Hormone-regulated expression and distribution of versican in mouse uterine tissues

Background  

Remodeling of the extracellular matrix is one of the most striking features observed in the uterus during the estrous cycle and after hormone replacement. Versican (VER) is a hyaluronan-binding proteoglycan that undergoes RNA alternative splicing, generating four distinct isoforms. This study analyzed the synthesis and distribution of VER in mouse uterine tissues during the estrous cycle, in ovariectomized (OVX) animals and after 17beta-estradiol (E2) and medroxyprogesterone (MPA) treatments, either alone or in combination.     

Evaluating the role of morphological characters in the phylogeny of some trypanosomatid genera (Excavata,Kinetoplastea, Trypanosomatida)

Over the last three decades, it has been progressively assumed that morphology has become obsolete for trypanosomatid systematics. Traditional taxonomy, based on the occurrence of specific kinds of cell morphotypes during life cycles and the morphometry of such cells, is often rejected by molecular phylogenies inferred mostly from 18S rDNA alone or combined with GAPDH. In such context, we hypothesized the affinities of 35 representatives of seven trypanosomatid genera from separated and combined cladistics analyses of morphological and 18S matrices. Morphology is shown to be more consistent and to have stronger synapomorphy retention than the 18S data. The strict consensus of cladograms from separated analyses was mostly unresolved, while combined analysis produced a meaningful and robust phylogenetic pattern, as evidenced by partition congruence index, Bremer support and frequencies of groups present/contradicted. The results (1) corroborate the separation of Angomonas and Strigomonas from Crithidia, which is now shown to be monophyletic, (2) support the revalidation of the genus Wallaceina, and (3) place the genera Herpetomonas, Leptomonas and Phytomonas within a single clade. Overall, we demonstrate the belief that morphological characters are inferior to molecular ones for trypanosomatid systematics is a consequence of neglecting their inclusion in phylogenetic analyses.     

A panel of the most suitable reference genes for RT-qPCR expression studies of coffee: screening their stability under different conditions

The reliability of analyses using real-time quantitative polymerase chain reaction (RT-qPCR) depends on the selection of appropriate reference genes to correct for sample-to-sample and run-to-run variations. The aim of the present study was to select the most suitable reference genes for gene expression analyses in tissue samples from coffee, Coffea arabica L. (Arabica) grown under well-watered (WW) and water-deficit (WD) conditions and C. canephora Pierre ex A. Froehner (Robusta) grown under WW conditions. Expression profiles and stabilities were evaluated for 12 reference genes in different tissues from C. arabica and for 8 genes in tissues from C. canephora. The web-based RefFinder tool, which combines the geNorm, NormFinder, Bestkeeper, and Delta-Ct algorithms, was employed to assess the stability of the tested genes. The most stable reference genes identified for all tissues grouped (WW/WD) of C. arabica were clathrin adaptor protein medium subunit (AP47), ubiquitin (UBQ), 60S ribosomal protein L39 (RPL39), and elongation factor 1α (EF1α), while class III alcohol dehydrogenase (ADH2), β-actin (ACT), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and ubiquitin (UBQ) genes were the most stable for all tissues grouped (WW) of C. canephora tissues. Validation by the expression level analysis of CaACO-like demonstrated that the use of the best and the worst set of reference genes produced different expression results. The results reinforce the general assumption that there is no universal reference gene and that it is essential to select the most appropriate gene for each individual experiment to apply adequate normalization procedures of RT-qPCR data.     

Optimization of arsenic phytoremediation using Eichhornia crassipes

This study aimed to evaluate the pH, phosphate, and nitrate in the process of arsenic absorption by Eichhornia crassipes (water hyacinth), using the surface response methodology, in order to optimize the process. The plants were exposed to a concentration of arsenic of 0.5 mg L^?1 (NaAsO₂) over a period of 10 days. The results indicated optimal levels for the absorption of arsenic by E. crassipes at pH equal to 7.5, absence of phosphate, and minimum nitrate level of 0.0887 mmol L^?1. For the tested concentration, E. crassipes was able to accumulate 498.4 mg kg^?1 of As (dry base) in its plant tissue and to reduce 83% of the initial concentration present in the aqueous medium where it was cultivated. The concentration of phosphorus in solution linearly increased the phosphorus content in the plants and negatively influenced the absorption of arsenic. The concentration of 0.5 mg L^?1 of As did not significantly affect the relative growth rate (RGR) and the tolerance index (TI). 94% of As (III) initially solubilized in water was converted by the end of the experiment period into As (V). The water hyacinth was important in the phytoremediation of arsenic when cultivated under optimal conditions for its removal.     

Glutamate-gated chloride channel subunit cDNA sequencing of Cochliomyia hominivorax (Diptera: Calliphoridae): cDNA variants and polymorphisms

The New World screwworm (NWS) Cochliomyia hominivorax (Coquerel) is one of the major myiasis-causing flies that injures livestock and leads to losses of ~US$ 2.7 billions/year in the Neotropics. Ivermectin (IVM), a macrocyclic lactone (ML), is the most used preventive insecticide for this parasite and targets the glutamate-gated chloride (GLUCLα) channels. Several authors have associated altered GluClα homologues to MLs resistance in invertebrates, although studies about resistance in NWS are limited to other genes. Here, we aimed to characterise the NWS GluClα (ChGluClα) cDNA and to search for alterations associated with IVM resistance in NWS larvae from a bioassay. The open reading frame of the ChGluClα comprised 1,359 bp and encoded a sequence of 452 amino acids. The ChGluClα cDNAs of the bioassay larvae showed different sequences that could be splice variants, which agree with the occurrence of alternative splicing in GluClα homologues. In addition, we found cDNAs with premature stop codons and the K242R SNP, which occurred more frequently in the surviving larvae and was located close to mutation (L256F) involved in ML resistance. Although these alterations were in low frequency, the ChGluClα sequencing will allow further studies to find alterations in the gene of resistant natural populations.     

