centrin 2 in primary ciliogenesis and nucleotide excision repair.

Centrin 2 is a small conserved calcium-binding protein that localizes to the centriolar distal lumen in human cells. It is required for efficient primary ciliogenesis and nucleotide excision repair (NER). Centrin 2 forms part of the xeroderma pigmentosum group C protein complex. To explore how centrin 2 contributes to these distinct processes, we mutated the four calcium-binding EF-hand domains of human centrin 2. Centrin 2 in which all four EF-hands had been mutated to ablate calcium binding (4DA mutant) was capable of supporting in vitro NER and was as effective as the wild-type protein in rescuing the UV sensitivity of centrin 2-null cells. However, we found that mutation of any of the EF-hand domains impaired primary ciliogenesis in human TERT-RPE1 cells to the same extent as deletion of centrin 2. Phenotypic analysis of the 4DA mutant revealed defects in centrosome localization, centriole satellite assembly, ciliary assembly and function and in interactions with POC5 and SFI1. These observations indicate that centrin 2 requires calcium-binding capacity for its primary ciliogenesis functions, but not for NER, and suggest that these functions require centrin 2 to be capable of forming complexes with partner proteins.This article has an associated First Person interview with the first author of the paper.

Chemically induced neurite-like outgrowth reveals a multicellular network function in patient-derived glioblastoma cells.

Tumor stem cells and malignant multicellular networks have been separately implicated in the therapeutic resistance of glioblastoma multiforme (GBM), the most aggressive type of brain cancer in adults. Here, we show that small-molecule inhibition of RHO-associated serine/threonine kinase proteins (ROCKi) significantly promoted the outgrowth of neurite-like cell projections in cultures of heterogeneous patient-derived GBM stem-like cells. These projections formed de novo-induced cellular network (iNet) 'webs', which regressed after withdrawal of ROCKi. Connected cells within the iNet web exhibited long range Ca2+ signal transmission, and significant lysosomal and mitochondrial trafficking. In contrast to their less-connected vehicle control counterparts, iNet cells remained viable and proliferative after high-dose radiation. These findings demonstrate a link between ROCKi-regulated cell projection dynamics and the formation of radiation-resistant multicellular networks. Our study identifies means to reversibly induce iNet webs ex vivo, and may thereby accelerate future studies into the biology of GBM cellular networks.

Chemotherapy selection pressure alters sphingolipid composition and mitochondrial bioenergetics in resistant HL-60 cells.

The combination of daunorubicin (dnr) and cytarabine (Ara-C) is a cornerstone of treatment for acute myelogenous leukemia (AML); resistance to these drugs is a major cause of treatment failure. Ceramide, a sphingolipid (SL), plays a critical role in cancer cell apoptosis in response to chemotherapy. Here, we investigated the effects of chemotherapy selection pressure with Ara-C and dnr on SL composition and enzyme activity in the AML cell line HL-60. Resistant cells, those selected for growth in Ara-C- and dnr-containing medium (HL-60/Ara-C and HL-60/dnr, respectively), demonstrated upregulated expression and activity of glucosylceramide synthase, acid ceramidase (AC), and sphingosine kinase 1 (SPHK1); were more resistant to ceramide than parental cells; and displayed sensitivity to inhibitors of SL metabolism. Lipidomic analysis revealed a general ceramide deficit and a profound upswing in levels of sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P) in HL-60/dnr cells versus parental and HL-60/Ara-C cells. Both chemotherapy-selected cells also exhibited comprehensive upregulations in mitochondrial biogenesis consistent with heightened reliance on oxidative phosphorylation, a property that was partially reversed by exposure to AC and SPHK1 inhibitors and that supports a role for the phosphorylation system in resistance. In summary, dnr and Ara-C selection pressure induces acute reductions in ceramide levels and large increases in S1P and C1P, concomitant with cell resilience bolstered by enhanced mitochondrial remodeling. Thus, strategic control of ceramide metabolism and further research to define mitochondrial perturbations that accompany the drug-resistant phenotype offer new opportunities for developing therapies that regulate cancer growth.

Investigating the Mitochondrial Permeability Transition Pore in Disease Phenotypes and Drug Screening.

