Publications by authors named "Noelia Lander"

33 Publications

Mitochondrial Pyruvate Carrier Subunits Are Essential for Pyruvate-Driven Respiration, Infectivity, and Intracellular Replication of Trypanosoma cruzi.

mBio 2021 04 6;12(2). Epub 2021 Apr 6.

Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, Georgia, USA

Pyruvate is the final metabolite of glycolysis and can be converted into acetyl coenzyme A (acetyl-CoA) in mitochondria, where it is used as the substrate for the tricarboxylic acid cycle. Pyruvate availability in mitochondria depends on its active transport through the heterocomplex formed by the mitochondrial pyruvate carriers 1 and 2 (MPC1/MPC2). We report here studies on MPC1/MPC2 of , the etiologic agent of Chagas disease. Endogenous tagging of () and with 3× showed that both encoded proteins colocalize with MitoTracker to the mitochondria of epimastigotes. Individual knockout (KO) of and genes using CRISPR/Cas9 was confirmed by PCR and Southern blot analyses. Digitonin-permeabilized -KO and -KO epimastigotes showed reduced O consumption rates when pyruvate, but not succinate, was used as the mitochondrial substrate, while α-ketoglutarate increased their O consumption rates due to an increase in α-ketoglutarate dehydrogenase activity. Defective mitochondrial pyruvate import resulted in decreased Ca uptake. The inhibitors UK5099 and malonate impaired pyruvate-driven oxygen consumption in permeabilized control cells. Inhibition of succinate dehydrogenase by malonate indicated that pyruvate needs to be converted into succinate to increase respiration. -KO and -KO epimastigotes showed little growth differences in standard or low-glucose culture medium. However, the ability of trypomastigotes to infect tissue culture cells and replicate as intracellular amastigotes was decreased in -KOs. Overall, MPC1 and MPC2 are essential for cellular respiration in the presence of pyruvate, invasion of host cells, and replication of amastigotes. is the causative agent of Chagas disease. Pyruvate is the end product of glycolysis, and its transport into the mitochondrion is mediated by the mitochondrial pyruvate carrier (MPC) subunits. Using the CRISPR/Cas9 technique, we generated individual () and knockouts and demonstrated that they are essential for pyruvate-driven respiration. Interestingly, although glycolysis was reported as not an important source of energy for the infective stages, MPC was essential for normal host cell invasion and intracellular replication.
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http://dx.doi.org/10.1128/mBio.00540-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092248PMC
April 2021

Signaling pathways involved in environmental sensing in Trypanosoma cruzi.

Mol Microbiol 2020 Oct 9. Epub 2020 Oct 9.

Center for Tropical and Emerging Global Diseases, and Department of Cellular Biology, University of Georgia, Athens, GA, USA.

Trypanosoma cruzi is a unicellular parasite and the etiologic agent of Chagas disease. The parasite has a digenetic life cycle alternating between mammalian and insect hosts, where it faces a variety of environmental conditions to which it must adapt in order to survive. The adaptation to these changes is mediated by signaling pathways that coordinate the cellular responses to the new environmental settings. Major environmental changes include temperature, nutrient availability, ionic composition, pH, osmolarity, oxidative stress, contact with host cells and tissues, host immune response, and intracellular life. Some of the signaling pathways and second messengers potentially involved in the response to these changes have been elucidated in recent years and will be the subject of this review.
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http://dx.doi.org/10.1111/mmi.14621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8032824PMC
October 2020

IP receptor-mediated Ca release from acidocalcisomes regulates mitochondrial bioenergetics and prevents autophagy in Trypanosoma cruzi.

Cell Calcium 2020 12 2;92:102284. Epub 2020 Sep 2.

Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, GA, 30602, USA. Electronic address:

In contrast to animal cells, the inositol 1,4,5-trisphosphate receptor of Trypanosoma cruzi (TcIPR) localizes to acidocalcisomes instead of the endoplasmic reticulum. Here, we present evidence that TcIPR is a Ca release channel gated by IP when expressed in DT40 cells knockout for all vertebrate IP receptors, and is required for Ca uptake by T. cruzi mitochondria, regulating pyruvate dehydrogenase dephosphorylation and mitochondrial O consumption, and preventing autophagy. Localization studies revealed its co-localization with an acidocalcisome marker in all life cycle stages of the parasite. Ablation of TcIPR by CRISPR/Cas9 genome editing caused: a) a reduction in O consumption rate and citrate synthase activity; b) decreased mitochondrial Ca transport without affecting the membrane potential; c) increased ammonia production and AMP/ATP ratio; d) stimulation of autophagosome formation, and e) marked defects in growth of culture forms (epimastigotes) and invasion of host cells by infective stages (trypomastigotes). Moreover, TcIPR overexpressing parasites showed decreased metacyclogenesis, trypomastigote host cell invasion and intracellular amastigote replication. In conclusion, the results suggest a modulatory activity of TcIPR-mediated acidocalcisome Ca release on cell bioenergetics in T. cruzi.
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http://dx.doi.org/10.1016/j.ceca.2020.102284DOI Listing
December 2020

Editorial: Unravelling Biology.

Front Cell Infect Microbiol 2020 28;10:382. Epub 2020 Jul 28.

Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil.

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http://dx.doi.org/10.3389/fcimb.2020.00382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7399052PMC
July 2020

Lathosterol Oxidase (Sterol C-5 Desaturase) Deletion Confers Resistance to Amphotericin B and Sensitivity to Acidic Stress in Leishmania major.

mSphere 2020 07 1;5(4). Epub 2020 Jul 1.