Clinical and Parasitological Protection in a Leishmania infantum-Macaque Model Vaccinated with Adenovirus and the Recombinant A2 Antigen

Background

Visceral leishmaniasis (VL) is a severe vector-born disease of humans and dogs caused by Leishmania donovani complex parasites. Approximately 0.2 to 0.4 million new human VL cases occur annually worldwide. In the new world, these alarming numbers are primarily due to the impracticality of current control methods based on vector reduction and dog euthanasia. Thus, a prophylactic vaccine appears to be essential for VL control. The current efforts to develop an efficacious vaccine include the use of animal models that are as close to human VL. We have previously reported a L. infantum-macaque infection model that is reliable to determine which vaccine candidates are most worthy for further development. Among the few amastigote antigens tested so far, one of specific interest is the recombinant A2 (rA2) protein that protects against experimental L. infantum infections in mice and dogs.

Methodology/Principal Findings

Primates were vaccinated using three rA2-based prime-boost immunization regimes: three doses of rA2 plus recombinant human interleukin-12 (rhIL-12) adsorbed in alum (rA2/rhIL-12/alum); two doses of non-replicative adenovirus recombinant vector encoding A2 (Ad5-A2) followed by two boosts with rA2/rhIL-12/alum (Ad5-A2+rA2/rhIL12/alum); and plasmid DNA encoding A2 gene (DNA-A2) boosted with two doses of Ad5-A2 (DNA-A2+Ad5-A2). Primates received a subsequent infectious challenge with L. infantum. Vaccines, apart from being safe, were immunogenic as animals responded with increased pre-challenge production of anti-A2-specific IgG antibodies, though with some variability in the response, depending on the vaccine formulation/protocol. The relative parasite load in the liver was significantly lower in immunized macaques as compared to controls. Protection correlated with hepatic granuloma resolution, and reduction of clinical symptoms, particularly when primates were vaccinated with the Ad5-A2+rA2/rhIL12/alum protocol.

Conclusions/Significance

The remarkable clinical protection induced by A2 in an animal model that is evolutionary close to humans qualifies this antigen as a suitable vaccine candidate against human VL.     

Mechanisms mediating the diuretic and natriuretic actions of the incretin hormone glucagon-like peptide-1

Glucagon-like peptide-1 (GLP-1) is a gut incretin hormone considered a promising therapeutic agent for type 2 diabetes because it stimulates beta cell proliferation and insulin secretion in a glucose-dependent manner. Cumulative evidence supports a role for GLP-1 in modulating renal function; however, the mechanisms by which GLP-1 induces diuresis and natriuresis have not been completely established. This study aimed to define the cellular and molecular mechanisms mediating the renal effects of GLP-1. GLP-1 (1 μg·kg(-1)·min(-1)) was intravenously administered in rats for the period of 60 min. GLP-1-infused rats displayed increased urine flow, fractional excretion of sodium, potassium, and bicarbonate compared with those rats that received vehicle (1% BSA/saline). GLP-1-induced diuresis and natriuresis were also accompanied by increases in renal plasma flow and glomerular filtration rate. Real-time RT-PCR in microdissected rat nephron segments revealed that GLP-1 receptor-mRNA expression was restricted to glomerulus and proximal convoluted tubule. In rat renal proximal tubule, GLP-1 significantly reduced Na(+)/H(+) exchanger isoform 3 (NHE3)-mediated bicarbonate reabsorption via a protein kinase A (PKA)-dependent mechanism. Reduced proximal tubular bicarbonate flux rate was associated with a significant increase of NHE3 phosphorylation at the PKA consensus sites in microvillus membrane vesicles. Taken together, these data suggest that GLP-1 has diuretic and natriuretic effects that are mediated by changes in renal hemodynamics and by downregulation of NHE3 activity in the renal proximal tubule. Moreover, our findings support the view that GLP-1-based agents may have a potential therapeutic use not only as antidiabetic drugs but also in hypertension and other disorders of sodium retention.     

The phylogeny and evolution of deoxyribonuclease II: an enzyme essential for lysosomal DNA degradation

Deoxyribonuclease II (DNase II) is an endonuclease with optimal activity at low pH, localized within the lysosomes of higher eukaryotes. The origin of this enzyme remains in dispute, and its phylogenetic distribution leaves many questions about its subsequent evolutionary history open. Earlier studies have documented its presence in various metazoans, as well as in Dictyostelium, Trichomonas and, anomalously, a single genus of bacteria (Burkholderia). This study makes use of searches of the genomes of various organisms against known DNase II query sequences, in order to determine the likely point of origin of this enzyme among cellular life forms. Its complete absence from any other bacteria makes prokaryotic origin unlikely. Convincing evidence exists for DNase II homologs in Alveolates such as Paramecium, Heterokonts such as diatoms and water molds, and even tentative matches in green algae. Apparent absences include red algae, plants, fungi, and a number of parasitic organisms. Based on this phylogenetic distribution and hypotheses of eukaryotic relationships, the most probable explanation is that DNase II has been subject to multiple losses. The point of origin is debatable, though its presence in Trichomonas and perhaps in other evolutionarily basal "Excavate" protists such as Reclinomonas, strongly support the hypothesis that DNase II arose as a plesiomorphic trait in eukaryotes. It probably evolved together with phagocytosis, specifically to facilitate DNA degradation and bacteriotrophy. The various absences in many eukaryotic lineages are accounted for by loss of phagotrophic function in intracellular parasites, in obligate autotrophs, and in saprophytes.