Skip to main content Accessibility help
×
Home
Hostname: page-component-5d6d958fb5-x8cck Total loading time: 0.49 Render date: 2022-11-28T06:03:52.115Z Has data issue: true Feature Flags: { "shouldUseShareProductTool": true, "shouldUseHypothesis": true, "isUnsiloEnabled": true, "useRatesEcommerce": false, "displayNetworkTab": true, "displayNetworkMapGraph": false, "useSa": true } hasContentIssue true

Article contents

High-throughput sequencing of kDNA amplicons for the analysis of Leishmania minicircles and identification of Neotropical species

Published online by Cambridge University Press:  16 November 2017

ARTHUR KOCHER*
Affiliation:
Laboratoire Evolution et Diversité Biologique, CNRS, Université Toulouse III Paul Sabatier, ENSFEA, IRD, UMR5174 EDB, Toulouse, France UMR MIVEGEC, IRD 224 – CNRS 5290 – Université de Montpellier, F34394 Montpellier, France
SOPHIE VALIÈRE
Affiliation:
GeT–PlaGe, Genotoul, INRA Auzeville, 31326 Castanet-Tolosan, France
ANNE-LAURE BAÑULS
Affiliation:
UMR MIVEGEC, IRD 224 – CNRS 5290 – Université de Montpellier, F34394 Montpellier, France
JÉRÔME MURIENNE
Affiliation:
Laboratoire Evolution et Diversité Biologique, CNRS, Université Toulouse III Paul Sabatier, ENSFEA, IRD, UMR5174 EDB, Toulouse, France
*
*Corresponding author. Laboratoire EDB, 118 route de Narbonne, 31062 Toulouse CEDEX 9, France. E-mail: arthur.kocher@gmail.com

Summary

Leishmania kinetoplast DNA contains thousands of small circular molecules referred to as kinetoplast DNA (kDNA) minicercles. kDNA minicircles are the preferred targets for sensitive Leishmania detection, because they are present in high copy number and contain conserved sequence blocks in which polymerase chain reaction (PCR) primers can be designed. On the other hand, the heterogenic nature of minicircle networks has hampered the use of this peculiar genomic region for strain typing. The characterization of Leishmania minicirculomes used to require isolation and cloning steps prior to sequencing. Here, we show that high-throughput sequencing of single minicircle PCR products allows bypassing these laborious laboratory tasks. The 120 bp long minicircle conserved region was amplified by PCR from 18 Leishmania strains representative of the major species complexes found in the Neotropics. High-throughput sequencing of PCR products enabled recovering significant numbers of distinct minicircle sequences from each strain, reflecting minicircle class diversity. Minicircle sequence analysis revealed patterns that are congruent with current hypothesis of Leishmania relationships. Then, we show that a barcoding-like approach based on minicircle sequence comparisons may allow reliable identifications of Leishmania spp. This work opens up promising perspectives for the study of kDNA minicercles and a variety of applications in Leishmania research.

