Multiplex PCR as a tool for the diagnosis of Leishmania spp. kDNA and the gapdh housekeeping gene of mammal hosts

Submitted by Sandra Infurna ([email protected]) on 2017-07-13T13:35:42Z No. of bitstreams: 1 renata_pires_etal_IOC_2017.pdf: 2389849 bytes, checksum: 52fabd62d73165e3c834a186be781fb0 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2017-07-13T13:47:25Z (GMT) No. of bitstreams: 1 renata_pires_etal_IOC_2017.pdf: 2389849 bytes, checksum: 52fabd62d73165e3c834a186be781fb0 (MD5)Made available in DSpace on 2017-07-13T13:47:25Z (GMT). No. of bitstreams: 1 renata_pires_etal_IOC_2017.pdf: 2389849 bytes, checksum: 52fabd62d73165e3c834a186be781fb0 (MD5) Previous issue date: 2017Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia de Tripanossomatídeos. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ. Brasil.Universidade Federal Fluminense. Instituto Biomédico. Centro de Ciências Médicas. Niterói, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia de Tripanossomatídeos. Rio de Janeiro, RJ. Brasil.The PCR assays usually employed for Leishmania diagnosis does not simultaneously detect a constitutive gene that would certify the viability of the DNA sample. We present a multiplex PCR approach for the simultaneous diagnosis of the Leishmania sp. kDNA fragment and a catalytic domain segment of a conserved region of the mammalian gapdh gene

Silver-stained electrophoresis polyacrylamide gel obtained from the sensitivity test for detecting <i>Leishmania</i> sp. DNA with a constant concentration of human DNA (100 ng).

<p>1: molecular-weight marker (50-bp DNA ladder); 2. empty; 3–8. mixture of human DNA and 100 (3), 50 (4), 10 (5), 5 (6), 1 (7) and 0.1 (8) ng of <i>L</i>. <i>tropica</i>; 9. uninfected rodent; 10. <i>Leishmania tropica</i> DNA; 11. <i>Leishmania tarentolae</i> DNA; and 12. Negative control for the PCR.</p

Details of the samples employed to validate the multiplex PCR system: Mammal order, geographic origin and tissue from wild mammalian hosts previously diagnosed with <i>Leishmania</i> spp. infection using a singleplex reaction.

<p>Details of the samples employed to validate the multiplex PCR system: Mammal order, geographic origin and tissue from wild mammalian hosts previously diagnosed with <i>Leishmania</i> spp. infection using a singleplex reaction.</p

Silver-stained electrophoresis polyacrylamide gel obtained from the amplification of gapdh and kDNA with the multiplex PCR system.

<p>(A) PCR products from the multiplex PCR system using Taq DNA polymerase enzymes (ABM^®) in the presence of 5% DMSO. (B) PCR products from the multiplex PCR system using the FideliTaq PCR Master Mix (Affymetrix, USB). In both figures: 1. molecular-weight marker (50-bp DNA ladder); 2. infected rodent; 3. uninfected rodent; 4. human DNA; and 5. negative PCR control.</p

DNA sequence and genomic region of molecular targets employed in the multiplex PCR system.

<p>DNA sequence and genomic region of molecular targets employed in the multiplex PCR system.</p

Human red blood cells release microvesicles with distinct sizes and protein composition that alter neutrophil phagocytosis

Abstract Extracellular vesicles (EVs) are membrane‐bound structures released by cells and tissues into biofluids, involved in cell‐cell communication. In humans, circulating red blood cells (RBCs), represent the most common cell‐type in the body, generating daily large numbers of microvesicles. In vitro, RBC vesiculation can be mimicked by stimulating RBCs with calcium ionophores, such as ionomycin and A23187. The fate of microvesicles released during in vivo aging of RBCs and their interactions with circulating cells is hitherto unknown. Using SEC plus DEG isolation methods, we have found that human RBCs generate microvesicles with two distinct sizes, densities and protein composition, identified by flow cytometry, and MRPS, and further validated by immune TEM. Furthermore, proteomic analysis revealed that RBC‐derived microvesicles (RBC‐MVs) are enriched in proteins with important functions in ion channel regulation, calcium homeostasis and vesicular transport, such as of sorcin, stomatin, annexin A7 and RAB proteins. Cryo‐electron microscopy identified two separate pathways of RBC‐MV‐neutrophil interaction, direct fusion with the plasma membrane and internalization, respectively. Functionally, RBC‐MVs decrease neutrophil ability to phagocytose Escherichia coli but do not affect their survival at 24 h. This work brings new insights regarding the complexity of the RBC‐MVs biogenesis, as well as their possible role in circulation