76 results on '"Lamping E"'
Search Results
2. MICROEVOLUTION OF CANDIDA ALBICANS STRAINS IN OLDER PEOPLE WITH LOW SALIVARY FLOW
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Thiyahuddin, N, primary, Rich, AM, additional, Lamping, E, additional, and Cannon, RD, additional
- Published
- 2021
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3. Milbemycins are broad-spectrum fungal multidrug efflux pump inhibitors that interact directly with the transmembrane domains of Candida albicans Cdr1p and Cdr2p: P110
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Niimi, M., Tanabe, K., Niimi, K., Lamping, E., Nagi, M., Holmes, A. R., Keniya, M. V., Monk, B. C., and Cannon, R. D.
- Published
- 2012
4. Clinically significant micafungin resistance in Candida albicans involves modification of a glucan synthase catalytic subunit GSC1 (FKS1) allele followed by loss of heterozygosity
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Niimi, K., Monk, B. C., Hirai, A., Hatakenaka, K., Umeyama, T., Lamping, E., Maki, K., Tanabe, K., Kamimura, T., Ikeda, F., Uehara, Y., Kano, R., Hasegawa, A., Cannon, R. D., and Niimi, M.
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- 2010
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5. Synthetic Organotellurium Compounds Sensitize Drug-Resistant Candida albicans Clinical Isolates to Fluconazole
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Reis de Sá, L. F., primary, Toledo, F. T., additional, Gonçalves, A. C., additional, Sousa, B. A., additional, dos Santos, A. A., additional, Brasil, P. F., additional, Duarte da Silva, V. A., additional, Tessis, A. C., additional, Ramos, J. A., additional, Carvalho, M. A., additional, Lamping, E., additional, and Ferreira-Pereira, A., additional
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- 2017
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6. A phase I, open-label, multiple-ascending-dose trial to investigate the safety, tolerability, pharmacokinetics, biological, and clinical activity of M7824, a novel bifunctional fusion protein targeting the PD-L1 and TGF-β pathways, in patients with metastatic or locally advanced solid tumors
- Author
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Strauss, J., primary, Heery, C., additional, Schlom, J., additional, Madan, R.A., additional, Lamping, E., additional, Marte, J., additional, Cordes, L., additional, Lan, Y., additional, Mahnke, L., additional, Helwig, C., additional, Lo, K.M., additional, and Gulley, J., additional
- Published
- 2016
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7. 314 - A phase I, open-label, multiple-ascending-dose trial to investigate the safety, tolerability, pharmacokinetics, biological, and clinical activity of M7824, a novel bifunctional fusion protein targeting the PD-L1 and TGF-β pathways, in patients with metastatic or locally advanced solid tumors
- Author
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Strauss, J., Heery, C., Schlom, J., Madan, R.A., Lamping, E., Marte, J., Cordes, L., Lan, Y., Mahnke, L., Helwig, C., Lo, K.M., and Gulley, J.
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- 2016
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8. Yeast membrane protein expression system and its application in drug screening.
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UCL - SSS/DDUV - Institut de Duve, Monk, Brian, Cannon, RD, Nakamura, K, Niimi, M, Niimi, K, Holmes, AR, Lamping, E, Harding, DRK, Goffeau, André, Decottignies, Anabelle, UCL - SSS/DDUV - Institut de Duve, Monk, Brian, Cannon, RD, Nakamura, K, Niimi, M, Niimi, K, Holmes, AR, Lamping, E, Harding, DRK, Goffeau, André, and Decottignies, Anabelle
- Abstract
This invention relates to a protein expression system, particularly although by no means exclusively, to a plasma membrane protein expression system, and the application of this system in understanding the basic biology of membrane bound proteins and in drug discovery.
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- 2003
9. Overexpression of Candida albicans CDR1 , CDR2 , or MDR1 Does Not Produce Significant Changes in Echinocandin Susceptibility
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Niimi, K., primary, Maki, K., additional, Ikeda, F., additional, Holmes, A. R., additional, Lamping, E., additional, Niimi, M., additional, Monk, B. C., additional, and Cannon, R. D., additional
- Published
- 2006
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10. Mas37p, a novel receptor subunit for protein import into mitochondria.
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Gratzer, S, primary, Lithgow, T, additional, Bauer, R E, additional, Lamping, E, additional, Paltauf, F, additional, Kohlwein, S D, additional, Haucke, V, additional, Junne, T, additional, Schatz, G, additional, and Horst, M, additional
- Published
- 1995
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11. Isolation and characterization of a mutant of Saccharomyces cerevisiae with pleiotropic deficiencies in transcriptional activation and repression.
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Lamping, E, primary, Lückl, J, additional, Paltauf, F, additional, Henry, S A, additional, and Kohlwein, S D, additional
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- 1994
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12. Coordinate regulation of phosphatidylserine decarboxylase in Saccharomyces cerevisiae
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Lamping, E, primary, Kohlwein, S D, additional, Henry, S A, additional, and Paltauf, F, additional
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- 1991
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13. Overexpression of Candida albicans CDR1, CDR2, or MDR1Does Not Produce Significant Changes in Echinocandin Susceptibility
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Niimi, K., Maki, K., Ikeda, F., Holmes, A. R., Lamping, E., Niimi, M., Monk, B. C., and Cannon, R. D.
- Abstract
ABSTRACTThe micafungin and caspofungin susceptibilities of Candida albicanslaboratory and clinical isolates and of Saccharomyces cerevisiaestrains stably hyperexpressing fungal ATP-binding cassette (ABC) or major facilitator superfamily (MFS) transporters involved in azole resistance were determined using three separate methods. Yeast strains hyperexpressing individual alleles of ABC transporters or an MFS transporter from C. albicansgave the expected resistance profiles for the azoles fluconazole, itraconazole, and voriconazole. The strains hyperexpressing CDR2showed slightly decreased susceptibility to caspofungin in agar plate drug resistance assays, as previously reported, but increased susceptibility to micafungin compared with either the strains hyperexpressing CDR1or the null parent deleted of seven ABC transporters. The strains hyperexpressing CDR1showed slightly decreased susceptibility to micafungin in these assays. A C. albicansclinical isolate overexpressing both Cdr1p and Cdr2p relative to its azole-sensitive isogenic progenitor acquired resistance to azole drugs and showed reduced susceptibility to caspofungin and slightly increased susceptibility to micafungin in agar plate drug resistance assays. None of the strains showed significant resistance to micafungin or caspofungin in liquid microdilution susceptibility assays. The antifungal activities of micafungin and caspofungin were similar in agarose diffusion assays, although the shape and size of the caspofungin inhibitory zones were affected by medium composition. The assessment of micafungin and caspofungin potency is therefore assay dependent; the differences seen with agar plate drug resistance assays occur over narrow ranges of echinocandin concentrations and are not of clinical significance.
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- 2006
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14. 1614P PSA responses and PSMA scan changes after immunotherapy for biochemically recurrent prostate cancer (BCR) without androgen deprivation therapy (ADT).
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Madan, R.A., Mena Gonzalez, E., Chandran, E.B.A., Aragon-Ching, J.B., Arlen, P.M., Lindenberg, L., Chen, C., Cordes, L., McMahon, S., Lamping, E., Hankin, A., Williams, N., McKinney, Y., Figg, W., Gulley, J.L., Choyke, P.L., and Karzai, F.
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ANDROGEN deprivation therapy , *PROSTATE cancer , *IMMUNOTHERAPY , *PROSTATE-specific antigen - Published
- 2024
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15. Synthetic Organotellurium Compounds Sensitize Drug-Resistant Candida albicansClinical Isolates to Fluconazole
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Reis de Sá, L. F., Toledo, F. T., Gonçalves, A. C., Sousa, B. A., dos Santos, A. A., Brasil, P. F., Duarte da Silva, V. A., Tessis, A. C., Ramos, J. A., Carvalho, M. A., Lamping, E., and Ferreira-Pereira, A.
- Abstract
ABSTRACTInvasive Candida albicansinfections are a serious health threat for immunocompromised individuals. Fluconazole is most commonly used to treat these infections, but resistance due to the overexpression of multidrug efflux pumps is of grave concern. This study evaluated the ability of five synthetic organotellurium compounds to reverse the fluconazole resistance of C. albicansclinical isolates. Compounds 1 to 4, at <10 μg/ml, ameliorated the fluconazole resistance of Saccharomyces cerevisiaestrains overexpressing the major C. albicansmultidrug efflux pumps Cdr1p and Mdr1p, whereas compound 5 only sensitized Mdr1p-overexpressing strains to fluconazole. Compounds 1 to 4 also inhibited efflux of the fluorescent substrate rhodamine 6G and the ATPase activity of Cdr1p, whereas all five of compounds 1 to 5 inhibited Nile red efflux by Mdr1p. Interestingly, all five compounds demonstrated synergy with fluconazole against efflux pump-overexpressing fluconazole-resistant C. albicansclinical isolates, isolate 95-142 overexpressing CDR1and CDR2, isolate 96-25 overexpressing MDR1and ERG11, and isolate 12-99 overexpressing CDR1, CDR2, MDR1, and ERG11. Overall, organotellurium compounds 1 and 2 were the most promising fluconazole chemosensitizers of fluconazole-resistant C. albicansisolates. Our data suggest that these novel organotellurium compounds inhibit pump efflux by two very important and distinct families of fungal multidrug efflux pumps: the ATP-binding cassette transporter Cdr1p and the major facilitator superfamily transporter Mdr1p.
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- 2016
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16. Efficacy, safety, and biomarker analyses of bintrafusp alfa, a bifunctional fusion protein targeting TGF-β and PD-L1, in patients with advanced non-small cell lung cancer.