Type
Special Issue Article
Copyright
Copyright © Cambridge University Press 2017 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Akhoundi, M., Downing, T., Votýpka, J., Kuhls, K., Lukeš, J., Cannet, A., Ravel, C., Marty, P., Delaunay, P., Kasbari, M., Granouillac, B., Gradoni, L. and Sereno, D. (2017). Leishmania infections: molecular targets and diagnosis. Molecular Aspects of Medicine 57, 129.CrossRefGoogle ScholarPubMed
Akhoundi, M., Kuhls, K., Cannet, A., Votýpka, J., Marty, P., Delaunay, P. and Sereno, D. (2016). A historical overview of the classification, evolution, and dispersion of Leishmania parasites and sandflies. PLoS Neglected Tropical Diseases 10, e0004349.CrossRefGoogle ScholarPubMed
Alvar, J., Vélez, I. D., Bern, C., Herrero, M., Desjeux, P., Cano, J., Jannin, J. and Boer, M. and the WHO Leishmaniasis Control Team (2012). Leishmaniasis worldwide and global estimates of its incidence. PLoS ONE 7, e35671.CrossRefGoogle ScholarPubMed
Angelici, M. C., Gramiccia, M. and Gradoni, L. (1989). Study on genetic polymorphism of Leishmania infantum through the analysis of restriction enzyme digestion patterns of kinetoplast DNA. Parasitology 99, 301309.CrossRefGoogle ScholarPubMed
Bandelt, H.-J., Forster, P. and Röhl, A. (1999). Median-joining networks for inferring intraspecific phylogenies. Molecular Biology and Evolution 16, 3748.CrossRefGoogle ScholarPubMed
Bañuls, A.-L., Guerrini, F., Pont, F. L., Barrera, C., Espinel, I., Guderian, R., Echeverria, R. and Tibayrenc, M. (1997). Evidence for hybridization by multilocus enzyme electrophoresis and random amplified polymorphic DNA between Leishmania braziliensis and Leishmania panamensis/guyanensis in Ecuador. Journal of Eukaryotic Microbiology 44, 408411.CrossRefGoogle ScholarPubMed
Bastrenta, B., Buitrago, R., Vargas, F., Le Pont, F., Torrez, M., Flores, M., Mita, N. and Brenière, S. F. (2002). First evidence of transmission of Leishmania (Viannia) lainsoni in a Sub Andean region of Bolivia. Acta Tropica 83, 249253.CrossRefGoogle Scholar
Berzunza-Cruz, M., Rodríguez-Moreno, Á, Gutiérrez-Granados, G., González-Salazar, C., Stephens, C. R., Hidalgo-Mihart, M., Marina, C. F., Rebollar-Téllez, E. A., Bailón-Martínez, D., Balcells, C. D., Ibarra-Cerdeña, C. N., Sánchez-Cordero, V. and Becker, I. (2015). Leishmania (L.) mexicana infected bats in Mexico: novel potential reservoirs. PLoS Neglected Tropical Diseases 9, e0003438.CrossRefGoogle ScholarPubMed
Binladen, J., Gilbert, M. T. P., Bollback, J. P., Panitz, F., Bendixen, C., Nielsen, R. and Willerslev, E. (2007). The use of coded PCR primers enables high-throughput sequencing of multiple homolog amplification products by 454 parallel sequencing. PLoS ONE 2, e197.CrossRefGoogle ScholarPubMed
Botilde, Y., Laurent, T., Quispe Tintaya, W., Chicharro, C., Cañavate, C., Cruz, I., Kuhls, K., Schönian, G. and Dujardin, J.-C. (2006). Comparison of molecular markers for strain typing of Leishmania infantum. Infection, Genetics and Evolution 6, 440446.CrossRefGoogle ScholarPubMed
Boyer, F., Mercier, C., Bonin, A., Le Bras, Y., Taberlet, P. and Coissac, E. (2016). Obitools: a unix-inspired software package for DNA metabarcoding. Molecular Ecology Resources 16, 176182.CrossRefGoogle ScholarPubMed
Brewster, S. and Barker, D. C. (2002). Analysis of minicircle classes in Leishmania (Viannia) species. Transactions of the Royal Society of Tropical Medicine and Hygiene 96, S55S63.CrossRefGoogle ScholarPubMed
Brown, S. D., Collins, R. A., Boyer, S., Lefort, M.-C., Malumbres-Olarte, J., Vink, C. J. and Cruickshank, R. H. (2012). Spider: an R package for the analysis of species identity and evolution, with particular reference to DNA barcoding. Molecular Ecology Resources 12, 562565.CrossRefGoogle Scholar
Cássia-Pires, R., Boité, M. C., D'Andrea, P. S., Herrera, H. M., Cupolillo, E., Jansen, A. M. and Roque, A. L. R. (2014). Distinct Leishmania species infecting wild caviomorph rodents (Rodentia: Hystricognathi) from Brazil. PLoS Neglected Tropical Diseases 8, e3389.CrossRefGoogle ScholarPubMed
Castresana, J. (2000). Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Molecular Biology and Evolution 17, 540552.CrossRefGoogle ScholarPubMed
