Lutzomyia umbratilis, the Main Vector of Leishmania guyanensis, Represents a Novel Species Complex?

BACKGROUND: Lutzomyia umbratilis is an important Leishmania guyanensis vector in South America. Previous studies have suggested differences in the vector competence between L. umbratilis populations situated on opposite banks of the Amazonas and Negro Rivers in the central Amazonian Brazil region, likely indicating a species complex. However, few studies have been performed on these populations and the taxonomic status of L. umbratilis remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Phylogeographic structure was estimated for six L. umbratilis samples from the central Amazonian region in Brazil by analyzing mtDNA using 1181 bp of the COI gene to assess whether the populations on opposite banks of these rivers consist of incipient or distinct species. The genetic diversity was fairly high and the results revealed two distinct clades ( = lineages) with 1% sequence divergence. Clade I consisted of four samples from the left bank of the Amazonas and Negro Rivers, whereas clade II comprised two samples from the right bank of Negro River. No haplotypes were shared between samples of two clades. Samples within clades exhibited low to moderate genetic differentiation (F(ST) = -0.0390-0.1841), whereas samples between clades exhibited very high differentiation (F(ST) = 0.7100-0.8497) and fixed differences. These lineages have diverged approximately 0.22 Mya in the middle Pleistocene. Demographic expansion was detected for the lineages I and II approximately 30,448 and 15,859 years ago, respectively, in the late Pleistocene. CONCLUSIONS/SIGNIFICANCE: The two genetic lineages may represent an advanced speciation stage suggestive of incipient or distinct species within L. umbratilis. These findings suggest that the Amazonas and Negro Rivers may be acting as effective barriers, thus preventing gene flow between populations on opposite sides. Such findings have important implications for epidemiological studies, especially those related to vector competence and anthropophily, and for vector control strategies. In addition, L. umbratilis represents an interesting example in speciation studies

Microfluidic devices for continuous production of pDNA/cationic liposome complexes for gene delivery and vaccine therapy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)To evaluate the process parameters for the production of plasmid DNA/cationic liposome (pDNA/CL) complexes in microfluidic systems, we studied two microfluidic devices: one with simple straight hydrodynamic flow focusing (SMD) and a second one with barriers in the mixing microchannel (patterned walls, PMD). A conventional bulk mixing method was used as a comparison to microfluidic mixing. The CL and the pDNA were combined at a molar positive/negative charge ratio of 6. The results showed that incorporating pDNA into the liposomal structures was different for the two microfluidic devices and that the temperature influenced the average size of complexes produced by the simple microfluidic device, while it did not influence the average complex size in the patterned wall device. Differences were also observed in pDNA probe accessibility in the complexes. The SMD yielded a similar quantity of non-electrostatic bound pDNA as that provided by the bulk mixing method. The complexes produced by the PMD had their pDNA probe accessibility decreased in 40% and achieved lower in vitro transfection levels in HeLa cells than the bulk mixing and simple microfluidic complexation methods. These differences are most likely due to different degrees of association between pDNA and CL, as controlled by the microfluidic devices. This study contributes to the development of rational strategies for controlling the formation of pDNA/CL complexes for further applications in gene and vaccine therapy. (C) 2013 Elsevier B.V. All rights reserved.111203210Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Estadual de Campinas - UNI-CAMP (Campinas, Brazil)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Continuous flow production of cationic liposomes at high lipid concentration in microfluidic devices for gene delivery applications

Microfluidics is a powerful technology that allows the production of cationic liposomes by the hydrodynamic focusing method. We first studied a single hydrodynamic focusing (SHF) device, which uses a central stream in which lipids dispersed in ethanol are injected and hydrodynamically compressed by the two aqueous streams. The ethanol diffusion from the inner stream to the aqueous stream encourages the formation of the liposomes. To intensify the mass diffusion and increase the surface area between the two fluids, a second device was designed with double hydrodynamic focusing (DHF). We investigated the influence of fluid flow velocity (V-f), Flow Rate Ratio (FRR) and total lipid concentration (C-lip) on the particle size of the CLs produced. The DHF microfluidic device had the ability of using higher V-f values than the SHF device, which resulted in a higher productivity level. Small Angle X-ray Scattering (SAXS) experiments were performed to structurally characterize the cationic liposomes produced by both microfluidic devices. The SAXS results revealed that both devices produce unilamellar cationic liposomes with a very small fraction of multilamellar liposomes; this finding is in agreement with the observations made in the analysis of the liposomes using Transmission Electron Microscopy (TEM). The biological efficacies of the cationic liposomes produced by both microfluidic devices were examined in vitro in HeLa cells, which confirmed their potential for gene delivery and vaccine therapy applications. (C) 2013 Elsevier B.V. All rights reserved.22642343