- Author
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Rajan A, Abdul Sater H, Rahma O, Agajanian R, Lassoued W, Marté JL, Tsai YT, Donahue RN, Lamping E, Bailey S, Weisman A, Walter-Rodriguez B, Ito R, Vugmeyster Y, Sato M, Machl A, Schlom J, and Gulley JL
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- Humans, B7-H1 Antigen, Immunologic Factors therapeutic use, Immunotherapy, Tumor Microenvironment, Carcinoma, Non-Small-Cell Lung pathology, Lung Neoplasms pathology
- Abstract
Background: Bintrafusp alfa, a first-in-class bifunctional fusion protein targeting transforming growth factor-β (TGF-β) and programmed cell death ligand 1, has demonstrated encouraging efficacy as second-line treatment in patients with non-small cell lung cancer (NSCLC) in a dose expansion cohort of the phase 1, open-label clinical trial (NCT02517398). Here, we report the safety, efficacy, and biomarker analysis of bintrafusp alfa in a second expansion cohort of the same trial (biomarker cohort)., Methods: Patients with stage IIIb/IV NSCLC who were either immune checkpoint inhibitor (ICI)-naïve (n=18) or ICI-experienced (n=23) were enrolled. The primary endpoint was the best overall response. Paired biopsies (n=9/41) and peripheral blood (n=14/41) pretreatment and on-treatment were studied to determine the immunological effects of treatment and for associations with clinical activity., Results: Per independent review committee assessment, objective responses were observed in the ICI-naïve group (overall response rate, 27.8%). No new or unexpected safety signals were identified. Circulating TGF-β levels were reduced (>97%; p<0.001) 2 weeks after initiation of treatment with bintrafusp alfa and remained reduced up to 12 weeks. Increases in lymphocytes and tumor-associated macrophages (TAMs) were observed in on-treatment biospies, with an increase in the M2 (tumor trophic TAMs)/M1 (inflammatory TAMs) ratio associated with poor outcomes. Specific peripheral immune analytes at baseline and early changes after treatment were associated with clinical response., Conclusions: Bintrafusp alfa was observed to have modest clinical activity and manageable safety, and was associated with notable immunologic changes involving modulation of the tumor immune microenvironment in patients with advanced NSCLC., Competing Interests: Competing interests: AR reports research funding to his institution from Promontory Therapeutics Inc. OR reports leadership roles as vice president and global clinical strategy head of gastrointestinal oncology at AstraZeneca and reports having stocks at AstraZeneca. RI reports employment with Merck Biopharma Co., Ltd., Tokyo, Japan, an affiliate of Merck KGaA and has attended the independent data monitoring committee/steering committee as part of employment responsibilities. YV reports employment with EMD Serono Research & Development Institute, Inc., Billerica, Massachusetts, USA, an affiliate of Merck KGaA and patent applications. MS reports employment with Merck Biopharma Co., Ltd., Tokyo, Japan, an affiliate of Merck KGaA. AM reports employment with EMD Serono Research & Development Institute, Inc., Billerica, Massachusetts, USA, an affiliate of Merck KGaA. JS reports patents. JLG reports patents and is vice president of the Society for Immunotherapy of Cancer. The remaining authors declare no conflicts of interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)
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- 2024
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17. Analysis of Time Between Skin Lesion and Lymph Node Biopsies and Lymph Node Metastasis in Patients With Melanoma.
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Le ELH, Lamping E, Helmkamp L, Bone J, McCarter M, Kounalakis N, and Stewart C
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- Humans, Lymphatic Metastasis pathology, Biopsy, Lymph Nodes pathology, Melanoma pathology, Skin Neoplasms pathology
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- 2023
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18. A Phase I Single-Arm Study of Biweekly NHS-IL12 in Patients With Metastatic Solid Tumors.
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Gatti-Mays ME, Tschernia NP, Strauss J, Madan RA, Karzai FH, Bilusic M, Redman J, Sater HA, Floudas CS, Toney NJ, Donahue RN, Jochems C, Marté JL, Francis D, McMahon S, Lamping E, Cordes L, Schlom J, and Gulley JL
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- Humans, State Medicine, Interleukin-12 therapeutic use, Recombinant Fusion Proteins therapeutic use, Neoplasms drug therapy, Neoplasms pathology, Neoplasms, Second Primary
- Abstract
Background: NHS-IL12 is a first-in-class, recombinant fusion protein composed of the human monoclonal antibody NHS76 (binds exposed DNA/histones at sites of intratumoral necrosis) fused to 2 IL-12 heterodimers. The maximum tolerated dose (MTD) and recommended phase II dose (RP2D) of NHS-IL12 monotherapy given subcutaneously (SC) every 4 weeks was previously reported. The study was expanded to include a high-exposure cohort with NHS-IL12 SC every 2 weeks (q2w)., Methods: This single-arm, phase I trial evaluated NHS-IL12 12 µg/kg SC q2w or 16.8µg/kg SC q2w in patients with metastatic solid tumors. The primary endpoint was safety., Results: Using a 3+3 design, 13 patients with advanced cancer were enrolled and 12 were dose-limiting toxicity (DLT) evaluable. There was 1 DLT (Grade 3 aspartate transaminase/alanine transaminase [AST/ALT] elevation). Other grade 3 toxicities included: flu-like symptoms 1/13 (8%), decreased absolute lymphocyte count (ALC) 1/13 (8%), decreased white blood cell count (WBC) 1/13 (8%), but most adverse events reported were low grade and self-limiting grade. Fifty percent of evaluable patients (6/12) experienced stable disease (SD) with 42% (5/12) developing progressive disease (PD) at the first restaging., Conclusion: Biweekly NHS-IL12 was well tolerated in this small phase I study. Additional studies incorporating NHS-IL12 with other immunomodulating agents are underway. (ClinicalTrials.gov Identifier: NCT01417546)., (Published by Oxford University Press 2023.)
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- 2023
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19. Correction: Residues forming the gating regions of asymmetric multidrug transporter Pdr5 also play roles in conformational switching and protein folding.
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Alhumaidi M, Nentwig LM, Rahman H, Schmitt L, Rudrow A, Harris A, Dillon C, Restrepo L, Lamping E, Arya N, Ambudkar SV, Choy JS, and Golin J
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- 2023
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20. Kinetics, Kinematics, and Fixed Postures: An Exploration of How Attentional Focus Manipulation Enhances Movement.
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Turner M, Hammer N, Lamping E, Wu WFW, and Becker J
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- Humans, Biomechanical Phenomena, Kinetics, Attention, Posture, Movement
- Abstract
There is debate in the literature regarding how manipulating the focus of attention (FOA) influences ground reaction forces during the standing long jump (SLJ) and gaps in understanding as to which phases of the SLJ are affected (takeoff, flight, and landing) and whether FOA manipulation benefits remain when tasks are performed in fixed body postures. P urpose: This study compared SLJ performance under external (EXT) and internal (INT) FOA conditions with free and fixed postures. M ethods: Twenty participants performed SLJs under EXT and INT FOA conditions while being allowed to swing arms freely and having to keep hands on their hips. Kinematics and kinetics were recorded using 3D-motion capture and force plates. Jump distances, projection angles, and ground reaction forces and impulses were compared across conditions using a 2 × 3 repeated measures ANOVA. R esults: Jump distances were significantly further with EXT FOA ( p < .001). These differences were due to increases in the takeoff distance ( p < .001) and landing distances ( p < .001), with flight distances not being different between the conditions ( p = .061). Peak horizontal ground reaction forces ( p < .001) and impulses ( p < .001) were both greater, while projection angles were lower ( p = .002) in the EXT FOA condition. Co nclusions: Improvements in SLJ distance with an EXT FOA are due to the takeoff and landing phases; manipulating FOA does change forces during the SLJ; and that benefits of an EXT FOA are realized even when movements are performed with constrained body postures.
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- 2023
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21. Residues forming the gating regions of asymmetric multidrug transporter Pdr5 also play roles in conformational switching and protein folding.
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Alhumaidi M, Nentwig LM, Rahman H, Schmitt L, Rudrow A, Harris A, Dillon C, Restrepo L, Lamping E, Arya N, Ambudkar SV, Choy JS, and Golin J
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- Protein Folding, Saccharomyces cerevisiae metabolism, ATP-Binding Cassette Transporters genetics, ATP-Binding Cassette Transporters metabolism, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism
- Abstract
ATP-binding cassette (ABC) multidrug transporters are large, polytopic membrane proteins that exhibit astonishing promiscuity for their transport substrates. These transporters unidirectionally efflux thousands of structurally and functionally distinct compounds. To preclude the reentry of xenobiotic molecules via the drug-binding pocket, these proteins contain a highly conserved molecular gate, essentially allowing the transporters to function as molecular diodes. However, the structure-function relationship of these conserved gates and gating regions are not well characterized. In this study, we combine recent single-molecule, cryo-EM data with genetic and biochemical analyses of residues in the gating region of the yeast multidrug transporter Pdr5, the founding member of a large group of clinically relevant asymmetric ABC efflux pumps. Unlike the symmetric ABCG2 efflux gate, the Pdr5 counterpart is highly asymmetric, with only four (instead of six) residues comprising the gate proper. However, other residues in the near vicinity are essential for the gating activity. Furthermore, we demonstrate that residues in the gate and in the gating regions have multiple functions. For example, we show that Ile-685 and Val-1372 are required not only for successful efflux but also for allosteric inhibition of Pdr5 ATPase activity. Our investigations reveal that the gating region residues of Pdr5, and possibly other ABCG transporters, play a role not only in molecular gating but also in allosteric regulation, conformational switching, and protein folding., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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22. Voriconazole Treatment Induces a Conserved Sterol/Pleiotropic Drug Resistance Regulatory Network, including an Alternative Ergosterol Biosynthesis Pathway, in the Clinically Important FSSC Species, Fusarium keratoplasticum .
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James JE, Santhanam J, Cannon RD, and Lamping E
- Abstract
Fusarium keratoplasticum is the Fusarium species most commonly associated with human infections (fusariosis). Antifungal treatment of fusariosis is often hampered by limited treatment options due to resistance towards azole antifungals. The mechanisms of antifungal resistance and sterol biosynthesis in fusaria are poorly understood. Therefore, in this study we assessed the transcriptional response of F. keratoplasticum when exposed to voriconazole. Our results revealed a group of dramatically upregulated ergosterol biosynthesis gene duplicates, most notably erg6A (912-fold), cyp51A (52-fold) and ebp1 (20-fold), which are likely part of an alternative ergosterol biosynthesis salvage pathway. The presence of human cholesterol biosynthesis gene homologs in F. keratoplasticum ( ebp1 , dhcr7 and dhcr24_1 , dhcr24_2 and dhcr24_3 ) suggests that additional sterol biosynthesis pathways may be induced in fusaria under other growth conditions or during host invasion. Voriconazole also induced the expression of a number of ABC efflux pumps. Further investigations suggested that the highly conserved master regulator of ergosterol biosynthesis, FkSR, and the pleiotropic drug resistance network that induces zinc-cluster transcription factor FkAtrR coordinate the response of FSSC species to azole antifungal exposure. In-depth genome mining also helped clarify the ergosterol biosynthesis pathways of moulds and provided a better understanding of antifungal drug resistance mechanisms in fusaria.
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- 2022
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23. Morphology, Phenotype, and Molecular Identification of Clinical and Environmental Fusarium solani Species Complex Isolates from Malaysia.
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James JE, Santhanam J, Zakaria L, Mamat Rusli N, Abu Bakar M, Suetrong S, Sakayaroj J, Abdul Razak MF, Lamping E, and Cannon RD
- Abstract
Fusarium infections in humans (fusariosis) and in economically important plants involve species of several Fusarium species complexes. Species of the Fusarium solani species complex (FSSC) are the most frequent cause of human fusariosis. The FSSC comprises more than 60 closely related species that can be separated into three major clades by multi-locus sequence typing (MLST) using translation elongation factor 1-alpha ( TEF1-α ) and RNA polymerase II ( RPB2 ) DNA sequences. The MLST nomenclature for clade 3 of the FSSC assigns numbers to species types (e.g., FSSC 2) and lowercase letters to identify unique haplotypes. The aim of this study was to analyse the genotypic and phenotypic characteristics of 15 environmental and 15 clinical FSSC isolates from Malaysia. MLST was used for the genotypic characterisation of FSSC isolates from various locations within Malaysia, which was complemented by their morphological characterisation on potato dextrose and carnation leaf agar. MLST identified eight different FSSC species: thirteen Fusarium keratoplasticum (i.e., FSSC 2), six Fusarium suttonianum (FSSC 20), five Fusarium falciforme (FSSC 3+4), two Fusarium cyanescens (FSSC 27) , and one each of Fusarium petroliphilum (FSSC 1), Fusarium waltergamsii (FSSC 7), Fusarium sp. (FSSC 12), and Fusarium striatum (FSSC 21). Consistent with previous reports from Malaysia, most (11 of 15) clinical FSSC isolates were F. keratoplasticum and the majority (9 of 15) of environmental isolates were F. suttonianum (5) or F. falciforme (4) strains. The taxonomic relationships of the isolates were resolved phylogenetically. The eight Fusarium species also showed distinct morphological characteristics, but these were less clearly defined and reached across species boundaries. Although TEF1-α and RPB2 sequences were sufficient for the species identification of most FSSC isolates, a more precise MLST scheme needs to be established to reliably assign individual isolates of the species-rich FSSC to their geographically-, epidemiologically-, and host-associated sub-lineages.