Ceccarelli, M., Galluzzi, L., Migliazzo, A. and Magnani, M. (2014). Detection and characterization of Leishmania (Leishmania) and Leishmania (Viannia) by SYBR green-based real-time PCR and high resolution melt analysis targeting kinetoplast minicircle DNA. PLoS ONE 9, e88845.CrossRefGoogle ScholarPubMed
Copeland, N. K. and Aronson, N. E. (2015). Leishmaniasis: treatment updates and clinical practice guidelines review. Current Opinion in Infectious Diseases 28, 426437.CrossRefGoogle ScholarPubMed
de Almeida, M. E., Steurer, F. J., Koru, O., Herwaldt, B. L., Pieniazek, N. J. and da Silva, A. J. (2011). Identification of Leishmania spp. by molecular amplification and DNA sequencing analysis of a fragment of rRNA internal transcribed spacer 2. Journal of Clinical Microbiology 49, 31433149.CrossRefGoogle ScholarPubMed
de Oliveira Ramos Pereira, L. and Brandão, A. (2013). An analysis of trypanosomatids kDNA minicircle by absolute dinucleotide frequency. Parasitology International 62, 397403.CrossRefGoogle ScholarPubMed
Dufour, P., Pons, J.-M., Collinson, J. M., Gernigon, J., Dies, J. I., Sourrouille, P. and Crochet, P.-A. (2017). Multilocus barcoding confirms the occurrence of elegant terns in Western Europe. Journal of Ornithology 158, 351361.CrossRefGoogle Scholar
Dujardin, J.-C., Bañuls, A.-L., Llanos-Cuentas, A., Alvarez, E., De Doncker, S., Jacquet, D., Le Ray, D., Arevalo, J. and Tibayrenc, M. (1995). Putative Leishmania hybrids in the Eastern Andean valley of Huanuco, Peru. Acta Tropica 59, 293307.CrossRefGoogle ScholarPubMed
Edgar, R. C. (2004). MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research 32, 17921797.CrossRefGoogle ScholarPubMed
Esling, P., Lejzerowicz, F. and Pawlowski, J. (2015). Accurate multiplexing and filtering for high-throughput amplicon-sequencing. Nucleic Acids Research 43, 25132524.CrossRefGoogle ScholarPubMed
Faye, B., Bañuls, A. L., Bucheton, B., Dione, M. M., Bassanganam, O., Hide, M., Dereure, J., Choisy, M., Ndiaye, J. L., Konaté, O., Claire, M., Senghor, M. W., Faye, M. N., Sy, I., Niang, A. A., Molez, J. F., Victoir, K., Marty, P., Delaunay, P., Knecht, R., Mellul, S., Diedhiou, S. and Gaye, O. (2010). Canine visceral leishmaniasis caused by Leishmania infantum in Senegal: risk of emergence in humans? Microbes and Infection 12, 12191225.CrossRefGoogle ScholarPubMed
Fazekas, A. J., Burgess, K. S., Kesanakurti, P. R., Graham, S. W., Newmaster, S. G., Husband, B. C., Percy, D. M., Hajibabaei, M. and Barrett, S. C. H. (2008). Multiple multilocus DNA barcodes from the plastid genome discriminate plant species equally well. PLoS ONE 3, e2802.CrossRefGoogle ScholarPubMed
Gao, G., Kapushoc, S. T., Simpson, A. M., Thiemann, O. H. and Simpson, L. (2001). Guide RNAs of the recently isolated LEM125 strain of Leishmania tarentolae: an unexpected complexity. RNA 7, 13351347.CrossRefGoogle ScholarPubMed
Gardener, P. J. and Howells, R. E. (1972). Isoenzyme variation in leishmanial parasites. Journal of Protozoology 19, 47.Google Scholar
Gibson, W., Crow, M. and Kearns, J. (1997). Kinetoplast DNA minicircles are inherited from both parents in genetic crosses of Trypanosoma brucei. Parasitology Research 83, 483488.CrossRefGoogle ScholarPubMed
Hajduk, S. and Ochsenreiter, T. (2010). RNA editing in kinetoplastids. RNA Biology 7, 229236.CrossRefGoogle ScholarPubMed
Harkins, K. M., Schwartz, R. S., Cartwright, R. A. and Stone, A. C. (2016). Phylogenomic reconstruction supports supercontinent origins for Leishmania. Infection, Genetics and Evolution 38, 101109.CrossRefGoogle ScholarPubMed
Jensen, R. E. and Englund, P. T. (2012). Network news: the replication of kinetoplast DNA. Annual Review of Microbiology 66, 473491.CrossRefGoogle ScholarPubMed
Koarashi, Y., Cáceres, A. G., Saca, F. M. Z., Flores, E. E. P., Trujillo, A. C., Alvares, J. L. A., Yoshimatsu, K., Arikawa, J., Katakura, K., Hashiguchi, Y. and Kato, H. (2016). Identification of causative Leishmania species in Giemsa-stained smears prepared from patients with cutaneous leishmaniasis in Peru using PCR–RFLP. Acta Tropica 158, 8387.CrossRefGoogle ScholarPubMed