Department of Biological Sciences, Texas Tech University, Lubbock, Texas, USA

Lathosterol oxidase (LSO) catalyzes the formation of the C-5-C-6 double bond in the synthesis of various types of sterols in mammals, fungi, plants, and protozoa. In parasites, mutations in or other sterol biosynthetic genes are associated with amphotericin B resistance. To investigate the biological roles of sterol C-5-C-6 desaturation, we generated an -null mutant line ( ) in , the causative agent for cutaneous leishmaniasis. parasites lacked the ergostane-based sterols commonly found in wild-type and instead accumulated equivalent sterol species without the C-5-C-6 double bond. These mutant parasites were replicative in culture and displayed heightened resistance to amphotericin B. However, they survived poorly after reaching the maximal density and were highly vulnerable to the membrane-disrupting detergent Triton X-100. In addition, mutants showed defects in regulating intracellular pH and were hypersensitive to acidic conditions. They also had potential alterations in the carbohydrate composition of lipophosphoglycan, a membrane-bound virulence factor in All these defects in were corrected upon the restoration of LSO expression. Together, these findings suggest that the C-5-C-6 double bond is vital for the structure of the sterol core, and while the loss of LSO can lead to amphotericin B resistance, it also makes parasites vulnerable to biologically relevant stress. Sterols are essential membrane components in eukaryotes, and sterol synthesis inhibitors can have potent effects against pathogenic fungi and trypanosomatids. Understanding the roles of sterols will facilitate the development of new drugs and counter drug resistance. LSO is required for the formation of the C-5-C-6 double bond in the sterol core structure in mammals, fungi, protozoans, plants, and algae. Functions of this C-5-C-6 double bond are not well understood. In this study, we generated and characterized a lathosterol oxidase-null mutant in Our data suggest that LSO is vital for the structure and membrane-stabilizing functions of leishmanial sterols. In addition, our results imply that while mutations in lathosterol oxidase can confer resistance to amphotericin B, an important antifungal and antiprotozoal agent, the alteration in sterol structure leads to significant defects in stress response that could be exploited for drug development.
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http://dx.doi.org/10.1128/mSphere.00380-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7333571PMC
July 2020

Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

Nat Methods 2020 05 6;17(5):481-494. Epub 2020 Apr 6.

Department of Biochemistry, University of Cambridge, Cambridge, UK.

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.
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http://dx.doi.org/10.1038/s41592-020-0796-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7200600PMC
May 2020

CRISPR/Cas9 Technology Applied to the Study of Proteins Involved in Calcium Signaling in Trypanosoma cruzi.

Methods Mol Biol 2020 ;2116:177-197

Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA, USA.

Chagas disease is a vector-borne tropical disease affecting millions of people worldwide, for which there is no vaccine or satisfactory treatment available. It is caused by the protozoan parasite Trypanosoma cruzi and considered endemic from North to South America. This parasite has unique metabolic and structural characteristics that make it an attractive organism for basic research. The genetic manipulation of T. cruzi has been historically challenging, as compared to other pathogenic protozoans. However, the use of the prokaryotic CRISPR/Cas9 system for genome editing has significantly improved the ability to generate genetically modified T. cruzi cell lines, becoming a powerful tool for the functional study of proteins in different stages of this parasite's life cycle, including infective trypomastigotes and intracellular amastigotes. Using the CRISPR/Cas9 method that we adapted to T. cruzi, it has been possible to perform knockout, complementation and in situ tagging of T. cruzi genes. In our system we cotransfect T. cruzi epimastigotes with an expression vector containing the Cas9 sequence and a single guide RNA, together with a donor DNA template to promote DNA break repair by homologous recombination. As a result, we have obtained homogeneous populations of mutant epimastigotes using a single resistance marker to modify both alleles of the gene. Mitochondrial Ca transport in trypanosomes is critical for shaping the dynamics of cytosolic Ca increases, for the bioenergetics of the cells, and for viability and infectivity. In this chapter we describe the most effective methods to achieve genome editing in T. cruzi using as example the generation of mutant cell lines to study proteins involved in calcium homeostasis. Specifically, we describe the methods we have used for the study of three proteins involved in the calcium signaling cascade of T. cruzi: the inositol 1,4,5-trisphosphate receptor (TcIPR), the mitochondrial calcium uniporter (TcMCU) and the calcium-sensitive pyruvate dehydrogenase phosphatase (TcPDP), using CRISPR/Cas9 technology as an approach to establish their role in the regulation of energy metabolism.
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http://dx.doi.org/10.1007/978-1-0716-0294-2_13DOI Listing
February 2021

A CRISPR/Cas9-riboswitch-Based Method for Downregulation of Gene Expression in .

Front Cell Infect Microbiol 2020 27;10:68. Epub 2020 Feb 27.

Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA, United States.

Few genetic tools were available to work with until the recent introduction of the CRISPR/Cas9 technique for gene knockout, gene knock-in, gene complementation, and endogenous gene tagging. Riboswitches are naturally occurring self-cleaving RNAs (ribozymes) that can be ligand-activated. Results from our laboratory recently demonstrated the usefulness of the ribozyme from , which has been shown to control reporter gene expression in response to exogenous glucosamine, for gene silencing in . In this work we used the CRISPR/Cas9 system for endogenously tagging glycoprotein 72 () and vacuolar proton pyrophosphatase () with the active () or inactive () ribozyme. Gene tagging was confirmed by PCR and protein downregulation was verified by western blot analyses. Further phenotypic characterization was performed by immunofluorescence analysis and quantification of growth . Our results indicate that the method was successful in silencing the expression of both genes without the need of glucosamine in the medium, suggesting that produces enough levels of endogenous glucosamine 6-phosphate to stimulate the ribozyme activity under normal growth conditions. This method could be useful to obtain knockdowns of essential genes in and to validate potential drug targets in this parasite.
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http://dx.doi.org/10.3389/fcimb.2020.00068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7056841PMC
April 2021

State-of-the-art CRISPR/Cas9 Technology for Genome Editing in Trypanosomatids.