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- 2022
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24. Inhibitor-Resistant Mutants Give Important Insights into Candida albicans ABC Transporter Cdr1 Substrate Specificity and Help Elucidate Efflux Pump Inhibition.
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Niimi M, Niimi K, Tanabe K, Cannon RD, and Lamping E
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- Antifungal Agents metabolism, Antifungal Agents pharmacology, Drug Resistance, Fungal genetics, Fluconazole metabolism, Fluconazole pharmacology, Fungal Proteins genetics, Fungal Proteins metabolism, Membrane Transport Proteins genetics, Membrane Transport Proteins metabolism, Substrate Specificity, ATP-Binding Cassette Transporters genetics, ATP-Binding Cassette Transporters metabolism, Candida albicans genetics, Candida albicans metabolism
- Abstract
Overexpression of ATP-binding cassette (ABC) transporters is a major cause of drug resistance in fungal pathogens. Milbemycins, enniatin B, beauvericin, and FK506 are promising leads for broad-spectrum fungal multidrug efflux pump inhibitors. The characterization of naturally generated inhibitor-resistant mutants is a powerful tool to elucidate structure-activity relationships in ABC transporters. We isolated 20 Saccharomyces cerevisiae mutants overexpressing Candida albicans ABC pump Cdr1 variants resistant to fluconazole efflux inhibition by milbemycin α25 (8 mutants), enniatin B (8), or beauvericin (4). The 20 mutations were in just 9 residues at the centers of transmembrane segment 1 (TMS1) (6 mutations), TMS4 (4), TMS5 (4), TMS8 (1), and TMS11 (2) and in A713P (3), a previously reported FK506-resistant "hot spot 1" mutation in extracellular loop 3. Six Cdr1-G521S/C/V/R (TMS1) variants were resistant to all four inhibitors, four Cdr1-M639I (TMS4) variants were resistant to milbemycin α25 and enniatin B, and two Cdr1-V668I/D (TMS5) variants were resistant to enniatin B and beauvericin. The eight milbemycin α25-resistant mutants were altered in four amino acids as follows: G521R, M639I, A713P, and T1355N (TMS11). These four Cdr1 variants responded differently to various types of inhibitors, and each exhibited altered substrate specificity and kinetic properties. The data infer an entry gate function for Cdr1-G521 and a role for Cdr1-A713 in the constitutively high Cdr1 ATPase activity. Cdr1-M639I and -T1355N possibly cause inhibitor resistance by altering TMS contacts near the substrate/inhibitor-binding pocket. Models for the interactions of substrates and different types of inhibitors with Cdr1 at various stages of the transport cycle are presented.
- Published
- 2022
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25. Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis.
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Madani G, Lamping E, Lee HJ, Niimi M, Mitra AK, and Cannon RD
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- Antifungal Agents, Cell Membrane, Candida albicans genetics, Fungal Proteins genetics, Membrane Transport Proteins, Saccharomyces cerevisiae genetics
- Abstract
The successful biochemical and biophysical characterization of ABC transporters depends heavily on the choice of the heterologous expression system. Over the past two decades, we have developed a yeast membrane protein expression platform that has been used to study many important fungal membrane proteins. The expression host Saccharomyces cerevisiae ADΔΔ is deleted in seven major endogenous ABC transporters and it contains the transcription factor Pdr1-3 with a gain-of-function mutation that enables the constitutive overexpression of heterologous membrane protein genes stably integrated as single copies at the genomic PDR5 locus. The creation of versatile plasmid vectors and the optimization of one-step cloning strategies enables the rapid and accurate cloning, mutagenesis, and expression of heterologous ABC transporters. Here, we describe the development and use of a novel protease-cleavable mGFPHis double tag (i.e., the monomeric yeast enhanced green fluorescent protein yEGFP3 fused to a six-histidine affinity purification tag) that was designed to avoid possible interference of the tag with the protein of interest and to increase the binding efficiency of the His tag to nickel-affinity resins. The fusion of mGFPHis to the membrane protein ORF (open reading frame) enables easy quantification of the protein by inspection of polyacrylamide gels and detection of degradation products retaining the mGFPHis tag. We demonstrate how this feature facilitates detergent screening for membrane protein solubilization. A protocol for the efficient, fast, and reliable isolation of the small-scale plasma membrane preparations of the C-terminally tagged Candida albicans multidrug efflux transporter Cdr1 overexpressed in S. cerevisiae ADΔΔ, is presented. This small-scale plasma membrane isolation protocol generates high-quality plasma membranes within a single working day. The plasma membrane preparations can be used to determine the enzyme activities of Cdr1 and Cdr1 mutant variants.
- Published
- 2021
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26. PDR Transporter ABC1 Is Involved in the Innate Azole Resistance of the Human Fungal Pathogen Fusarium keratoplasticum .
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James JE, Lamping E, Santhanam J, and Cannon RD
- Abstract
Fusarium keratoplasticum is arguably the most common Fusarium solani species complex (FSSC) species associated with human infections. Invasive fusariosis is a life-threatening fungal infection that is difficult to treat with conventional azole antifungals. Azole drug resistance is often caused by the increased expression of pleiotropic drug resistance (PDR) ATP-binding cassette (ABC) transporters of the ABCG sub-family. Most investigations of Fusarium ABC transporters associated with azole antifungal drug resistance are limited to plant pathogens. Through the manual curation of the entire ABCG protein family of four FSSC species including the fully annotated genome of the plant pathogen Nectria haematococca we identified PDR transporters ABC1 and ABC2 as the efflux pump candidates most likely to be associated with the innate azole resistance phenotype of Fusarium keratoplasticum . An initial investigation of the transcriptional response of logarithmic phase F. keratoplasticum cells to 16 mg/L voriconazole confirmed strong upregulation (372-fold) of ABC1 while ABC2 mRNA levels were unaffected by voriconazole exposure over a 4 h time-period. Overexpression of F. keratoplasticum ABC1 and ABC2 in the genetically modified Saccharomyces cerevisiae host ADΔΔ caused up to ∼1,024-fold increased resistance to a number of xenobiotics, including azole antifungals. Although ABC1 and ABC2 were only moderately (20% and 10%, respectively) expressed compared to the Candida albicans multidrug efflux pump CDR1 , overexpression of F. keratoplasticum ABC1 caused even higher resistance levels to certain xenobiotics (e.g., rhodamine 6G and nigericin) than CDR1 . Our investigations suggest an important role for ABC1 orthologues in the innate azole resistance phenotype of FSSC species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 James, Lamping, Santhanam and Cannon.)
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- 2021
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27. Engineering a Cysteine-Deficient Functional Candida albicans Cdr1 Molecule Reveals a Conserved Region at the Cytosolic Apex of ABCG Transporters Important for Correct Folding and Trafficking of Cdr1.
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Madani G, Lamping E, and Cannon RD
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- ATP Binding Cassette Transporter, Subfamily G metabolism, Cysteine genetics, Mutation, Protein Folding, Protein Transport, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, ATP Binding Cassette Transporter, Subfamily G genetics, Candida albicans genetics, Candida albicans metabolism, Cysteine metabolism, Fungal Proteins genetics, Fungal Proteins metabolism, Membrane Transport Proteins genetics, Membrane Transport Proteins metabolism
- Abstract
Pleiotropic drug resistance (PDR) ATP-binding cassette (ABC) transporters of the ABCG family are eukaryotic membrane proteins that pump an array of compounds across organelle and cell membranes. Overexpression of the archetype fungal PDR transporter Cdr1 is a major cause of azole antifungal drug resistance in Candida albicans , a significant fungal pathogen that can cause life-threatening invasive infections in immunocompromised individuals. To date, no structure for any PDR transporter has been solved. The objective of this project was to investigate the role of the 23 Cdr1 cysteine residues in the stability, trafficking, and function of the protein when expressed in the eukaryotic model organism, Saccharomyces cerevisiae The biochemical characterization of 18 partially cysteine-deficient Cdr1 variants revealed that the six conserved extracellular cysteines were critical for proper expression, localization, and function of Cdr1. They are predicted to form three covalent disulfide bonds that stabilize the large extracellular domains of fungal PDR transporters. Our investigations also revealed a novel nucleotide-binding domain motif, GX
2[3] CPX3 NPAD/E, at the peripheral cytosolic apex of ABCG transporters that possibly contributes to the unique ABCG transport cycle. With this knowledge, we engineered an "almost cysteine-less," yet fully functional, Cdr1 variant, Cdr1P-CID, that had all but the six extracellular cysteines replaced with serine, alanine, or isoleucine (C1106I of the new motif). It is now possible to perform cysteine-cross-linking studies that will enable more detailed biochemical investigations of fungal PDR transporters and confirm any future structure(s) solved for this important protein family. IMPORTANCE Overexpression of the fungal pleiotropic drug resistance (PDR) transporter Cdr1 is a major cause of antifungal drug resistance in Candida albicans , a significant fungal pathogen that can cause life-threatening invasive infections in immunocompromised individuals. To date, no structure for any PDR ABC transporter has been solved. Cdr1 contains 23 cysteines; 10 are cytosolic and 13 are predicted to be in the transmembrane or the extracellular domains. The objective of this project was to create, and biochemically characterize, CDR1 mutants to reveal which cysteines are most important for Cdr1 stability, trafficking, and function. During this process we discovered a novel motif at the cytosolic apex of PDR transporters that ensures the structural and functional integrity of the ABCG transporter family. The creation of a functional Cys-deficient Cdr1 molecule opens new avenues for cysteine-cross-linking studies that will facilitate the detailed characterization of an important ABCG transporter family member., (Copyright © 2021 Madani et al.)- Published
- 2021
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28. Bintrafusp alfa, a bifunctional fusion protein targeting TGF-β and PD-L1, in patients with human papillomavirus-associated malignancies.