Kocher, A., Thoisy, B. D., Catzeflis, F., Huguin, M., Valiere, S., Zinger, L., Bañuls, A.-L. and Murienne, J. (2017 a). Evaluation of short mitochondrial metabarcodes for the identification of amazonian mammals. Methods in Ecology and Evolution 8, 12761283.CrossRefGoogle Scholar
Kocher, A., Gantier, J.-C., Gaborit, P., Zinger, L., Holota, H., Valiere, S., Dusfour, I., Girod, R., Bañuls, A.-L. and Murienne, J. (2017 b) Vector soup: high-throughput identification of Neotropical phlebotomine sand flies using metabarcoding. Molecular Ecology Resources 17, 172182.CrossRefGoogle ScholarPubMed
Kreutzer, R. D., Semko, M. E., Hendricks, L. D. and Wright, N. (1983). Identification of Leishmania spp. by multiple isozyme analysis. The American Journal of Tropical Medicine and Hygiene 32, 703715.CrossRefGoogle ScholarPubMed
Lainson, R. and Shaw, J. J. (2010). New world leishmaniasis. Topley and Wilson's Microbiology and Microbial Infections. doi: 10.1002/9780470688618.taw0182.CrossRefGoogle Scholar
Lee, S.-T., Tarn, C. and Chang, K.-P. (1993). Characterization of the switch of kinetoplast DNA minicircle dominance during development and reversion of drug resistance in Leishmania. Molecular and Biochemical Parasitology 58, 187203.CrossRefGoogle ScholarPubMed
Marco, J. D., Uezato, H., Mimori, T., Barroso, P. A., Korenaga, M., Nonaka, S., Basombrío, M. A., Taranto, N. J. and Hashiguchi, Y. (2006). Are cytochrome B gene sequencing and polymorphism-specific polymerase chain reaction as reliable as multilocus enzyme electrophoresis for identifying Leishmania spp. from Argentina? The American Journal of Tropical Medicine and Hygiene 75, 256260.Google ScholarPubMed
Marfurt, J., Nasereddin, A., Niederwieser, I., Jaffe, C. L., Beck, H.-P. and Felger, I. (2003). Identification and differentiation of Leishmania species in clinical samples by PCR amplification of the miniexon sequence and subsequent restriction fragment length polymorphism analysis. Journal of Clinical Microbiology 41, 31473153.CrossRefGoogle ScholarPubMed
Mariette, J., Escudié, F., Allias, N., Salin, G., Noirot, C., Thomas, S. and Klopp, C. (2012). NG6: integrated next generation sequencing storage and processing environment. BMC Genomics 13, 462.CrossRefGoogle ScholarPubMed
Maroli, M., Feliciangeli, M. D., Bichaud, L., Charrel, R. N. and Gradoni, L. (2013). Phlebotomine sandflies and the spreading of leishmaniases and other diseases of public health concern. Medical and Veterinary Entomology 27, 123147.CrossRefGoogle ScholarPubMed
Noyes, H. A., Reyburn, H., Bailey, J. W. and Smith, D. (1998). A nested-PCR-based schizodeme method for identifying Leishmania kinetoplast minicircle classes directly from clinical samples and its application to the study of the epidemiology of Leishmania tropica in Pakistan. Journal of Clinical Microbiology 36, 28772881.Google Scholar
Paradis, E., Claude, J. and Strimmer, K. (2004). APE: analyses of phylogenetics and evolution in R language. Bioinformatics (Oxford, England) 20, 289290.CrossRefGoogle ScholarPubMed
Pereira Júnior, A. M., Teles, C. B. G., de Azevedo dos Santos, A. P., de Souza Rodrigues, M., Marialva, E. F., Pessoa, F. A. C. and Medeiros, J. F. (2015). Ecological aspects and molecular detection of Leishmania DNA Ross (Kinetoplastida: Trypanosomatidae) in Phlebotomine sandflies (Diptera: Psychodidae) in terra firme and várzea environments in the Middle Solimões region, Amazonas State, Brazil. Parasites & Vectors 8, 180.CrossRefGoogle ScholarPubMed
R Core team (2014). R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing.Google Scholar
Ravel, C., Cortes, S., Pratlong, F., Morio, F., Dedet, J.-P. and Campino, L. (2006). First report of genetic hybrids between two very divergent Leishmania species: Leishmania infantum and Leishmania major. International Journal for Parasitology 36, 13831388.CrossRefGoogle ScholarPubMed
Read, L. K., Lukeš, J. and Hashimi, H. (2015). Trypanosome RNA editing: the complexity of getting U in and taking U out. Wiley Interdisciplinary Reviews: RNA 7, 3351.CrossRefGoogle ScholarPubMed