J Eukaryot Microbiol 2019 11 7;66(6):981-991. Epub 2019 Jul 7.

Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, Georgia, 30602.

CRISPR/Cas9 technology has revolutionized biology. This prokaryotic defense system against foreign DNA has been repurposed for genome editing in a broad range of cell tissues and organisms. Trypanosomatids are flagellated protozoa belonging to the order Kinetoplastida. Some of its most representative members cause important human diseases affecting millions of people worldwide, such as Chagas disease, sleeping sickness and different forms of leishmaniases. Trypanosomatid infections represent an enormous burden for public health and there are no effective treatments for most of the diseases they cause. Since the emergence of the CRISPR/Cas9 technology, the genetic manipulation of these parasites has notably improved. As a consequence, genome editing is now playing a key role in the functional study of proteins, in the characterization of metabolic pathways, in the validation of alternative targets for antiparasitic interventions, and in the study of parasite biology and pathogenesis. In this work we review the different strategies that have been used to adapt the CRISPR/Cas9 system to Trypanosoma cruzi, Trypanosoma brucei, and Leishmania spp., as well as the research progress achieved using these approaches. Thereby, we will present the state-of-the-art molecular tools available for genome editing in trypanosomatids to finally point out the future perspectives in the field.
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http://dx.doi.org/10.1111/jeu.12747DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6842398PMC
November 2019

Direct determination of anaerobe contributions to the energy metabolism of Trypanosoma cruzi by chip calorimetry.

Anal Bioanal Chem 2019 Jul 15;411(17):3763-3768. Epub 2019 May 15.

Department of Clinical Pathology, University of Campinas (UNICAMP), Campinas, SP, 13083-877, Brazil.

We describe a chip calorimetric technique that allows the investigation of biological material under anoxic conditions in a micro-scale and in real time. Due to the fast oxygen exchange through the sample flow channel wall, the oxygen concentration inside the samples could be switched between atmospheric oxygen partial pressure to an oxygen concentration of 0.5% within less than 2 h. Using this technique, anaerobic processes in the energy metabolism of Trypanosoma cruzi could be studied directly. The comparison of the calorimetric and respirometric response of T. cruzi cells to the treatment with the mitochondrial inhibitors oligomycin and antimycin A and the uncoupler FCCP revealed that the respiration-related heat rate is superimposed by strong anaerobic contributions. Calorimetric measurements under anoxic conditions and with glycolytic inhibitors showed that anaerobic metabolic processes contribute from 30 to 40% to the overall heat production rate. Similar basal and antimycin A heat rates with cells under anoxic conditions indicated that the glycolytic rates are independent of the oxygen concentration which confirms the absence of the "Pasteur effect" in Trypanosomes. Graphical abstract.
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http://dx.doi.org/10.1007/s00216-019-01882-3DOI Listing
July 2019

Functional analysis and importance for host cell infection of the Ca-conducting subunits of the mitochondrial calcium uniporter of .

Mol Biol Cell 2019 07 15;30(14):1676-1690. Epub 2019 May 15.

Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo 13083, Brazil.

We report here that the etiologic agent of Chagas disease, possesses two unique paralogues of the mitochondrial calcium uniporter complex subunit that we named and . The predicted structure of the proteins indicates that, as predicted for the and paralogues, they are composed of two helical membrane-spanning domains and contain a WDXXEPXXY motif. Overexpression of each gene led to a significant increase in mitochondrial Ca uptake, while knockout (KO) of either or led to a loss of mitochondrial Ca uptake, without affecting the mitochondrial membrane potential. -KO and -KO epimastigotes exhibited reduced growth rate in low-glucose medium and alterations in their respiratory rate, citrate synthase activity, and AMP/ATP ratio, while trypomastigotes had reduced ability to efficiently infect host cells and replicate intracellularly as amastigotes. By gene complementation of KO cell lines or by a newly developed CRISPR/Cas9-mediated knock-in approach, we also studied the importance of critical amino acid residues of the four paralogues on mitochondrial Ca uptake. In conclusion, the results predict a hetero-oligomeric structure for the MCU complex, with structural and functional differences, as compared with those in the mammalian complex.
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http://dx.doi.org/10.1091/mbc.E19-03-0152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6727756PMC
July 2019

MICU1 and MICU2 Play an Essential Role in Mitochondrial Ca Uptake, Growth, and Infectivity of the Human Pathogen Trypanosoma cruzi.

mBio 2019 05 7;10(3). Epub 2019 May 7.

Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil

The mitochondrial Ca uptake in trypanosomatids, which belong to the eukaryotic supergroup Excavata, shares biochemical characteristics with that of animals, which, together with fungi, belong to the supergroup Opisthokonta. However, the composition of the mitochondrial calcium uniporter (MCU) complex in trypanosomatids is quite peculiar, suggesting lineage-specific adaptations. In this work, we used to study the role of orthologs for mitochondrial calcium uptake 1 (MICU1) and MICU2 in mitochondrial Ca uptake. MICU1 (TcMICU1) and TcMICU2 have mitochondrial targeting signals, two canonical EF-hand calcium-binding domains, and localize to the mitochondria. Using the CRISPR/Cas9 system (i.e., clustered regularly interspaced short palindromic repeats with Cas9), we generated and knockout (-KO) cell lines. Ablation of either or showed a significantly reduced mitochondrial Ca uptake in permeabilized epimastigotes without dissipation of the mitochondrial membrane potential or effects on the AMP/ATP ratio or citrate synthase activity. However, none of these proteins had a gatekeeper function at low cytosolic Ca concentrations ([Ca]), as occurs with their mammalian orthologs. -KO and -KO epimastigotes had a lower growth rate and impaired oxidative metabolism, while infective trypomastigotes have a reduced capacity to invade host cells and to replicate within them as amastigotes. The findings of this work, which is the first to study the role of MICU1 and MICU2 in organisms evolutionarily distant from animals, suggest that, although these components were probably present in the last eukaryotic common ancestor (LECA), they developed different roles during evolution of different eukaryotic supergroups. The work also provides new insights into the adaptations of trypanosomatids to their particular life styles. is the etiologic agent of Chagas disease and belongs to the early-branching eukaryotic supergroup Excavata. Its mitochondrial calcium uniporter (MCU) subunit shares similarity with the animal ortholog that was important to discover its encoding gene. In animal cells, the MICU1 and MICU2 proteins act as Ca sensors and gatekeepers of the MCU, preventing Ca uptake under resting conditions and favoring it at high cytosolic Ca concentrations ([Ca]). Using the CRISPR/Cas9 technique, we generated and knockout cell lines and showed that MICU1 and -2 do not act as gatekeepers at low [Ca] but are essential for normal growth, host cell invasion, and intracellular replication, revealing lineage-specific adaptations.
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http://dx.doi.org/10.1128/mBio.00348-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6509184PMC
May 2019

Genome Editing by CRISPR/Cas9 in Trypanosoma cruzi.

Methods Mol Biol 2019 ;1955:61-76

Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA, USA.

The genetic manipulation of the human parasite Trypanosoma cruzi has been significantly improved since the implementation of the CRISPR/Cas9 system for genome editing in this organism. The system was initially used for gene knockout in T. cruzi, later on for endogenous gene tagging and more recently for gene complementation. Mutant cell lines obtained by CRISPR/Cas9 have been used for the functional characterization of proteins in different stages of this parasite's life cycle, including infective trypomastigotes and intracellular amastigotes. In this chapter we describe the methodology to achieve genome editing by CRISPR/Cas9 in T. cruzi. Our method involves the utilization of a template cassette (donor DNA) to promote double-strand break repair by homologous directed repair (HDR). In this way, we have generated homogeneous populations of genetically modified parasites in 4-5 weeks without the need of cell sorting, selection of clonal populations, or insertion of more than one resistance marker to modify both alleles of the gene. The methodology has been organized according to three main genetic purposes: gene knockout, gene complementation of knockout cell lines generated by CRISPR/Cas9, and C-terminal tagging of endogenous genes in T. cruzi. In addition, we refer to the specific results that have been published using each one of these strategies.
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http://dx.doi.org/10.1007/978-1-4939-9148-8_5DOI Listing
July 2019

Calcium-sensitive pyruvate dehydrogenase phosphatase is required for energy metabolism, growth, differentiation, and infectivity of .

J Biol Chem 2018 11 19;293(45):17402-17417. Epub 2018 Sep 19.

From the Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, 13083, Brazil and

In vertebrate cells, mitochondrial Ca uptake by the mitochondrial calcium uniporter (MCU) leads to Ca-mediated stimulation of an intramitochondrial pyruvate dehydrogenase phosphatase (PDP). This enzyme dephosphorylates serine residues in the E1α subunit of pyruvate dehydrogenase (PDH), thereby activating PDH and resulting in increased ATP production. Although a phosphorylation/dephosphorylation cycle for the E1α subunit of PDH from nonvertebrate organisms has been described, the Ca-mediated PDP activation has not been studied. In this work, we investigated the Ca sensitivity of two recombinant PDPs from the protozoan human parasites (TcPDP) and (TbPDP) and generated a -KO cell line to establish TcPDP's role in cell bioenergetics and survival. Moreover, the mitochondrial localization of the TcPDP was studied by CRISPR/Cas9-mediated endogenous tagging. Our results indicate that TcPDP and TbPDP both are Ca-sensitive phosphatases. Of note, -KO epimastigotes exhibited increased levels of phosphorylated TcPDH, slower growth and lower oxygen consumption rates than control cells, an increased AMP/ATP ratio and autophagy under starvation conditions, and reduced differentiation into infective metacyclic forms. Furthermore, -KO trypomastigotes were impaired in infecting cultured host cells. We conclude that TcPDP is a Ca-stimulated mitochondrial phosphatase that dephosphorylates TcPDH and is required for normal growth, differentiation, infectivity, and energy metabolism in Our results support the view that one of the main roles of the MCU is linked to the regulation of intramitochondrial dehydrogenases.
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http://dx.doi.org/10.1074/jbc.RA118.004498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6231137PMC
November 2018

Inorganic polyphosphate interacts with nucleolar and glycosomal proteins in trypanosomatids.

Mol Microbiol 2018 12 18;110(6):973-994. Epub 2018 Oct 18.

Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA, 30602, USA.