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Strauss J, Gatti-Mays ME, Cho BC, Hill A, Salas S, McClay E, Redman JM, Sater HA, Donahue RN, Jochems C, Lamping E, Burmeister A, Marté JL, Cordes LM, Bilusic M, Karzai F, Ojalvo LS, Jehl G, Rolfe PA, Hinrichs CS, Madan RA, Schlom J, and Gulley JL
- Subjects
- Female, Humans, Male, Middle Aged, Neoplasms virology, Papillomavirus Infections pathology, B7-H1 Antigen drug effects, Neoplasms drug therapy, Papillomaviridae drug effects, Papillomavirus Infections complications, Papillomavirus Infections drug therapy, Transforming Growth Factor beta drug effects
- Abstract
Background: Bintrafusp alfa is a first-in-class bifunctional fusion protein composed of the extracellular domain of transforming growth factor (TGF)-βRII (a TGF-β 'trap') fused to a human IgG1 mAb blocking programmed cell death ligand 1. This is the largest analysis of patients with advanced, pretreated human papillomavirus (HPV)-associated malignancies treated with bintrafusp alfa., Methods: In these phase 1 (NCT02517398) and phase 2 trials (NCT03427411), 59 patients with advanced, pretreated, checkpoint inhibitor-naive HPV-associated cancers received bintrafusp alfa intravenously every 2 weeks until progressive disease, unacceptable toxicity, or withdrawal. Primary endpoint was best overall response per Response Evaluation Criteria in Solid Tumors (RECIST) V.1.1; other endpoints included safety., Results: As of April 17, 2019 (phase 1), and October 4, 2019 (phase 2), the confirmed objective response rate per RECIST V.1.1 in the checkpoint inhibitor-naive, full-analysis population was 30.5% (95% CI, 19.2% to 43.9%; five complete responses); eight patients had stable disease (disease control rate, 44.1% (95% CI, 31.2% to 57.6%)). In addition, three patients experienced a delayed partial response after initial disease progression, for a total clinical response rate of 35.6% (95% CI, 23.6% to 49.1%). An additional patient with vulvar cancer had an unconfirmed response. Forty-nine patients (83.1%) experienced treatment-related adverse events, which were grade 3/4 in 16 patients (27.1%). No treatment-related deaths occurred., Conclusion: Bintrafusp alfa showed clinical activity and manageable safety and is a promising treatment in HPV-associated cancers. These findings support further investigation of bintrafusp alfa in patients with advanced, pretreated HPV-associated cancers., Competing Interests: Competing interests: JStrauss, CSH, and JLG disclose inventorship on a National Institutes of Health patent related to the work. JSchlom and JLG disclose that the National Cancer Institute has a Cooperative Research and Development Agreement (CRADA) with EMD Serono Research & Development Institute; an affiliate of Merck KGaA, Darmstadt, Germany. BCC discloses research funding from Novartis, Bayer, AstraZeneca, MOGAM Institute, Dong-A ST, Champions Oncology, Janssen, Yuhan, Ono, Dizal Pharma, and Merck Sharp & Dohme; discloses consultancy with Novartis, AstraZeneca, Boehringer-Ingelheim, Roche, Bristol Myers Squibb, Ono, Yuhan, Pfizer, Eli Lilly, Janssen, Takeda, and Merck Sharp & Dohme; discloses stock ownership with TheraCanVac; and discloses royalty from Champions Oncology. CSH is an employee of Merck KGaA. LSO and PAR are employees of EMD Serono Research and Development Institute. CSH discloses CRADA with Kite Pharma., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)
- Published
- 2020
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29. Decreased working memory capacity among individuals with a mood disorder who have increased metabolic burden.
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Peterman JS, Marshall DF, Lamping E, Easter RE, Babu P, Langenecker SA, McInnis MG, and Ryan KA
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- Adolescent, Adult, Cognition, Humans, Memory, Short-Term, Mood Disorders epidemiology, Young Adult, Bipolar Disorder, Depressive Disorder, Major
- Abstract
Background: Individuals with mood disorders experience a higher rate of obesity than the general population, putting them at risk for poorer outcomes. The relationship between obesity and a core feature of the mood disorders, neurocognition, is less understood. We examined the interaction of obesity as indexed by body mass index (BMI) and working memory performance in a large sample of individuals with bipolar disorder (BD), major depressive disorder (MDD), and healthy controls (HC)., Methods: Participants with BD (n = 133), MDD (n = 78), and HC (n = 113) (age range 18-40) completed a spatial working memory (SWM) task that included three-graded increases in the number of target locations. Participants were subdivided by BMI classification into six diagnostic-BMI (BMI groups: Normal Weight, Overweight/Obese) subgroups. Performance on the task was indexed by number of errors within each difficulty level., Results: The number of errors, across all groups, increased with task difficulty. There was an interaction between errors and diagnostic-BMI group. Post-hoc analyses indicated that while the Normal Weight-BD group did not differ in performance from the other groups, the Overweight/Obese-BD group performed significantly worse than HC groups., Limitations: Metabolic effects of psychotropic medications due to the naturalistic nature of the study, younger age of the MDD sample, and utilizing self-reported indicators of obesity may limit generalizability., Conclusions: Individuals with BD with increased metabolic burden exhibit increased working memory errors than non-psychiatric controls who also have increased metabolic burden. Future work could address prevention and amelioration of such difficulties to reduce associated functional morbidity., Competing Interests: Declaration of Competing Interest Dr. Marshall, Dr. Ryan, Ms. Lamping, Ms. Easter, and Ms. Babu report no competing interests Dr. Langenecker has served as a consultant for Cogstate, Ltd, EPI-Q, and Easter Seals, Inc, in work unrelated to the present work. Dr. McInnis has affiliations with Janssen Pharmaceuticals, (Copyright © 2020 Elsevier B.V. All rights reserved.)
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- 2020
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30. A 23 bp cyp51A Promoter Deletion Associated With Voriconazole Resistance in Clinical and Environmental Isolates of Neocosmospora keratoplastica .
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James JE, Lamping E, Santhanam J, Milne TJ, Abd Razak MF, Zakaria L, and Cannon RD
- Abstract
In the fungal pathogen Aspergillus fumigatus , resistance to azole antifungals is often linked to mutations in CYP51A , a gene that encodes the azole antifungal drug target lanosterol 14α-demethylase. The aim of this study was to investigate whether similar changes could be associated with azole resistance in a Malaysian Fusarium solani species complex (FSSC) isolate collection. Most (11 of 15) clinical FSSC isolates were Neocosmospora keratoplastica and the majority (6 of 10) of environmental isolates were Neocosmospora suttoniana strains. All 25 FSSC isolates had high minimum inhibitory concentrations (MICs) for itraconazole and posaconazole, low MICs for amphotericin B, and various (1 to >32 mg/l) voriconazole susceptibilities. There was a tight association between a 23 bp CYP51A promoter deletion and high (>32 mg/l) voriconazole MICs; of 19 FSSC strains sequenced, nine isolates had voriconazole MICs > 32 mg/l, and they all contained the 23 bp CYP51A promoter deletion, although it was absent in the ten remaining isolates with low (≤12 mg/l) voriconazole MICs. Surprisingly, this association between voriconazole resistance and the 23 bp CYP51A promoter deletion held true across species boundaries. It was randomly distributed within and across species boundaries and both types of FSSC isolates were found among environmental and clinical isolates. Three randomly selected N. keratoplastica isolates with low (≤8 mg/l) voriconazole MICs had significantly lower (1.3-7.5 times) CYP51A mRNA expression levels than three randomly selected N. keratoplastica isolates with high (>32 mg/l) voriconazole MICs. CYP51A expression levels, however, were equally strongly induced (~6,500-fold) by voriconazole in two representative strains reaching levels, after 80 min of induction, that were comparable to those of CYP51B . Our results suggest that FSSC isolates with high voriconazole MICs have a 23 bp CYP51A promoter deletion that provides a potentially useful marker for voriconazole resistance in FSSC isolates. Early detection of possible voriconazole resistance is critical for choosing the correct treatment option for patients with invasive fusariosis., (Copyright © 2020 James, Lamping, Santhanam, Milne, Abd Razak, Zakaria and Cannon.)
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- 2020
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31. A Phase I Dose-Escalation Trial of BN-CV301, a Recombinant Poxviral Vaccine Targeting MUC1 and CEA with Costimulatory Molecules.
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Gatti-Mays ME, Strauss J, Donahue RN, Palena C, Del Rivero J, Redman JM, Madan RA, Marté JL, Cordes LM, Lamping E, Orpia A, Burmeister A, Wagner E, Pico Navarro C, Heery CR, Schlom J, and Gulley JL
- Subjects
- Animals, CD58 Antigens, Humans, Intercellular Adhesion Molecule-1, Mucin-1, T-Lymphocytes immunology, Vaccinia virus immunology, Cancer Vaccines, Carcinoembryonic Antigen
- Abstract
Purpose: BN-CV301 is a poxviral-based vaccine comprised of recombinant (rec.) modified vaccinia Ankara (MVA-BN-CV301; prime) and rec. fowlpox (FPV-CV301; boost). Like its predecessor PANVAC, BN-CV301 contains transgenes encoding tumor-associated antigens MUC1 and CEA as well as costimulatory molecules (B7.1, ICAM-1, and LFA-3). PANVAC was reengineered to make it safer and more antigenic., Patients and Methods: This open-label, 3+3 design, dose-escalation trial evaluated three dose levels (DL) of MVA-BN-CV301: one, two, or four subcutaneous injections of 4 × 10
8 infectious units (Inf.U)/0.5 mL on weeks 0 and 4. All patients received FPV-CV301 subcutaneously at 1 × 109 Inf.U/0.5 mL every 2 weeks for 4 doses, then every 4 weeks. Clinical and immune responses were evaluated., Results: There were no dose-limiting toxicities. Twelve patients enrolled on trial [dose level (DL) 1 = 3, DL2 = 3, DL3 = 6). Most side effects were seen with the prime doses and lessened with subsequent boosters. All treatment-related adverse events were temporary, self-limiting, grade 1/2, and included injection-site reactions and flu-like symptoms. Antigen-specific T cells to MUC1 and CEA, as well as to a cascade antigen, brachyury, were generated in most patients. Single-agent BN-CV301 produced a confirmed partial response (PR) in 1 patient and prolonged stable disease (SD) in multiple patients, most notably in KRAS -mutant gastrointestinal tumors. Furthermore, 2 patients with KRAS -mutant colorectal cancer had prolonged SD when treated with an anti-PD-L1 antibody following BN-CV301., Conclusions: The BN-CV301 vaccine can be safely administered to patients with advanced cancer. Further studies of the vaccine in combination with other agents are planned. See related commentary by Repáraz et al., p. 4871 ., (©2019 American Association for Cancer Research.)- Published
- 2019
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32. Yeast Species in the Oral Cavities of Older People: A Comparison between People Living in Their Own Homes and Those in Rest Homes.
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Thiyahuddin NM, Lamping E, Rich AM, and Cannon RD
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Oral candidiasis is prevalent among older people due to predisposing factors such as impaired immune defenses, medications and denture use. An increasing number of older people live in rest home facilities and it is unclear how this institutionalized living affects the quantity and type of fungi colonizing these people's oral cavities. Smears and swabs of the palate and tongue and saliva samples were taken from participants residing in rest homes (RH; n = 20) and older people living in their own homes (OH; n = 20). Yeast in samples were quantified and identified by culturing on CHROMagar Candida and sequencing the ITS2 region of rDNA. A higher proportion of RH residents had Candida hyphae present in smears compared to OH participants (35% vs. 30%) although this difference was not statistically significant ( p = 0.74). RH residents had, on average, 23 times as many yeast per mL saliva as OH participants ( p = 0.01). Seven yeast species were identified in OH samples and only five in RH samples, with Candida albicans and Candida glabrata being the most common species isolated from both participant groups. The results indicate that older people living in aged-care facilities were more likely to have candidiasis and have a higher yeast carriage rate than similarly aged people living at home. This may be due to morbidities which led to the need for residential care and/or related to the rest home environment.