Ready, P. D. (2013). Biology of phlebotomine sand flies as vectors of disease agents. Annual Review of Entomology 58, 227250.CrossRefGoogle ScholarPubMed
Richini-Pereira, V. B., Marson, P. M., Hayasaka, E. Y., Victoria, C., da Silva, R. C. and Langoni, H. (2014). Molecular detection of Leishmania spp. in road-killed wild mammals in the Central Western area of the state of São Paulo, Brazil. Journal of Venomous Animals and Toxins Including Tropical Diseases 20, 17.CrossRefGoogle ScholarPubMed
Rioux, J. A., Lanotte, G., Serres, E., Pratlong, F., Bastien, P., Perieres, J. (1990). Taxonomy of Leishmania. Use of isoenzymes. Suggestions for a new classification. Annales de Parasitologie Humaine et Comparee 65, 111125.CrossRefGoogle ScholarPubMed
Rodrigues, E. H. G., da Silva Soares, F. C., Werkhäuser, R. P., de Brito, M. E. F., Fernandes, O., Abath, F. G. C. and Brandão, A. (2013). The compositional landscape of minicircle sequences isolated from active lesions and scars of American cutaneous leishmaniasis. Parasites & Vectors 6, 1.CrossRefGoogle ScholarPubMed
Roque, A. L. R., Cupolillo, E., Marchevsky, R. S. and Jansen, A. M. (2010). Thrichomys laurentius (Rodentia; Echimyidae) as a putative reservoir of Leishmania infantum and L. braziliensis: patterns of experimental infection. PLoS Neglected Tropical Diseases 4, e589.CrossRefGoogle ScholarPubMed
Rotureau, B. (2006). Ecology of the Leishmania species in the Guianan ecoregion complex. The American Journal of Tropical Medicine and Hygiene 74, 8196.Google ScholarPubMed
Rougeron, V., De Meeûs, T. and Bañuls, A.-L. (2017). Reproduction in Leishmania: a focus on genetic exchange. Infection, Genetics and Evolution 50, 128132.CrossRefGoogle ScholarPubMed
Saitou, N. and Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4, 406425.Google ScholarPubMed
Schnell, I. B., Bohmann, K. and Gilbert, M. T. P. (2015). Tag jumps illuminated – reducing sequence-to-sample misidentifications in metabarcoding studies. Molecular Ecology Resources 15, 12891303.CrossRefGoogle ScholarPubMed
Simon, S., Veron, V. and Carme, B. (2010). Leishmania spp. Identification by polymerase chain reaction–restriction fragment length polymorphism analysis and its applications in French Guiana. Diagnostic Microbiology and Infectious Disease 66, 175180.CrossRefGoogle ScholarPubMed
Simpson, L. (1987). The mitochondrial genome of kinetoplastid protozoa: genomic organization, transcription, replication, and evolution. Annual Review of Microbiology 41, 363380.CrossRefGoogle ScholarPubMed
Simpson, L. (1997). The genomic organization of guide RNA genes in kinetoplastid protozoa: several conundrums and their solutions. Molecular and Biochemical Parasitology 86, 133141.CrossRefGoogle ScholarPubMed
Telleria, J., Lafay, B., Virreira, M., Barnabé, C., Tibayrenc, M. and Svoboda, M. (2006). Trypanosoma cruzi: sequence analysis of the variable region of kinetoplast minicircles. Experimental Parasitology 114, 279288.CrossRefGoogle ScholarPubMed
Thomaz-Soccol, V., Lanotte, G., Rioux, J. A., Pratlong, F., Martini-Dumas, A. and Serres, E. (1993). Monophyletic origin of the genus Leishmania Ross, 1903. Annales de Parasitologie Humaine et Comparée 68, 107107.Google ScholarPubMed
Victoir, K., Bañuls, A. L., Arevalo, J., Llanos-Cuentas, A., Hamers, R., Noël, S., Doncker, S. D., Ray, D. L., Tibayrenc, M. and Dujardin, J. C. (1998). The gp63 gene locus, a target for genetic characterization of Leishmania belonging to subgenus Viannia. Parasitology 117, 113.Google ScholarPubMed
Weirather, J. L., Jeronimo, S. M., Gautam, S., Sundar, S., Kang, M., Kurtz, M. A., Haque, R., Schriefer, A., Talhari, S., Carvalho, E. M., Donelson, J. E. and Wilson, M. E. (2011). Serial quantitative PCR assay for detection, species discrimination, and quantification of Leishmania spp. in human samples. Journal of Clinical Microbiology 49, 38923904.CrossRefGoogle ScholarPubMed
Zelazny, A. M., Fedorko, D. P., Li, L., Neva, F. A. and Fischer, S. H. (2005). Evaluation of 7sl RNA gene sequences for the identification of Leishmania spp. The American Journal of Tropical Medicine and Hygiene 72, 415420.Google ScholarPubMed
Supplementary material: File

