Characterization of Albendazole-Randomly Methylated-β-Cyclodextrin Inclusion Complex and In Vivo Evaluation of Its Antihelmitic Activity in a Murine Model of Trichinellosis

Albendazole is a benzimidazole carbamate extensively used in oral chemotherapy against intestinal parasites, due to its broad spectrum activity, good tolerance and low cost. However, the drug has the disadvantage of poor bioavailability due to its very low solubility in water; as a consequence, a very active area of research focuses on the development of new pharmaceutical formulations to increase its solubility, dissolution rate, and bioavailability. The primary objective of this study was to prepare randomly methylated β-cyclodextrins inclusion complexes to increase albendazole dissolution rate, in order to enhance its antiparasitic activity. This formulation therapeutic efficacy was contrasted with that of the pure drug by treating Trichinella spiralis infected mice during the intestinal phase of the parasite cycle, on days five and six post-infection. This protocol significantly decreased muscle larval burden measured in the parenteral stage on day 30 post-infection, when compared with the untreated control. Thus, it could be demonstrated that the inclusion complexes improve the in vivo therapeutic activity of albendazole.     

Genome-wide analyses of Liberibacter species provides insights into evolution,phylogenetic relationships,and virulence factors

‘Candidatus Liberibacter’ species are insect-transmitted, phloem-limited α-Proteobacteria in the order of Rhizobiales. The citrus industry is facing significant challenges due to huanglongbing, associated with infection from ‘Candidatus Liberibacter asiaticus’ (Las). In order to gain greater insight into ‘Ca. Liberibacter’ biology and genetic diversity, we have performed genome sequencing and comparative analyses of diverse ‘Ca. Liberibacter’ species, including those that can infect citrus. Our phylogenetic analysis differentiates ‘Ca. Liberibacter’ species and Rhizobiales in separate clades and suggests stepwise evolution from a common ancestor splitting first into nonpathogenic Liberibacter crescens followed by diversification of pathogenic ‘Ca. Liberibacter’ species. Further analysis of Las genomes from different geographical locations revealed diversity among isolates from the United States. Our phylogenetic study also indicates multiple Las introduction events in California and spread of the pathogen from Florida to Texas. Texan Las isolates were closely related, while Florida and Asian isolates exhibited the most genetic variation. We have identified conserved Sec translocon (SEC)-dependent effectors likely involved in bacterial survival and virulence of Las and analysed their expression in their plant host (citrus) and insect vector (Diaphorina citri). Individual SEC-dependent effectors exhibited differential expression patterns between host and vector, indicating that Las uses its effector repertoire to differentially modulate diverse organisms. Collectively, this work provides insights into the evolution of ‘Ca. Liberibacter’ species, the introduction of Las in the United States and identifies promising Las targets for disease management.     

DNA Qualification Workflow for Next Generation Sequencing of Histopathological Samples

Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results. The qualification process consists of the quantification of double strand DNA (dsDNA) and the assessment of its suitability for downstream applications, such as high-throughput next-generation sequencing. We tested the two most frequently used instrumentations to define their role in this process: NanoDrop, based on UV spectroscopy, and Qubit 2.0, which uses fluorochromes specifically binding dsDNA. Quantitative PCR (qPCR) was used as the reference technique as it simultaneously assesses DNA concentration and suitability for PCR amplification. We used 17 genomic DNAs from 6 fresh-frozen (FF) tissues, 6 formalin-fixed paraffin-embedded (FFPE) tissues, 3 cell lines, and 2 commercial preparations. Intra- and inter-operator variability was negligible, and intra-methodology variability was minimal, while consistent inter-methodology divergences were observed. In fact, NanoDrop measured DNA concentrations higher than Qubit and its consistency with dsDNA quantification by qPCR was limited to high molecular weight DNA from FF samples and cell lines, where total DNA and dsDNA quantity virtually coincide. In partially degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements. Multiplex PCR amplifying 191 regions of 46 cancer-related genes was designated the downstream application, using 40 ng dsDNA from FFPE samples calculated by Qubit. All but one sample produced amplicon libraries suitable for next-generation sequencing. NanoDrop UV-spectrum verified contamination of the unsuccessful sample. In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard workflow for qualification of DNA preparations should include the sequential combination of NanoDrop and Qubit to assess the purity and quantity of dsDNA, respectively.     

Diversity of ferns and lycophytes from Reserve Natural Punta Lara, Buenos Aires, Argentina

Punta Lara Natural Reserve shelters the relict of the Southernmost riparian forest in the world, where the flora is threatened by the anthropic expansion and by the introduction of exotic species. In this area the ferns and lycophytes grow in particular environments as marshland, grassland and marginal forest. The aim of this work was to evaluate the diversity of ferns and lycophytes that inhabit in Punta Lara Natural Reserve. Field trips were conducted from 2006 to 2009 and local herbaria and specific bibliography were also revised. As a result of our study twenty four taxa of ferns and lycophytes were recognized. Their habit was mainly terrestrial (70%), fewer aquatic (17%) and epiphytic (13%). Five native taxa were scarcely recorded whereas two native species previously documented were not found. We assume that the main threats on ferns and lycophytes populations in Punta Lara Reserve are: removal for comercialization, invasion by exotic species and climatic fluctuations (drought periods). Keys for taxa identification are given and strategies for the conservation of threatened taxa are proposed.     