Inorganic polyphosphate (polyP) is a polymer of three to hundreds of phosphate units bound by high-energy phosphoanhydride bonds and present from bacteria to humans. Most polyP in trypanosomatids is concentrated in acidocalcisomes, acidic calcium stores that possess a number of pumps, exchangers, and channels, and are important for their survival. In this work, using polyP as bait we identified > 25 putative protein targets in cell lysates of both Trypanosoma cruzi and Trypanosoma brucei. Gene ontology analysis of the binding partners found a significant over-representation of nucleolar and glycosomal proteins. Using the polyphosphate-binding domain (PPBD) of Escherichia coli exopolyphosphatase (PPX), we localized long-chain polyP to the nucleoli and glycosomes of trypanosomes. A competitive assay based on the pre-incubation of PPBD with exogenous polyP and subsequent immunofluorescence assay of procyclic forms (PCF) of T. brucei showed polyP concentration-dependent and chain length-dependent decrease in the fluorescence signal. Subcellular fractionation experiments confirmed the presence of polyP in glycosomes of T. brucei PCF. Targeting of yeast PPX to the glycosomes of PCF resulted in polyP hydrolysis, alteration in their glycolytic flux and increase in their susceptibility to oxidative stress.
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http://dx.doi.org/10.1111/mmi.14131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6281790PMC
December 2018

The mitochondrial calcium uniporter complex in trypanosomes.

Cell Biol Int 2018 Jun 25;42(6):656-663. Epub 2018 Jan 25.

Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil.

The presence of a conserved mechanism for mitochondrial calcium uptake in trypanosomatids was crucial for the molecular identification of the mitochondrial calcium uniporter (MCU), a long-sought channel present in most eukaryotic organisms. Since then, research efforts to elucidate the role of MCU and its regulatory elements in different biological models have multiplied. MCU is the pore-forming subunit of a multimeric complex (the MCU complex or MCUC) and its predicted structure in trypanosomes is simpler than in mammalian cells, lacking two of its subunits and probably possessing other unidentified components. MCU protein has been characterized in Trypanosoma brucei and Trypanosoma cruzi, the causative agents of African and American trypanosomiasis, respectively. Contrary to its mammalian homolog, TbMCU was found to be essential for cell growth and survival, while its paralog MCUb is an essential protein in T. cruzi. These findings could be further exploited for chemotherapeutic purposes. The emergence of new molecular tools for the genetic manipulation of trypanosomatids has been determinant for the functional characterization of the MCUC components in these organisms. However, further research has to be done to determine the role of each component in intracellular calcium signaling and cell bioenergetics. In this mini-review we summarize the original results on mitochondrial calcium uptake in trypanosomes, how did they contribute to the molecular identification of the MCU, and the functional characterization of the MCUC subunits that has so far been studied in these peculiar eukaryotes.
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http://dx.doi.org/10.1002/cbin.10928DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5980684PMC
June 2018

Endogenous C-terminal Tagging by CRISPR/Cas9 in .

Bio Protoc 2017 May;7(10)

Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, 13083, Brazil.

To achieve the C-terminal tagging of endogenous proteins in we use the Cas9/pTREX-n vector (Lander ., 2015) to insert a specific tag sequence (3xHA or 3xc-Myc) at the 3' end of a specific gene of interest (GOI). Chimeric sgRNA targeting the 3' end of the GOI is PCR-amplified and cloned into Cas9/pTREX-n vector. Then a DNA donor molecule to induce DNA repair by homologous recombination is amplified. This donor sequence contains the tag sequence and a marker for antibiotic resistance, plus 100 bp homology arms corresponding to regions located right upstream of the stop codon and downstream of the Cas9 target site at the GOI locus. Vectors pMOTag23M (Oberholzer ., 2006) or pMOHX1Tag4H (Lander ., 2016b) are used as PCR templates for DNA donor amplification. Epimastigotes co-transfected with the sgRNA/Cas9/pTREX-n construct and the DNA donor cassette are then cultured for 5 weeks with antibiotics for selection of double resistant parasites. Endogenous gene tagging is finally verified by PCR and Western blot analysis.
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http://dx.doi.org/10.21769/BioProtoc.2299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5531189PMC
May 2017

Different Roles of Mitochondrial Calcium Uniporter Complex Subunits in Growth and Infectivity of .

mBio 2017 05 9;8(3). Epub 2017 May 9.

Departamento de Patología Clínica, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil

is the agent of Chagas disease, and the finding that this parasite possesses a mitochondrial calcium uniporter (TcMCU) with characteristics similar to that of mammalian mitochondria was fundamental for the discovery of the molecular nature of MCU in eukaryotes. We report here that ablation of , or its paralog , by clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 led to a marked decrease in mitochondrial Ca uptake without affecting the membrane potential of these cells, whereas overexpression of each gene caused a significant increase in the ability of mitochondria to accumulate Ca While knockout (KO) epimastigotes were viable and able to differentiate into trypomastigotes, infect host cells, and replicate normally, ablation of resulted in epimastigotes having an important growth defect, lower rates of respiration and metacyclogenesis, more pronounced autophagy changes under starvation, and significantly reduced infectivity. Overexpression of , in contrast to what was proposed for its mammalian ortholog, did not result in a dominant negative effect on TcMCU. The finding of a mitochondrial calcium uniporter (MCU) in was essential for the discovery of the molecular nature of this transporter in mammals. In this work, we used the CRISPR/Cas9 technique that we recently developed for to knock out two components of the uniporter: MCU, the pore subunit, and MCUb, which was proposed as a negative regulator of MCU in human cells. In contrast to what occurs in human cells, MCU is not essential, while MCUb is essential for growth, differentiation, and infectivity; has a bioenergetic role; and does not act as a dominant negative subunit of MCU.
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http://dx.doi.org/10.1128/mBio.00574-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5424207PMC
May 2017

Pravastatin Chronic Treatment Sensitizes Hypercholesterolemic Mice Muscle to Mitochondrial Permeability Transition: Protection by Creatine or Coenzyme Q.