Mitochondria act as 'sinks' for Ca2+ signaling, with mitochondrial Ca2+ uptake linking physiological stimuli to increased ATP production. However, mitochondrial Ca2+ overload can induce a cellular catastrophe by opening of the mitochondrial permeability transition pore (mPTP). This pore is a large conductance pathway in the inner mitochondrial membrane that causes bioenergetic collapse and appears to represent a final common path to cell death in many diseases. The role of the mPTP as a determinant of disease outcome is best established in ischemia/reperfusion injury in the heart, brain, and kidney, and it is also implicated in neurodegenerative disorders and muscular dystrophies. As the probability of pore opening can be modulated by drugs, it represents a useful pharmacological target for translational research in drug discovery. Described in this unit is a protocol utilizing isolated mitochondria to quantify this phenomenon and to develop a high-throughput platform for phenotypic screens for Ca2+ dyshomeostasis. © 2019 The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Localization of Four Allergens in Artemisia Pollen by Immunofluorescent Antibodies.

BACKGROUND: Artemisia pollens have a high potential to induce allergic symptoms. Seven allergen components have been identified, but only Art v 7 has been localized in the pollen grain. This study aimed to localize the allergens in the pollen grains of 4 Artemisia spp. METHODS: Pollen extracts from 2 Chinese Artemisia spp., A. argyi and A. annua, were used to immunize BALB/c mice. Recombinant Art v 1 and Art v 3 allergens were used to select specific monoclonal antibodies (mAbs). Three mAbs were used to purify the natural allergens and were then analyzed by mass spectrometry. As reported previously, polyclonal antibodies were obtained from rabbits immunized with 3 synthesized peptides of Art an 7. Using conventional histology procedures with pollens from 4 Artemisia spp. (A. argyi, A. annua, A. capilaris, and A. sieversiana), allergen images were observed and recorded by fluorescence and confocal laser microscopy. RESULTS: We obtained 2 specific mAbs against Art v 1, 1 against Art v 2, and 4 against Art v 3 homologs. The Art v 1 and Art v 3 homologs were mainly located on the pollen walls, and the Art v 7 homologous protein was localized intracellularly around nuclei. The location of the Art v 2 homologous protein varied across species, being intracellular around nuclei for A. annua and A. argyi, and in both the pollen wall and around nuclei for A. capilaris and A. sieversiana. CONCLUSIONS: Four mugwort allergens were localized in the pollen, and the major Art v 1 and Art v 3 allergens were located mainly in the pollen wall.

Causes of hOCT1-Dependent Cholangiocarcinoma Resistance to Sorafenib and Sensitization by Tumor-Selective Gene Therapy.

Although the multi-tyrosine kinase inhibitor sorafenib is useful in the treatment of several cancers, cholangiocarcinoma (CCA) is refractory to this drug. Among other mechanisms of chemoresistance, impaired uptake through human organic cation transporter type 1 (hOCT1) (gene SLC22A1) has been suggested. Here we have investigated the events accounting for this phenotypic characteristic and have evaluated the interest of selective gene therapy strategies to overcome this limitation. Gene expression and DNA methylation of SLC22A1 were analyzed using intrahepatic (iCCA) and extrahepatic (eCCA) biopsies (Copenhagen and Salamanca cohorts; n = 132) and The Cancer Genome Atlas (TCGA)-CHOL (n = 36). Decreased hOCT1 mRNA correlated with hypermethylation status of the SLC22A1 promoter. Treatment of CCA cells with decitabine (demethylating agent) or butyrate (histone deacetylase inhibitor) restored hOCT1 expression and increased sorafenib uptake. MicroRNAs able to induce hOCT1 mRNA decay were analyzed in paired samples of TCGA-CHOL (n = 9) and Copenhagen (n = 57) cohorts. Consistent up-regulation in tumor tissue was found for miR-141 and miR-330. High proportion of aberrant hOCT1 mRNA splicing in CCA was also seen. Lentiviral-mediated transduction of eCCA (EGI-1 and TFK-1) and iCCA (HuCCT1) cells with hOCT1 enhanced sorafenib uptake and cytotoxic effects. In chemically induced CCA in rats, reduced rOct1 expression was accompanied by impaired sorafenib uptake. In xenograft models of eCCA cells implanted in mouse liver, poor response to sorafenib was observed. However, tumor growth was markedly reduced by cotreatment with sorafenib and adenoviral vectors encoding hOCT1 under the control of the BIRC5 promoter, a gene highly up-regulated in CCA. Conclusion: The reason for impaired hOCT1-mediated sorafenib uptake by CCA is multifactorial. Gene therapy capable of selectively inducing hOCT1 in tumor cells can be considered a potentially useful chemosensitization strategy to improve the response of CCA to sorafenib.