The influence of habitat, season and tidal regime in the activity of the intertidal crab Neohelice (=Chasmagnathus) granulata

The activity pattern of intertidal crabs is influenced by factors that usually change rhythmically following tidal and/or diel cycles, and is often associated with the use of refuges. The movement activity of the burrowing crab Neohelice granulata was compared among three populations from SW Atlantic coastal areas where they face different tidal regimes, water salinities, substrata and biological factors. At each site, we examined the seasonal activity of the crabs (individuals collected in pitfall traps) in two types of habitat: mudflat and salt marsh. The working hypothesis is that the activity would vary according to the diverse environmental conditions encountered at geographical and local scales. Crab activity varied between sites and seasons showing to be more intense when habitats were covered by water. The most active groups were large males, followed by large non-ovigerous females. Ovigerous females were almost inactive. Most crabs were near or inside burrows at low tides in Mar Chiquita and Bahía Blanca, but they were active at both low and high tides in San Antonio during spring and summer. N. granulata were active in a wide range of temperatures: from 10 to 37 °C at low tides and at temperatures as low as 2 °C when covered by water. Differences of activity between mudflat and salt marsh varied among sites depending on flooding frequencies. Movement activity of N. granulata varied both in space and in time; crabs move under very different abiotic conditions (e.g., low or high tide, daylight or night, low and high temperature) and their movement may also be prevented or elicited by biotic conditions like burrow complexity, food quality and predation pressure. The wide set of conditions under which N. granulata can be active may explain why this is the only semiterrestrial crab inhabiting latitudes higher than 40°S in South America.     

Glutamine Synthetase Sensitivity to Oxidative Modification during Nutrient Starvation in Prochlorococcus marinus PCC 9511

Glutamine synthetase plays a key role in nitrogen metabolism, thus the fine regulation of this enzyme in Prochlorococcus, which is especially important in the oligotrophic oceans where this marine cyanobacterium thrives. In this work, we studied the metal-catalyzed oxidation of glutamine synthetase in cultures of Prochlorococcus marinus strain PCC 9511 subjected to nutrient limitation. Nitrogen deprivation caused glutamine synthetase to be more sensitive to metal-catalyzed oxidation (a 36% increase compared to control, non starved samples). Nutrient starvation induced also a clear increase (three-fold in the case of nitrogen) in the concentration of carbonyl derivatives in cell extracts, which was also higher (22%) upon addition of the inhibitor of electron transport, DCMU, to cultures. Our results indicate that nutrient limitations, representative of the natural conditions in the Prochlorococcus habitat, affect the response of glutamine synthetase to oxidative inactivating systems. Implications of these results on the regulation of glutamine synthetase by oxidative alteration prior to degradation of the enzyme in Prochlorococcus are discussed.     

Reproductive traits of a nonindigenous amphipod associated with alternative habitat structures in presence of an invasive ecosystem-engineering polychaete

Hydrobiologia - Understanding reproductive traits of invasive species and their modulation by different invaded habitats is crucial for monitoring invasive processes. The invasive ecosystem...     

Expansion of CD11b+Ly6G+Ly6Cint cells driven by medroxyprogesterone acetate in mice bearing breast tumors restrains NK cell effector functions

The progesterone analog medroxyprogesterone acetate (MPA) is widely used as a hormone replacement therapy in postmenopausal women and as contraceptive. However, prolonged administration of MPA is associated with increased incidence of breast cancer through ill-defined mechanisms. Here, we explored whether exposure to MPA during mammary tumor growth affects myeloid-derived suppressor cells (MDSCs; CD11b⁺Gr-1⁺, mostly CD11b⁺Ly6G⁺Ly6C^int and CD11b⁺Ly6G^?Ly6C^high cells) and natural killer (NK) cells, potentially restraining tumor immunosurveillance. We used the highly metastatic 4T1 breast tumor (which does not express the classical progesterone receptor and expands MDSCs) to challenge BALB/c mice in the absence or in the presence of MPA. We observed that MPA promoted the accumulation of NK cells in spleens of tumor-bearing mice, but with reduced degranulation ability and in vivo cytotoxic activity. Simultaneously, MPA induced a preferential expansion of CD11b⁺Ly6G⁺Ly6C^int cells in spleen and bone marrow of 4T1 tumor-bearing mice. In vitro, MPA promoted nuclear mobilization of the glucocorticoid receptor (GR) in 4T1 cells and endowed these cells with the ability to promote a preferential differentiation of bone marrow cells into CD11b⁺Ly6G⁺Ly6C^int cells that displayed suppressive activity on NK cell degranulation. Sorted CD11b⁺Gr-1⁺ cells from MPA-treated tumor-bearing mice exhibited higher suppressive activity on NK cell degranulation than CD11b⁺Gr-1⁺ cells from vehicle-treated tumor-bearing mice. Thus, MPA, acting through the GR, endows tumor cells with an enhanced capacity to expand CD11b⁺Ly6G⁺Ly6C^int cells that subsequently display a stronger suppression of NK cell-mediated anti-tumor immunity. Our results describe an alternative mechanism by which MPA may affect immunosurveillance and have potential implication in breast cancer incidence.