Front Pharmacol 2017 5;8:185. Epub 2017 Apr 5.

Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de CampinasSão Paulo, Brazil.

Statins are efficient cholesterol-lowering medicines utilized worldwide. However, 10% of patients suffer from adverse effects specially related to skeletal muscle function. Pro- or anti-oxidant effects of statins have been reported. Here we hypothesized that statins induce muscle mitochondrial oxidative stress leading to mitochondrial permeability transition (MPT) which may explain statin muscle toxicity. Thus, our aims were to investigate the effects of statin chronic treatment on muscle mitochondrial respiration rates, MPT and redox state indicators in the context of hypercholesterolemia. For this purpose, we studied muscle biopsies of the hypercholesterolemic LDL receptor knockout mice ( treated with pravastatin during 3 months. Plantaris, but not soleus muscle of treated mice showed significant inhibition of respiration rates induced by ADP (-14%), oligomycin (-20%) or FCCP (-40%). Inhibitions of respiratory rates were sensitive to EGTA (Ca chelator), cyclosporin A (MPT inhibitor), ruthenium red (inhibitor of mitochondria Ca uptake) and coenzyme Q (antioxidant), indicating that pravastatin treatment favors Ca induced MPT. Diet supplementation with creatine (antioxidant) also protected treated mice against pravastatin sensitization to Ca induced MPT. Among several antioxidant enzymes analyzed, only catalase activity was increased by 30% in plantaris muscle of pravastatin treated mice. Oxidized lipids, but not proteins biomarkers were identified in treated plantaris muscle. Taken together, the present results suggest that chronic pravastatin administration to a model of familial hypercholesterolemia promotes mitochondrial dysfunctions in plantaris muscle that can be counteracted by antioxidants administered either (CoQ) or (creatine). Therefore, we propose that inhibition of muscle mitochondrial respiration by pravastatin leads to an oxidative stress that, in the presence of calcium, opens the permeability transition pore. This mitochondrial oxidative stress caused by statin treatment also signals for cellular antioxidant system responses such as catalase upregulation. These results suggest that the detrimental effects of statins on muscle mitochondria could be prevented by co-administration of a safe antioxidant such as creatine or CoQ10.
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http://dx.doi.org/10.3389/fphar.2017.00185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5380726PMC
April 2017

CRISPR/Cas9-mediated endogenous C-terminal Tagging of Trypanosoma cruzi Genes Reveals the Acidocalcisome Localization of the Inositol 1,4,5-Trisphosphate Receptor.

J Biol Chem 2016 Dec 28;291(49):25505-25515. Epub 2016 Oct 28.

From the Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo 13083, Brazil and

Methods for genetic manipulation of Trypanosoma cruzi, the etiologic agent of Chagas disease, have been highly inefficient, and no endogenous tagging of genes has been reported to date. We report here the use of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system for endogenously tagging genes in this parasite. The utility of the method was established by tagging genes encoding proteins of known localization such as TcFCaBP (flagellar calcium binding protein) and TcVP1 (vacuolar proton pyrophosphatase), and two proteins of undefined or disputed localization, the TcMCU (mitochondrial calcium uniporter) and TcIPR (inositol 1,4,5-trisphosphate receptor). We confirmed the flagellar and acidocalcisome localization of TcFCaBP and TcVP1 by co-localization with antibodies to the flagellum and acidocalcisomes, respectively. As expected, TcMCU was co-localized with the voltage-dependent anion channel to the mitochondria. However, in contrast to previous reports and our own results using overexpressed TcIPR, endogenously tagged TcIPR showed co-localization with antibodies against VP1 to acidocalcisomes. These results are also in agreement with our previous reports on the localization of this channel to acidocalcisomes of Trypanosoma brucei and suggest that caution should be exercised when overexpression of tagged genes is done to localize proteins in T. cruzi.
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http://dx.doi.org/10.1074/jbc.M116.749655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5207250PMC
December 2016

Genome Editing by CRISPR/Cas9: A Game Change in the Genetic Manipulation of Protists.

J Eukaryot Microbiol 2016 09 15;63(5):679-90. Epub 2016 Jul 15.

Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, 13083, Brazil.

Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system has been transformative in biology. Originally discovered as an adaptive prokaryotic immune system, CRISPR/Cas9 has been repurposed for genome editing in a broad range of model organisms, from yeast to mammalian cells. Protist parasites are unicellular organisms producing important human diseases that affect millions of people around the world. For many of these diseases, such as malaria, Chagas disease, leishmaniasis and cryptosporidiosis, there are no effective treatments or vaccines available. The recent adaptation of the CRISPR/Cas9 technology to several protist models will be playing a key role in the functional study of their proteins, in the characterization of their metabolic pathways, and in the understanding of their biology, and will facilitate the search for new chemotherapeutic targets. In this work we review recent studies where the CRISPR/Cas9 system was adapted to protist parasites, particularly to Apicomplexans and trypanosomatids, emphasizing the different molecular strategies used for genome editing of each organism, as well as their advantages. We also discuss the potential usefulness of this technology in the green alga Chlamydomonas reinhardtii.
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http://dx.doi.org/10.1111/jeu.12338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5012946PMC
September 2016

Polyphosphate and acidocalcisomes.