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- 2019
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33. First-in-Human Phase I Trial of a Tumor-Targeted Cytokine (NHS-IL12) in Subjects with Metastatic Solid Tumors.
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Strauss J, Heery CR, Kim JW, Jochems C, Donahue RN, Montgomery AS, McMahon S, Lamping E, Marté JL, Madan RA, Bilusic M, Silver MR, Bertotti E, Schlom J, and Gulley JL
- Subjects
- Adult, Aged, Cell Line, Tumor, DNA Fragmentation drug effects, Drug-Related Side Effects and Adverse Reactions immunology, Drug-Related Side Effects and Adverse Reactions pathology, Female, Humans, Immunoglobulin G adverse effects, Influenza, Human chemically induced, Influenza, Human pathology, Interleukin-12 administration & dosage, Interleukin-12 adverse effects, Killer Cells, Natural drug effects, Killer Cells, Natural immunology, Lymphocytes, Tumor-Infiltrating drug effects, Lymphocytes, Tumor-Infiltrating immunology, Male, Maximum Tolerated Dose, Middle Aged, Natural Killer T-Cells drug effects, Natural Killer T-Cells immunology, Neoplasms immunology, Neoplasms pathology, Neoplasms, Second Primary immunology, Neoplasms, Second Primary pathology, Receptors, Antigen, T-Cell genetics, Receptors, Antigen, T-Cell immunology, Recombinant Fusion Proteins adverse effects, Transaminases metabolism, Immunoglobulin G administration & dosage, Interleukin-12 immunology, Neoplasms drug therapy, Neoplasms, Second Primary drug therapy, Recombinant Fusion Proteins administration & dosage
- Abstract
Purpose: The NHS-IL12 immunocytokine is composed of two IL12 heterodimers fused to the NHS76 antibody. Preclinical studies have shown that this antibody targets IL12 to regions of tumor necrosis by binding histones on free DNA fragments in these areas, resulting in enhanced antitumor activity. The objectives of this phase I study were to determine the maximum tolerated dose (MTD) and pharmacokinetics of NHS-IL12 in subjects with advanced solid tumors., Patients and Methods: Subjects ( n = 59) were treated subcutaneously with NHS-IL12 in a single ascending-dose cohort followed by a multiple ascending-dose cohort ( n = 37 with every 4-week dosing)., Results: The most frequently observed treatment-related adverse events (TRAE) included decreased circulating lymphocytes, increased liver transaminases, and flu-like symptoms. Of the grade ≥3 TRAEs, all were transient and only one was symptomatic (hyperhidrosis). The MTD is 16.8 μg/kg. A time-dependent rise in IFNγ and an associated rise in IL10 were observed following NHS-IL12. Of peripheral immune cell subsets evaluated, most noticeable were increases in frequencies of activated and mature natural killer (NK) cells and NKT cells. Based on T-cell receptor sequencing analysis, increases in T-cell receptor diversity and tumor-infiltrating lymphocyte density were observed after treatment where both biopsies and peripheral blood mononuclear cells were available. Although no objective tumor responses were observed, 5 subjects had durable stable disease (range, 6-30+ months)., Conclusions: NHS-IL12 was well tolerated up to a dose of 16.8 μg/kg, which is the recommended phase II dose. Early clinical immune-related activity warrants further studies, including combination with immune checkpoint inhibitors. See related commentary by Lyerly et al., p. 9 ., (©2018 American Association for Cancer Research.)
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- 2019
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34. FK506 Resistance of Saccharomyces cerevisiae Pdr5 and Candida albicans Cdr1 Involves Mutations in the Transmembrane Domains and Extracellular Loops.
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Tanabe K, Bonus M, Tomiyama S, Miyoshi K, Nagi M, Niimi K, Chindamporn A, Gohlke H, Schmitt L, Cannon RD, Niimi M, and Lamping E
- Subjects
- Biological Transport drug effects, Biological Transport genetics, Candida albicans drug effects, Depsipeptides pharmacology, Drug Resistance, Fungal genetics, Saccharomyces cerevisiae drug effects, ATP-Binding Cassette Transporters genetics, Antifungal Agents pharmacology, Candida albicans genetics, Fungal Proteins genetics, Membrane Transport Proteins genetics, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Tacrolimus pharmacology
- Abstract
The 23-membered-ring macrolide tacrolimus, a commonly used immunosuppressant, also known as FK506, is a broad-spectrum inhibitor and an efflux pump substrate of pleiotropic drug resistance (PDR) ATP-binding cassette (ABC) transporters. Little, however, is known about the molecular mechanism by which FK506 inhibits PDR transporter drug efflux. Thus, to obtain further insights we searched for FK506-resistant mutants of Saccharomyces cerevisiae cells overexpressing either the endogenous multidrug efflux pump Pdr5 or its Candida albicans orthologue, Cdr1. A simple but powerful screen gave 69 FK506-resistant mutants with, between them, 72 mutations in either Pdr5 or Cdr1. Twenty mutations were in just three Pdr5/Cdr1 equivalent amino acid positions, T550/T540 and T552/S542 of extracellular loop 1 (EL1) and A723/A713 of EL3. Sixty of the 72 mutations were either in the ELs or the extracellular halves of individual transmembrane spans (TMSs), while 11 mutations were found near the center of individual TMSs, mostly in predicted TMS-TMS contact points, and only two mutations were in the cytosolic nucleotide-binding domains of Pdr5. We propose that FK506 inhibits Pdr5 and Cdr1 drug efflux by slowing transporter opening and/or substrate release, and that FK506 resistance of Pdr5/Cdr1 drug efflux is achieved by modifying critical intramolecular contact points that, when mutated, enable the cotransport of FK506 with other pump substrates. This may also explain why the 35 Cdr1 mutations that caused FK506 insensitivity of fluconazole efflux differed from the 13 Cdr1 mutations that caused FK506 insensitivity of cycloheximide efflux., (Copyright © 2018 American Society for Microbiology.)
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- 2018
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35. Phase I Trial of M7824 (MSB0011359C), a Bifunctional Fusion Protein Targeting PD-L1 and TGFβ, in Advanced Solid Tumors.
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Strauss J, Heery CR, Schlom J, Madan RA, Cao L, Kang Z, Lamping E, Marté JL, Donahue RN, Grenga I, Cordes L, Christensen O, Mahnke L, Helwig C, and Gulley JL
- Subjects
- Adult, Aged, Antibodies, Bispecific pharmacology, Antineoplastic Agents, Immunological pharmacology, Combined Modality Therapy, Female, Humans, Male, Middle Aged, Molecular Targeted Therapy adverse effects, Molecular Targeted Therapy methods, Neoplasm Metastasis, Neoplasm Staging, Neoplasms metabolism, Neoplasms mortality, Neoplasms pathology, Retreatment, Treatment Outcome, Antibodies, Bispecific therapeutic use, Antineoplastic Agents, Immunological therapeutic use, B7-H1 Antigen antagonists & inhibitors, Neoplasms drug therapy, Transforming Growth Factor beta antagonists & inhibitors
- Abstract
Purpose: M7824 (MSB0011359C) is an innovative first-in-class bifunctional fusion protein composed of a mAb against programmed death ligand 1 (PD-L1) fused to a TGFβ "trap." Experimental Design: In the 3+3 dose-escalation component of this phase I study (NCT02517398), eligible patients with advanced solid tumors received M7824 at 1, 3, 10, or 20 mg/kg once every 2 weeks until confirmed progression, unacceptable toxicity, or trial withdrawal; in addition, a cohort received an initial 0.3 mg/kg dose to evaluate pharmacokinetics/pharmacodynamics, followed by 10 mg/kg dosing. The primary objective is to determine the safety and maximum tolerated dose (MTD); secondary objectives include pharmacokinetics, immunogenicity, and best overall response. Results: Nineteen heavily pretreated patients with ECOG 0-1 have received M7824. Grade ≥3 treatment-related adverse events occurred in four patients (skin infection secondary to localized bullous pemphigoid, asymptomatic lipase increase, colitis with associated anemia, and gastroparesis with hypokalemia). The MTD was not reached. M7824 saturated peripheral PD-L1 and sequestered any released plasma TGFβ1, -β2, and -β3 throughout the dosing period at >1 mg/kg. There were signs of efficacy across all dose levels, including one ongoing confirmed complete response (cervical cancer), two durable confirmed partial responses (PR; pancreatic cancer; anal cancer), one near-PR (cervical cancer), and two cases of prolonged stable disease in patients with growing disease at study entry (pancreatic cancer; carcinoid). Conclusions: M7824 has a manageable safety profile in patients with heavily pretreated advanced solid tumors. Early signs of efficacy are encouraging, and multiple expansion cohorts are ongoing in a range of tumors. Clin Cancer Res; 24(6); 1287-95. ©2018 AACR ., (©2018 American Association for Cancer Research.)
- Published
- 2018
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36. Avelumab for metastatic or locally advanced previously treated solid tumours (JAVELIN Solid Tumor): a phase 1a, multicohort, dose-escalation trial.