Kocher et al supplementary material

Kocher et al supplementary material 1

Download Kocher et al supplementary material(File)
File 2 MB
Supplementary material: File

Kocher et al. supplementary material

Kocher et al. supplementary material 1

Download Kocher et al. supplementary material(File)
File 2 MB
Supplementary material: File

Kocher et al supplementary material

Kocher et al supplementary material 2

Download Kocher et al supplementary material(File)
File 17 KB
Supplementary material: File

Kocher et al supplementary material

Kocher et al supplementary material 3

Download Kocher et al supplementary material(File)
File 16 KB
Supplementary material: File

Kocher et al supplementary material

Kocher et al supplementary material 4

Download Kocher et al supplementary material(File)
File 177 KB
13
Cited by

Save article to Kindle

To save this article to your Kindle, first ensure coreplatform@cambridge.org is added to your Approved Personal Document E-mail List under your Personal Document Settings on the Manage Your Content and Devices page of your Amazon account. Then enter the ‘name’ part of your Kindle email address below. Find out more about saving to your Kindle.

Note you can select to save to either the @free.kindle.com or @kindle.com variations. ‘@free.kindle.com’ emails are free but can only be saved to your device when it is connected to wi-fi. ‘@kindle.com’ emails can be delivered even when you are not connected to wi-fi, but note that service fees apply.

Find out more about the Kindle Personal Document Service.

High-throughput sequencing of kDNA amplicons for the analysis of Leishmania minicircles and identification of Neotropical species
Available formats
×

Save article to Dropbox

To save this article to your Dropbox account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you used this feature, you will be asked to authorise Cambridge Core to connect with your Dropbox account. Find out more about saving content to Dropbox.

High-throughput sequencing of kDNA amplicons for the analysis of Leishmania minicircles and identification of Neotropical species
Available formats
×

Save article to Google Drive

To save this article to your Google Drive account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you used this feature, you will be asked to authorise Cambridge Core to connect with your Google Drive account. Find out more about saving content to Google Drive.

High-throughput sequencing of kDNA amplicons for the analysis of Leishmania minicircles and identification of Neotropical species
Available formats
×
×

Reply to: Submit a response

Please enter your response.

Your details

Please enter a valid email address.

Conflicting interests

Do you have any conflicting interests? *