Biochem Soc Trans 2016 Feb;44(1):1-6

Center for Tropical and Global Emerging Diseases and Department of Cellular Biology, University of Georgia, Athens 30602, U.S.A. Departamento de Patología Clínica, Universidade Estadual de Campinas, São Paulo 13083-877, Brazil

Inorganic polyphosphate (polyP) accumulates in acidocalcisomes, acidic calcium stores that have been found from bacteria to human cells. Proton pumps, such as the vacuolar proton pyrophosphatase (V-H(+)-PPase or VP1), the vacuolar proton ATPase (V-H(+)-ATPase) or both, maintain their acidity. A vacuolar transporter chaperone (VTC) complex is involved in the synthesis and translocation of polyP to these organelles in several eukaryotes, such as yeast, trypanosomatids, Apicomplexan and algae. Studies in trypanosomatids have revealed the role of polyP and acidocalcisomes in osmoregulation and calcium signalling.
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http://dx.doi.org/10.1042/BST20150193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4879816PMC
February 2016

CRISPR/Cas9-Induced Disruption of Paraflagellar Rod Protein 1 and 2 Genes in Trypanosoma cruzi Reveals Their Role in Flagellar Attachment.

mBio 2015 Jul 21;6(4):e01012. Epub 2015 Jul 21.

Unlabelled: Trypanosoma cruzi is the etiologic agent of Chagas disease, and current methods for its genetic manipulation have been highly inefficient. We report here the use of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system for disrupting genes in the parasite by three different strategies. The utility of the method was established by silencing genes encoding the GP72 protein, which is required for flagellar attachment, and paraflagellar rod proteins 1 and 2 (PFR1, PFR2), key components of the parasite flagellum. We used either vectors containing single guide RNA (sgRNA) and Cas9, separately or together, or one vector containing sgRNA and Cas9 plus donor DNA for homologous recombination to rapidly generate mutant cell lines in which the PFR1, PFR2, and GP72 genes have been disrupted. We demonstrate that genome editing of these endogenous genes in T. cruzi is successful without detectable toxicity of Cas9. Our results indicate that PFR1, PFR2, and GP72 contribute to flagellar attachment to the cell body and motility of the parasites. Therefore, CRISPR/Cas9 allows efficient gene disruption in an almost genetically intractable parasite and suggest that this method will improve the functional analyses of its genome.

Importance: Trypanosoma cruzi is the agent of Chagas disease, which affects millions of people worldwide. Vaccines to prevent this disease are not available, and drug treatments are not completely effective. The study of the biology of this parasite through genetic approaches will make possible the development of new preventive or treatment options. Previous attempts to use the CRISPR/Cas9 in T. cruzi found a detectable but low frequency of Cas9-facilitated homologous recombination and fluorescent marker swap between exogenous genes, while Cas9 was toxic to the cells. In this report, we describe new approaches that generate complete disruption of an endogenous gene without toxicity to the parasites and establish the relevance of several proteins for flagellar attachment and motility.
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http://dx.doi.org/10.1128/mBio.01012-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4513075PMC
July 2015

The acidocalcisome vacuolar transporter chaperone 4 catalyzes the synthesis of polyphosphate in insect-stages of Trypanosoma brucei and T. cruzi.

J Eukaryot Microbiol 2014 Mar-Apr;61(2):155-65. Epub 2014 Jan 3.

Department of Cellular Biology, Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA, 30602, USA; Department of Biology, Georgia State University, Atlanta, GA, 30303, USA.

Polyphosphate is a polymer of inorganic phosphate found in both prokaryotes and eukaryotes. Polyphosphate typically accumulates in acidic, calcium-rich organelles known as acidocalcisomes, and recent research demonstrated that vacuolar transporter chaperone 4 catalyzes its synthesis in yeast. The human pathogens Trypanosoma brucei and T. cruzi possess vacuolar transporter chaperone 4 homologs. We demonstrate that T. cruzi vacuolar transporter chaperone 4 localizes to acidocalcisomes of epimastigotes by immunofluorescence and immuno-electron microscopy and that the recombinant catalytic region of the T. cruzi enzyme is a polyphosphate kinase. RNA interference of the T. brucei enzyme in procyclic form parasites reduced short chain polyphosphate levels and resulted in accumulation of pyrophosphate. These results suggest that this trypanosome enzyme is an important component of a polyphosphate synthase complex that utilizes ATP to synthesize and translocate polyphosphate to acidocalcisomes in insect stages of these parasites.
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http://dx.doi.org/10.1111/jeu.12093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3959231PMC
November 2014

Trypanosoma brucei vacuolar transporter chaperone 4 (TbVtc4) is an acidocalcisome polyphosphate kinase required for in vivo infection.

J Biol Chem 2013 Nov 10;288(47):34205-34216. Epub 2013 Oct 10.

Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, Georgia 30602. Electronic address:

Polyphosphate (polyP) is an anionic polymer of orthophosphate groups linked by high energy bonds that typically accumulates in acidic, calcium-rich organelles known as acidocalcisomes. PolyP synthesis in eukaryotes was unclear until it was demonstrated that the protein named Vtc4p (vacuolar transporter chaperone 4) is a long chain polyP kinase that localizes to the yeast vacuole. Here, we report that TbVtc4 (Vtc4 ortholog of Trypanosoma brucei) encodes, in contrast, a short chain polyP kinase that localizes to acidocalcisomes. The subcellular localization of TbVtc4 was demonstrated by fluorescence and electron microscopy of cell lines expressing TbVtc4 in its endogenous locus fused to an epitope tag and by purified polyclonal antibodies against TbVtc4. Recombinant TbVtc4 was expressed in bacteria, and polyP kinase activity was assayed in vitro. The in vitro growth of conditional knock-out bloodstream form trypanosomes (TbVtc4-KO) was significantly affected relative to the parental cell line. This mutant had reduced polyP kinase activity and short chain polyP content and was considerably less virulent in mice. The wild-type phenotype was recovered when an ectopic copy of the TbVtc4 gene was expressed in the presence of doxycycline. The mutant also exhibited a defect in volume recovery under osmotic stress conditions in vitro, underscoring the relevance of polyP in osmoregulation.
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http://dx.doi.org/10.1074/jbc.M113.518993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3837161PMC
November 2013

New insights into roles of acidocalcisomes and contractile vacuole complex in osmoregulation in protists.