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Heery CR, O'Sullivan-Coyne G, Madan RA, Cordes L, Rajan A, Rauckhorst M, Lamping E, Oyelakin I, Marté JL, Lepone LM, Donahue RN, Grenga I, Cuillerot JM, Neuteboom B, Heydebreck AV, Chin K, Schlom J, and Gulley JL
- Subjects
- Abdominal Pain chemically induced, Aged, Amylases blood, Antibodies blood, Antibodies, Monoclonal immunology, Antibodies, Monoclonal pharmacokinetics, Antibodies, Monoclonal, Humanized, Antineoplastic Agents immunology, Antineoplastic Agents pharmacokinetics, Aspartate Aminotransferases blood, Autoimmune Diseases chemically induced, Chills chemically induced, Creatine Kinase blood, Dysphonia chemically induced, Fatigue chemically induced, Female, Fever chemically induced, Half-Life, Humans, Male, Middle Aged, Myositis chemically induced, Response Evaluation Criteria in Solid Tumors, Antibodies, Monoclonal administration & dosage, Antibodies, Monoclonal adverse effects, Antineoplastic Agents administration & dosage, Antineoplastic Agents adverse effects, Neoplasms drug therapy
- Abstract
Background: Avelumab (MSB0010718C) is a human IgG1 monoclonal antibody that binds to PD-L1, inhibiting its binding to PD-1, which inactivates T cells. We aimed to establish the safety and pharmacokinetics of avelumab in patients with solid tumours while assessing biological correlatives for future development., Methods: This open-label, single-centre, phase 1a, dose-escalation trial (part of the JAVELIN Solid Tumor trial) assessed four doses of avelumab (1 mg/kg, 3 mg/kg, 10 mg/kg, and 20 mg/kg), with dose-level cohort expansions to provide additional safety, pharmacokinetics, and target occupancy data. This study used a standard 3 + 3 cohort design and assigned patients sequentially at trial entry according to the 3 + 3 dose-escalation algorithm and depending on the number of dose-limiting toxicities during the first 3-week assessment period (the primary endpoint). Patient eligibility criteria included age 18 years or older, Eastern Cooperative Oncology Group performance status 0-1, metastatic or locally advanced previously treated solid tumours, and adequate end-organ function. Avelumab was given as a 1-h intravenous infusion every 2 weeks. Patients in the dose-limiting toxicity analysis set were assessed for the primary endpoint of dose-limiting toxicity, and all patients enrolled in the dose-escalation part were assessed for the secondary endpoints of safety (treatment-emergent and treatment-related adverse events according to National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0), pharmacokinetic and pharmacodynamic profiles (immunological effects), best overall response by Response Evaluation Criteria, and antidrug antibody formation. The population for the pharmacokinetic analysis included a subset of patients with rich pharmacokinetic samples from two selected disease-specific expansion cohorts at the same study site who had serum samples obtained at multiple early timepoints. This trial is registered with ClinicalTrials.gov, number NCT01772004. Patient recruitment to the dose-escalation part reported here is closed., Findings: Between Jan 31, 2013, and Oct 8, 2014, 53 patients were enrolled (four patients at 1 mg/kg, 13 at 3 mg/kg, 15 at 10 mg/kg, and 21 at 20 mg/kg). 18 patients were analysed in the dose-limiting toxicity analysis set: three at dose level 1 (1 mg/kg), three at dose level 2 (3 mg/kg), six at dose level 3 (10 mg/kg), and six at dose level 4 (20 mg/kg). Only one dose-limiting toxicity occurred, at the 20 mg/kg dose, and thus the maximum tolerated dose was not reached. In all 53 enrolled patients (the safety analysis set), common treatment-related adverse events (occurring in >10% of patients) included fatigue (21 patients [40%]), influenza-like symptoms (11 [21%]), fever (8 [15%]), and chills (6 [11%]). Grade 3-4 treatment-related adverse events occurred in nine (17%) of 53 patients, with autoimmune disorder (n=3), increased blood creatine phosphokinase (n=2), and increased aspartate aminotransferase (n=2) each occurring in more than one patient (autoimmune disorder in two patients at 10 mg/kg and one patient at 20 mg/kg, increased blood creatine phosphokinase in two patients at 20 mg/kg, and increased aspartate aminotransferase in one patient at 1 mg/kg, and one patient at 10 mg/kg). Six (11%) of 53 patients had a serious treatment-related adverse event: autoimmune disorder (two [13%]), lower abdominal pain (one [7%]), fatigue (one [7%]), and influenza-like illness (one [7%]) in three patients treated at 10 mg/kg dose level, and autoimmune disorder (one [5%]), increased amylase (one [5%]), myositis (one [5%]), and dysphonia (one [5%]) in three patients who received the 20 mg/kg dose. We recorded some evidence of clinical activity in various solid tumours, with partial confirmed or unconfirmed responses in four (8%) of 53 patients; 30 (57%) additional patients had stable disease. Pharmacokinetic analysis (n=86) showed a dose-proportional exposure between doses of 3 mg/kg and 20 mg/kg and a half-life of 95-99 h (3·9-4·1 days) at the 10 mg/kg and 20 mg/kg doses. Target occupancy was greater than 90% at doses of 3 mg/kg and 10 mg/kg. Antidrug antibodies were detected in two (4%) of 53 patients. No substantial differences were found in absolute lymphocyte count or multiple immune cell subsets, including those expressing PD-L1, after treatment with avelumab. 31 (58%) of 53 patients in the overall safety population died; no deaths were related to treatment on study., Interpretation: Avelumab has an acceptable toxicity profile up to 20 mg/kg and the maximum tolerated dose was not reached. Based on pharmacokinetics, target occupancy, and immunological analysis, we chose 10 mg/kg every 2 weeks as the dose for further development and phase 3 trials are ongoing., Funding: National Cancer Institute and Merck KGaA., (Copyright © 2017 Elsevier Ltd. All rights reserved.)
- Published
- 2017
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37. Role of Ectopic Gene Conversion in the Evolution of a Candida krusei Pleiotropic Drug Resistance Transporter Family.
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Lamping E, Zhu JY, Niimi M, and Cannon RD
- Subjects
- Candida drug effects, DNA Copy Number Variations, Drug Resistance, Fungal, Candida genetics, Evolution, Molecular, Fungal Proteins genetics, Gene Conversion, Genetic Pleiotropy, Multidrug Resistance-Associated Proteins genetics
- Abstract
Gene duplications enable the evolution of novel gene function, but strong positive selection is required to preserve advantageous mutations in a population. This is because frequent ectopic gene conversions (EGCs) between highly similar, tandem-duplicated, sequences, can rapidly remove fate-determining mutations by replacing them with the neighboring parent gene sequences. Unfortunately, the high sequence similarities between tandem-duplicated genes severely hamper empirical studies of this important evolutionary process, because deciphering their correct sequences is challenging. In this study, we employed the eukaryotic model organism Saccharomyces cerevisiae to clone and functionally characterize all 30 alleles of an important pair of tandem-duplicated multidrug efflux pump genes, ABC1 and ABC11 , from seven strains of the diploid pathogenic yeast Candida krusei Discovery and functional characterization of their closest ancestor, C. krusei ABC12 , helped elucidate the evolutionary history of the entire gene family. Our data support the proposal that the pleiotropic drug resistance (PDR) transporters Abc1p and Abc11p have evolved by concerted evolution for ∼134 MY. While >90% of their sequences remained identical, very strong purifying selection protected six short DNA patches encoding just 18 core amino acid (aa) differences in particular trans membrane span (TMS) regions causing two distinct efflux pump functions. A proline-kink change at the bottom of Abc11p TMS3 was possibly fate determining. Our data also enabled the first empirical estimates for key parameters of eukaryotic gene evolution, they provided rare examples of intron loss, and PDR transporter phylogeny confirmed that C. krusei belongs to a novel, yet unnamed, third major Saccharomycotina lineage., (Copyright © 2017 by the Genetics Society of America.)
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- 2017
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38. Synthetic Organotellurium Compounds Sensitize Drug-Resistant Candida albicans Clinical Isolates to Fluconazole.
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Reis de Sá LF, Toledo FT, Gonçalves AC, Sousa BA, Dos Santos AA, Brasil PF, Duarte da Silva VA, Tessis AC, Ramos JA, Carvalho MA, Lamping E, and Ferreira-Pereira A
- Subjects
- Candida albicans genetics, Candida albicans metabolism, Drug Resistance, Fungal genetics, Fungal Proteins genetics, Fungal Proteins metabolism, Gene Expression Regulation, Fungal drug effects, Gene Expression Regulation, Fungal genetics, Microbial Sensitivity Tests, Organotechnetium Compounds pharmacology, Saccharomyces cerevisiae drug effects, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Antifungal Agents pharmacology, Candida albicans drug effects, Fluconazole pharmacology
- Abstract
Invasive Candida albicans infections are a serious health threat for immunocompromised individuals. Fluconazole is most commonly used to treat these infections, but resistance due to the overexpression of multidrug efflux pumps is of grave concern. This study evaluated the ability of five synthetic organotellurium compounds to reverse the fluconazole resistance of C. albicans clinical isolates. Compounds 1 to 4, at <10 μg/ml, ameliorated the fluconazole resistance of Saccharomyces cerevisiae strains overexpressing the major C. albicans multidrug efflux pumps Cdr1p and Mdr1p, whereas compound 5 only sensitized Mdr1p-overexpressing strains to fluconazole. Compounds 1 to 4 also inhibited efflux of the fluorescent substrate rhodamine 6G and the ATPase activity of Cdr1p, whereas all five of compounds 1 to 5 inhibited Nile red efflux by Mdr1p. Interestingly, all five compounds demonstrated synergy with fluconazole against efflux pump-overexpressing fluconazole-resistant C. albicans clinical isolates, isolate 95-142 overexpressing CDR1 and CDR2, isolate 96-25 overexpressing MDR1 and ERG11, and isolate 12-99 overexpressing CDR1, CDR2, MDR1, and ERG11 Overall, organotellurium compounds 1 and 2 were the most promising fluconazole chemosensitizers of fluconazole-resistant C. albicans isolates. Our data suggest that these novel organotellurium compounds inhibit pump efflux by two very important and distinct families of fungal multidrug efflux pumps: the ATP-binding cassette transporter Cdr1p and the major facilitator superfamily transporter Mdr1p., (Copyright © 2016 American Society for Microbiology.)
- Published
- 2016
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39. Identification and functional characterization of Penicillium marneffei pleiotropic drug resistance transporters ABC1 and ABC2.
- Author
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Panapruksachat S, Iwatani S, Oura T, Vanittanakom N, Chindamporn A, Niimi K, Niimi M, Lamping E, Cannon RD, and Kajiwara S
- Subjects
- Asia, Southeastern, Cloning, Molecular, Gene Expression, Genome, Fungal, Humans, Penicillium isolation & purification, Protein Transport, Saccharomyces cerevisiae drug effects, Saccharomyces cerevisiae genetics, Antifungal Agents metabolism, Antifungal Agents pharmacology, Drug Resistance, Fungal, Membrane Transport Proteins genetics, Membrane Transport Proteins metabolism, Penicillium genetics, Penicillium metabolism
- Abstract
Penicilliosis caused by the dimorphic fungus Penicillium marneffei is an endemic, AIDS-defining illness and, after tuberculosis and cryptococcosis, the third most common opportunistic infection of AIDS patients in tropical Southeast Asia. Untreated, patients have poor prognosis; however, primary amphotericin B treatment followed by prolonged itraconazole prophylaxis is effective. To identify ATP-binding cassette (ABC) transporters that may play a role in potential multidrug resistance of P. marneffei, we identified and classified all 46 P. marneffei ABC transporters from the genome sequence. PmABC1 and PmABC2 were most similar to the archetype Candida albicans multidrug efflux pump gene CDR1. P. marneffei Abc1p (PmAbc1p) was functionally expressed in Saccharomyces cerevisiae, although at rather low levels, and correctly localized to the plasma membrane, causing cells to be fourfold to eightfold more resistant to azoles and many other xenobiotics than untransformed cells. P. marneffei Abc2p (PmAbc2p) was expressed at similarly low levels, but it had no efflux activity and did not properly localize to the plasma membrane. Interestingly, PmAbc1p mislocalized and lost its transport activity when cells were shifted to 37 °C. We conclude that expression of PmAbc1p in S. cerevisiae confers resistance to several xenobiotics indicating that PmAbc1p may be a multidrug efflux pump., (© The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2016
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40. Identification and characterization of Candida utilis multidrug efflux transporter CuCdr1p.
- Author
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Watanasrisin W, Iwatani S, Oura T, Tomita Y, Ikushima S, Chindamporn A, Niimi M, Niimi K, Lamping E, Cannon RD, and Kajiwara S
- Subjects
- Antifungal Agents pharmacology, Cloning, Molecular, Drug Resistance, Multiple, Fungal, Gene Expression, Recombinant Proteins genetics, Recombinant Proteins metabolism, Saccharomyces cerevisiae drug effects, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, ATP-Binding Cassette Transporters genetics, ATP-Binding Cassette Transporters metabolism, Candida genetics, Candida metabolism
- Abstract
The edible, nitrate assimilating, yeast Candida utilis is a commercial food additive, and it is a potentially useful host for heterologous protein expression. A number of ATP-binding cassette (ABC) transporters are multidrug efflux pumps that can cause multidrug resistance in opportunistic pathogens. In order to develop optimal novel antimicrobial agents it is imperative to understand the structure, function and expression of these transporters. With the ultimate aim of developing an alternative yeast host for the heterologous expression of eukaryotic membrane transporters, and to identify ABC transporters potentially associated with C. utilis multidrug resistance, we classified the entire repertoire of 30 C. utilis ABC proteins. We named the open reading frame most similar to the archetype multidrug efflux pump gene C. albicans CDR1 as CuCDR1 Overexpression of CuCDR1 in Saccharomyces cerevisiae ADΔ caused multidrug resistance similar to that of cells overexpressing CaCDR1 Unlike CaCdr1p, however, the C-terminally green fluorescent protein (GFP) tagged CuCdr1p-GFP was functionally impaired and did not properly localize to the plasma membrane. CuCdr1p function could be recovered however by adding a 15 amino acid linker -GAGGSAGGSGGAGAG- between CuCdr1p and the C-terminal GFP tag., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2016
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41. Bone marrow abnormalities and early bone lesions in multiple myeloma and its precursor disease: a prospective study using functional and morphologic imaging.
- Author
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Bhutani M, Turkbey B, Tan E, Korde N, Kwok M, Manasanch EE, Tageja N, Mailankody S, Roschewski M, Mulquin M, Carpenter A, Lamping E, Minter AR, Weiss BM, Mena E, Lindenberg L, Calvo KR, Maric I, Usmani SZ, Choyke PL, Kurdziel K, and Landgren O
- Subjects
- Adult, Aged, Aged, 80 and over, Biomarkers, Female, Fluorodeoxyglucose F18, Humans, Magnetic Resonance Imaging, Male, Middle Aged, Monoclonal Gammopathy of Undetermined Significance diagnostic imaging, Positron Emission Tomography Computed Tomography, Bone Marrow pathology, Bone and Bones pathology, Multiple Myeloma diagnostic imaging, Precancerous Conditions
- Abstract
The incidence and importance of bone marrow involvement and/or early bone lesions in multiple myeloma (MM) precursor diseases is largely unknown. This study prospectively compared the sensitivity of several imaging modalities in monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM) and MM. Thirty patients (10 each with MGUS, SMM and MM) were evaluated with skeletal survey, [18F]FDG-PET/CT, [18F]NaF-PET/CT and morphologic dynamic contrast enhanced (DCE)-MRI. An additional 16 SMM patients had skeletal surveys and FDG-PET/CT. Among MGUS patients, DCE-MRI found only one focal marrow abnormality; other evaluations were negative. Among 26 SMM patients, five (19%) were re-classified as MM based on lytic bone lesions on CT and six had unifocal or diffuse marrow abnormality. Among MM, marrow abnormalities were observed on FDG-PET/CT in 8/10 patients and on DCE-MRI in nine evaluable patients. Abnormal NaF uptake was observed only in MM patients with lytic lesions on CT, providing no additional clinical information.
- Published
- 2016
- Full Text
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42. Drug resistance is conferred on the model yeast Saccharomyces cerevisiae by expression of full-length melanoma-associated human ATP-binding cassette transporter ABCB5.
- Author
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Keniya MV, Holmes AR, Niimi M, Lamping E, Gillet JP, Gottesman MM, and Cannon RD
- Subjects
- ATP Binding Cassette Transporter, Subfamily B, ATP Binding Cassette Transporter, Subfamily B, Member 1 genetics, Clorgyline pharmacology, Daunorubicin pharmacology, Humans, Rhodamine 123 pharmacology, Rhodamines pharmacology, Saccharomyces cerevisiae genetics, Tacrolimus pharmacology, ATP Binding Cassette Transporter, Subfamily B, Member 1 metabolism, Melanoma metabolism, Saccharomyces cerevisiae drug effects, Saccharomyces cerevisiae metabolism
- Abstract
ABCB5, an ATP-binding cassette (ABC) transporter, is highly expressed in melanoma cells, and may contribute to the extreme resistance of melanomas to chemotherapy by efflux of anti-cancer drugs. Our goal was to determine whether we could functionally express human ABCB5 in the model yeast Saccharomyces cerevisiae, in order to demonstrate an efflux function for ABCB5 in the absence of background pump activity from other human transporters. Heterologous expression would also facilitate drug discovery for this important target. DNAs encoding ABCB5 sequences were cloned into the chromosomal PDR5 locus of a S. cerevisiae strain in which seven endogenous ABC transporters have been deleted. Protein expression in the yeast cells was monitored by immunodetection using both a specific anti-ABCB5 antibody and a cross-reactive anti-ABCB1 antibody. ABCB5 function in recombinant yeast cells was measured by determining whether the cells possessed increased resistance to known pump substrates, compared to the host yeast strain, in assays of yeast growth. Three ABCB5 constructs were made in yeast. One was derived from the ABCB5-β mRNA, which is highly expressed in human tissues but is a truncation of a canonical full-size ABC transporter. Two constructs contained full-length ABCB5 sequences: either a native sequence from cDNA or a synthetic sequence codon-harmonized for S. cerevisiae. Expression of all three constructs in yeast was confirmed by immunodetection. Expression of the codon-harmonized full-length ABCB5 DNA conferred increased resistance, relative to the host yeast strain, to the putative substrates rhodamine 123, daunorubicin, tetramethylrhodamine, FK506, or clorgyline. We conclude that full-length ABCB5 can be functionally expressed in S. cerevisiae and confers drug resistance.
- Published
- 2014
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43. Small, synthetic, GC-rich mRNA stem-loop modules 5' proximal to the AUG start-codon predictably tune gene expression in yeast.
- Author
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Lamping E, Niimi M, and Cannon RD
- Subjects
- 5' Untranslated Regions, ATP-Binding Cassette Transporters genetics, ATP-Binding Cassette Transporters metabolism, Base Pairing, Candida albicans metabolism, Cloning, Molecular, Codon, Initiator, Fungal Proteins genetics, Fungal Proteins metabolism, Gene Expression, Genetic Vectors genetics, Genetic Vectors metabolism, Metabolic Engineering, Nucleic Acid Conformation, RNA, Messenger chemistry, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Thermodynamics, RNA, Messenger biosynthesis, Saccharomyces cerevisiae metabolism
- Abstract
Background: A large range of genetic tools has been developed for the optimal design and regulation of complex metabolic pathways in bacteria. However, fewer tools exist in yeast that can precisely tune the expression of individual enzymes in novel metabolic pathways suitable for industrial-scale production of non-natural compounds. Tuning expression levels is critical for reducing the metabolic burden of over-expressed proteins, the accumulation of toxic intermediates, and for redirecting metabolic flux from native pathways involving essential enzymes without negatively affecting the viability of the host. We have developed a yeast membrane protein hyper-expression system with critical advantages over conventional, plasmid-based, expression systems. However, expression levels are sometimes so high that they adversely affect protein targeting/folding or the growth and/or phenotype of the host. Here we describe the use of small synthetic mRNA control modules that allowed us to predictably tune protein expression levels to any desired level. Down-regulation of expression was achieved by engineering small GC-rich mRNA stem-loops into the 5' UTR that inhibited translation initiation of the yeast ribosomal 43S preinitiation complex (PIC)., Results: Exploiting the fact that the yeast 43S PIC has great difficulty scanning through GC-rich mRNA stem-loops, we created yeast strains containing 17 different RNA stem-loop modules in the 5' UTR that expressed varying amounts of the fungal multidrug efflux pump reporter Cdr1p from Candida albicans. Increasing the length of mRNA stem-loops (that contained only GC-pairs) near the AUG start-codon led to a surprisingly large decrease in Cdr1p expression; ~2.7-fold for every additional GC-pair added to the stem, while the mRNA levels remained largely unaffected. An mRNA stem-loop of seven GC-pairs (∆G = -15.8 kcal/mol) reduced Cdr1p expression levels by >99%, and even the smallest possible stem-loop of only three GC-pairs (∆G = -4.4 kcal/mol) inhibited Cdr1p expression by ~50%., Conclusion: We have developed a simple cloning strategy to fine-tune protein expression levels in yeast that has many potential applications in metabolic engineering and the optimization of protein expression in yeast. This study also highlights the importance of considering the use of multiple cloning-sites carefully to preclude unwanted effects on gene expression.
- Published
- 2013
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44. Specific interactions between the Candida albicans ABC transporter Cdr1p ectodomain and a D-octapeptide derivative inhibitor.
- Author
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Niimi K, Harding DR, Holmes AR, Lamping E, Niimi M, Tyndall JD, Cannon RD, and Monk BC
- Subjects
- Drug Resistance, Fungal, Fungal Proteins genetics, Membrane Transport Proteins genetics, Microbial Sensitivity Tests, Models, Molecular, Protein Binding, Protein Conformation, Suppression, Genetic, Candida albicans enzymology, Enzyme Inhibitors metabolism, Fungal Proteins antagonists & inhibitors, Fungal Proteins metabolism, Membrane Transport Proteins metabolism, Oligopeptides metabolism
- Abstract
Overexpression of the Candida albicans ATP-binding cassette transporter CaCdr1p causes clinically significant resistance to azole drugs including fluconazole (FLC). Screening of a ~1.89 × 10(6) member D-octapeptide combinatorial library that concentrates library members at the yeast cell surface identified RC21v3, a 4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative of the D-octapeptide D-NH(2) -FFKWQRRR-CONH(2) , as a potent and stereospecific inhibitor of CaCdr1p. RC21v3 chemosensitized Saccharomyces cerevisiae strains overexpressing CaCdr1p but not other fungal ABC transporters, the C. albicans MFS transporter CaMdr1p or the azole target enzyme CaErg11p, to FLC. RC21v3 also chemosensitized clinical C. albicans isolates overexpressing CaCDR1 to FLC, even when CaCDR2 was overexpressed. Specific targeting of CaCdr1p by RC21v3 was confirmed by spontaneous RC21v3 chemosensitization-resistant suppressor mutants of S. cerevisiae expressing CaCdr1p. The suppressor mutations introduced a positive charge beside, or within, extracellular loops 1, 3, 4 and 6 of CaCdr1p or an aromatic residue near the extracytoplasmic end of transmembrane segment 5. The mutations did not affect CaCdr1p localization or CaCdr1p ATPase activity but some increased susceptibility to the CaCdr1p substrates FLC, rhodamine 6G, rhodamine 123 and cycloheximide. The suppressor mutations showed that the drug-like CaCdr1p inhibitors FK506, enniatin, milbemycin α11 and milbemycin β9 have modes of action similar to RC21v3., (© 2012 Blackwell Publishing Ltd.)
- Published
- 2012
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45. The monoamine oxidase A inhibitor clorgyline is a broad-spectrum inhibitor of fungal ABC and MFS transporter efflux pump activities which reverses the azole resistance of Candida albicans and Candida glabrata clinical isolates.