Int Rev Cell Mol Biol 2013 ;305:69-113

Department of Cellular Biology and Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia, USA.

While free-living protists are usually subjected to hyposmotic environments, parasitic protists are also in contact with hyperosmotic habitats. Recent work in one of these parasites, Trypanosoma cruzi, has revealed that its contractile vacuole complex, which usually collects and expels excess water as a mechanism of regulatory volume decrease after hyposmotic stress, has also a role in cell shrinking when the cells are submitted to hyperosmotic stress. Trypanosomes also have an acidic calcium store rich in polyphosphate (polyP), named the acidocalcisome, which is involved in their response to osmotic stress. Here, we review newly emerging insights on the role of acidocalcisomes and the contractile vacuole complex in the cellular response to hyposmotic and hyperosmotic stresses. We also review the current state of knowledge on the composition of these organelles and their other roles in calcium homeostasis and protein trafficking.
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http://dx.doi.org/10.1016/B978-0-12-407695-2.00002-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3818246PMC
September 2014

Localization and developmental regulation of a dispersed gene family 1 protein in Trypanosoma cruzi.

Infect Immun 2010 Jan 19;78(1):231-40. Epub 2009 Oct 19.

Centro de Biotecnología, Instituto de Estudios Avanzados, Caracas 1080, Venezuela.

The dispersed gene family 1 (DGF-1) is the fifth largest gene family in the Trypanosoma cruzi genome, with over 500 members (11). Many of the predicted DGF-1 protein products have several transmembrane domains and N-glycosylation and phosphorylation sites and were thought to localize in the plasma membrane. Here, we report that affinity-purified antibodies against a region of one of these proteins (DGF-1.2) localized it intracellularly in different stages of the parasite. DGF-1.2 is more abundant in the amastigote stage than in trypomastigotes and epimastigotes, as detected by immunofluorescence and Western blot analyses. The protein changed localization during intracellular or extracellular differentiation from the trypomastigote to the amastigote stage, where it finally localized to small bodies in close contact with the inner side of the amastigote plasma membrane. DGF-1.2 did not colocalize with markers of other subcellular organelles, such as acidocalcisomes, glycosomes, reservosomes, lipid droplets, or endocytic vesicles. During extracellular differentiation, the protein was detected in the culture medium from 0 to 22 h, peaking at 14 h. The presence of DGF-1.2 in the differentiation culture medium was confirmed by mass spectrometry analysis. Finally, when epimastigotes were subjected to starvation, there was a decrease in the labeling of the cells and, in Western blots, the appearance of bands of lower molecular mass, suggesting its cleavage. These results represent the first report of direct immunodetection and developmental expression and secretion of a DGF-1 protein.
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http://dx.doi.org/10.1128/IAI.00780-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798230PMC
January 2010

Molecular and biochemical characterisation of Trypanosoma cruzi phosphofructokinase.

Mem Inst Oswaldo Cruz 2009 Aug;104(5):745-8

Centro de Biotecnología, Instituto de Estudios Avanzados, Caracas, Venezuela.

The characterisation of the gene encoding Trypanosoma cruzi CL Brener phosphofructokinase (PFK) and the biochemical properties of the expressed enzyme are reported here. In contradiction with previous reports, the PFK genes of CL Brener and YBM strain T. cruzi were found to be similar to their Leishmania mexicana and Trypanosoma brucei homologs in terms of both kinetic properties and size, with open reading frames encoding polypeptides with a deduced molecular mass of 53,483. The predicted amino acid sequence contains the C-terminal glycosome-targeting tripeptide SKL; this localisation was confirmed by immunofluorescence assays. In sequence comparisons with the genes of other eukaryotes, it was found that, despite being an adenosine triphosphate-dependent enzyme, T. cruzi PFK shows significant sequence similarity with inorganic pyrophosphate-dependent PFKs.
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http://dx.doi.org/10.1590/s0074-02762009000500014DOI Listing
August 2009

Usefulness of 12 Y-STRs for forensic genetics evaluation in two populations from Venezuela.

Leg Med (Tokyo) 2008 Mar 5;10(2):107-12. Epub 2007 Nov 5.

Unidad de Genética Médica, Facultad de Medicina, Universidad del Zulia, Maracaibo, Venezuela.

The distribution of allele frequencies and haplotypes for 12 STRs loci, (DYS19, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS437, DYS438 and DYS439) on the Y-chromosome from two Venezuelan populations were determined in 173 DNA samples of unrelated males living in Caracas (62) and Maracaibo (111). Some parameters of forensic importance were calculated. AMOVA and genetic distances between these populations were estimated. The results confirmed Y-STR genotypes as useful markers for forensic genetics analysis.
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http://dx.doi.org/10.1016/j.legalmed.2007.08.005DOI Listing
March 2008