- Author
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Holmes AR, Keniya MV, Ivnitski-Steele I, Monk BC, Lamping E, Sklar LA, and Cannon RD
- Subjects
- ATP-Binding Cassette Transporters genetics, ATP-Binding Cassette Transporters metabolism, Biological Transport, Candida albicans enzymology, Candida albicans isolation & purification, Candida glabrata enzymology, Candida glabrata isolation & purification, Drug Resistance, Fungal, Drug Synergism, Flow Cytometry, Fluconazole pharmacology, Fluorescent Dyes, Gene Expression, High-Throughput Screening Assays, Humans, Microbial Sensitivity Tests, Monoamine Oxidase genetics, Monoamine Oxidase metabolism, Organisms, Genetically Modified, Rhodamines, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Small Molecule Libraries, ATP-Binding Cassette Transporters antagonists & inhibitors, Antifungal Agents pharmacology, Candida albicans genetics, Candida glabrata genetics, Clorgyline pharmacology, Monoamine Oxidase Inhibitors pharmacology
- Abstract
Resistance to the commonly used azole antifungal fluconazole (FLC) can develop due to overexpression of ATP-binding cassette (ABC) and major facilitator superfamily (MFS) plasma membrane transporters. An approach to overcoming this resistance is to identify inhibitors of these efflux pumps. We have developed a pump assay suitable for high-throughput screening (HTS) that uses recombinant Saccharomyces cerevisiae strains hyperexpressing individual transporters from the opportunistic fungal pathogen Candida albicans. The recombinant strains possess greater resistance to azoles and other pump substrates than the parental host strain. A flow cytometry-based HTS, which measured increased intracellular retention of the fluorescent pump substrate rhodamine 6G (R6G) within yeast cells, was used to screen the Prestwick Chemical Library (PCL) of 1,200 marketed drugs. Nine compounds were identified as hits, and the monoamine oxidase A inhibitor (MAOI) clorgyline was identified as an inhibitor of two C. albicans ABC efflux pumps, CaCdr1p and CaCdr2p. Secondary in vitro assays confirmed inhibition of pump-mediated efflux by clorgyline. Clorgyline also reversed the FLC resistance of S. cerevisiae strains expressing other individual fungal ABC transporters (Candida glabrata Cdr1p or Candida krusei Abc1p) or the C. albicans MFS transporter Mdr1p. Recombinant strains were also chemosensitized by clorgyline to other azoles (itraconazole and miconazole). Importantly, clorgyline showed synergy with FLC against FLC-resistant C. albicans clinical isolates and a C. glabrata strain and inhibited R6G efflux from a FLC-resistant C. albicans clinical isolate. Clorgyline is a novel broad-spectrum inhibitor of two classes of fungal efflux pumps that acts synergistically with azoles against azole-resistant C. albicans and C. glabrata strains.
- Published
- 2012
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46. Chimeras of Candida albicans Cdr1p and Cdr2p reveal features of pleiotropic drug resistance transporter structure and function.
- Author
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Tanabe K, Lamping E, Nagi M, Okawada A, Holmes AR, Miyazaki Y, Cannon RD, Monk BC, and Niimi M
- Subjects
- ATP-Binding Cassette Transporters genetics, Antifungal Agents metabolism, Antifungal Agents pharmacology, Biological Transport, Candida albicans chemistry, Candida albicans drug effects, Candida albicans genetics, Drug Resistance, Fungal, Fungal Proteins genetics, Protein Structure, Tertiary, Protein Transport, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Saccharomyces cerevisiae drug effects, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, ATP-Binding Cassette Transporters chemistry, ATP-Binding Cassette Transporters metabolism, Candida albicans metabolism, Fungal Proteins chemistry, Fungal Proteins metabolism
- Abstract
Members of the pleiotropic drug resistance (PDR) family of ATP binding cassette (ABC) transporters consist of two homologous halves, each containing a nucleotide binding domain (NBD) and a transmembrane domain (TMD). The PDR transporters efflux a variety of hydrophobic xenobiotics and despite the frequent association of their overexpression with the multidrug resistance of fungal pathogens, the transport mechanism of these transporters is poorly understood. Twenty-eight chimeric constructs between Candida albicans Cdr1p (CaCdr1p) and Cdr2p (CaCdr2p), two closely related but functionally distinguishable PDR transporters, were expressed in Saccharomyces cerevisiae. All chimeras expressed equally well, localized properly at the plasma membrane, retained their transport ability, but their substrate and inhibitor specificities differed significantly between individual constructs. A detailed characterization of these proteins revealed structural features that contribute to their substrate specificities and their transport mechanism. It appears that most transmembrane spans of CaCdr1p and CaCdr2p provide or affect multiple, probably overlapping, substrate and inhibitor binding site(s) similar to mammalian ABC transporters. The NBDs, in particular NBD1 and/or the ∼150 amino acids N-terminal to NBD1, can also modulate the substrate specificities of CaCdr1p and CaCdr2p., (© 2011 Blackwell Publishing Ltd.)
- Published
- 2011
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47. Fungal PDR transporters: Phylogeny, topology, motifs and function.
- Author
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Lamping E, Baret PV, Holmes AR, Monk BC, Goffeau A, and Cannon RD
- Subjects
- ATP-Binding Cassette Transporters chemistry, ATP-Binding Cassette Transporters metabolism, ATP-Binding Cassette Transporters physiology, Amino Acid Motifs, Antifungal Agents pharmacology, DNA-Binding Proteins chemistry, Drug Resistance, Fungal genetics, Drug Resistance, Multiple genetics, Fungal Proteins metabolism, Fungi metabolism, Humans, Drug Resistance, Fungal physiology, Drug Resistance, Multiple physiology, Fungal Proteins chemistry, Fungal Proteins physiology, Fungi classification, Fungi physiology, Phylogeny
- Abstract
The overexpression of pleiotropic drug resistance (PDR) efflux pumps of the ATP-binding cassette (ABC) transporter superfamily frequently correlates with multidrug resistance. Phylogenetic analysis of 349 full-size ( approximately 160kDa) PDR proteins (Pdrps) from 55 fungal species, including major fungal pathogens, identified nine separate protein clusters (A-G, H1a/H1b and H2). Fungal, plant and human ABCG-family Pdrps possess a nucleotide-binding domain [NBD] and a transmembrane domain [TMD] in a family-defining 'reverse' ABC transporter topology [NBD-TMD] that is duplicated [NBD-TMD](2) in full-size fungal and plant Pdrps. Although full-size Pdrps have similar halves indicating early gene duplication/fusion, they show asymmetry of their NBDs and extracellular loops (ELs). Members of cluster F are most symmetric and may be closely related to the evolutionary ancestor of Pdrps. Unique structural elements are predicted, new PDR-specific motifs identified, and the significance of these and other structural features discussed., (Copyright 2009 Elsevier Inc. All rights reserved.)
- Published
- 2010
- Full Text
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48. Use of a yeast-based membrane protein expression technology to overexpress drug resistance efflux pumps.
- Author
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Lamping E and Cannon RD
- Subjects
- ATP-Binding Cassette Transporters, Antifungal Agents metabolism, Antifungal Agents pharmacology, Azoles metabolism, Azoles pharmacology, Cytochrome P-450 Enzyme System genetics, Drug Resistance, Fungal genetics, Drug Resistance, Fungal physiology, Fungal Proteins genetics, Membrane Proteins genetics, Models, Genetic, Polymerase Chain Reaction, Saccharomyces cerevisiae drug effects, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Fungal Proteins metabolism, Membrane Proteins metabolism, Saccharomyces cerevisiae metabolism
- Abstract
Azole antifungal drugs are used widely to treat people with oral fungal infections. Unfortunately, fungi can develop resistance to these drugs. This resistance can be due to the overexpression or mutation of cytochrome P450 14alpha-lanosterol demethylase, also known as ERG11 or CYP51, and/or the overexpression of membrane-located multidrug efflux pumps. We have developed a heterologous membrane protein expression system that can be used to study the structure and function of these proteins in the non-pathogenic, genetically stable, and versatile eukaryotic model organism, Saccharomyces cerevisiae. In this chapter we describe the techniques used to express the Candida albicans efflux pump Cdr1p in S. cerevisiae.
- Published
- 2010
- Full Text
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49. Identification of Nile red as a fluorescent substrate of the Candida albicans ATP-binding cassette transporters Cdr1p and Cdr2p and the major facilitator superfamily transporter Mdr1p.
- Author
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Ivnitski-Steele I, Holmes AR, Lamping E, Monk BC, Cannon RD, and Sklar LA
- Subjects
- ATP Binding Cassette Transporter, Subfamily B, Member 1 antagonists & inhibitors, Biological Transport drug effects, Depsipeptides pharmacology, Fungal Proteins antagonists & inhibitors, Oxazines analysis, Rhodamines metabolism, ATP Binding Cassette Transporter, Subfamily B, Member 1 metabolism, Candida albicans, Fluorescent Dyes metabolism, Fungal Proteins metabolism, Oxazines metabolism
- Abstract
Clinically relevant azole resistance in the fungal pathogen Candida albicans is most often associated with the increased expression of plasma membrane efflux pumps, specifically the ATP-binding cassette (ABC) transporters CaCdr1p and CaCdr2p and the major facilitator superfamily (MFS) transporter CaMdr1p. Development of potent pump inhibitors that chemosensitize cells to azoles is a promising approach to overcome antifungal resistance. Here we identify Nile red as a new fluorescent substrate for CaCdr1p, CaCdr2p, and CaMdr1p. Nile red was effluxed efficiently from Saccharomyces cerevisiae cells heterologously expressing these transporters. Enniatin selectively inhibited the efflux of Nile red from S. cerevisiae cells expressing CaCdr1p or CaMdr1p but not from cells expressing CaCdr2p. This indicates that Nile red can be used for the identification of inhibitors specific for particular transporters mediating antifungal resistance in pathogenic yeast.
- Published
- 2009
- Full Text
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50. Efflux-mediated antifungal drug resistance.
- Author
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Cannon RD, Lamping E, Holmes AR, Niimi K, Baret PV, Keniya MV, Tanabe K, Niimi M, Goffeau A, and Monk BC
- Subjects
- ATP-Binding Cassette Transporters genetics, ATP-Binding Cassette Transporters metabolism, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Fungal Proteins genetics, Fungal Proteins metabolism, Fungi genetics, Humans, Membrane Proteins genetics, Membrane Proteins metabolism, Membrane Transport Proteins metabolism, Transcription Factors genetics, Transcription Factors metabolism, Antifungal Agents metabolism, Antifungal Agents pharmacology, Drug Resistance, Fungal physiology, Fungi drug effects, Fungi metabolism, Mycoses diagnosis, Mycoses drug therapy, Mycoses microbiology
- Abstract
Fungi cause serious infections in the immunocompromised and debilitated, and the incidence of invasive mycoses has increased significantly over the last 3 decades. Slow diagnosis and the relatively few classes of antifungal drugs result in high attributable mortality for systemic fungal infections. Azole antifungals are commonly used for fungal infections, but azole resistance can be a problem for some patient groups. High-level, clinically significant azole resistance usually involves overexpression of plasma membrane efflux pumps belonging to the ATP-binding cassette (ABC) or the major facilitator superfamily class of transporters. The heterologous expression of efflux pumps in model systems, such Saccharomyces cerevisiae, has enabled the functional analysis of efflux pumps from a variety of fungi. Phylogenetic analysis of the ABC pleiotropic drug resistance family has provided a new view of the evolution of this important class of efflux pumps. There are several ways in which the clinical significance of efflux-mediated antifungal drug resistance can be mitigated. Alternative antifungal drugs, such as the echinocandins, that are not efflux pump substrates provide one option. Potential therapeutic approaches that could overcome azole resistance include targeting efflux pump transcriptional regulators and fungal stress response pathways, blockade of energy supply, and direct inhibition of efflux pumps.
- Published
- 2009
- Full Text
- View/download PDF
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