Evaluation of diuron tolerance and biotransformation by the white-rot fungus Ganoderma lucidum

The white rot basidiomycete Ganoderma lucidum was evaluated for its capability to tolerate and to degrade the herbicide diuron. Diuron at a subtoxic concentration was added at the start of the cultivation in glucose liquid stationary cultures. Under this condition diuron was a laccase inducer. Almost 50% of the initially present diuron was removed after 15 d of cultivation. Two diuron metabolites were found N′-(3,4-dichlorophenyl)-N-methylurea (DCPMU) and 3,4-dichlorophenylurea (DCPU). The addition of the cytochrome P450 inhibitors 1-aminobenzotriazole and piperonyl butoxide reduced significantly the capability of the fungus in degrading diuron. The activities of superoxide dismutase and catalase were significantly increased in the mycelial extracts by the presence of diuron. On the other hand, diuron did not cause any significant alteration in the levels of reactive oxygen species. Additionally, laccase could also degrade diuron in vitro and this degradation was increased by the addition of synthetic mediators, 3-ethylbenzthiazoline-6-sulphonic acid and acetylacetone. Significant reduction in the toxicity, as evaluated by the Lactuca sativa bioassay, was observed after G. lucidum treatment. In conclusion, G. lucidum is able to metabolize diuron by intra- and extracellular mechanisms, without the accumulation of toxic products.     

Role of leucine zipper-like motifs in the oligomerization of Pseudomonas putida phasins

Background

Phasins are low molecular mass proteins that accumulate strongly in bacterial cells in response to the intracellular storage of polyhydroxyalkanoates (PHA). Although lacking catalytic activity, phasins are the major components of the surface of the PHA granules and could be potentially involved in the formation of a network-like protein layer surrounding the polyester inclusions. Structural models revealed phasins to possess coiled-coil regions that might be important in the establishment of protein-protein interactions. However, there is not experimental evidence of a coiled-coil mediated oligomerization in these proteins.

H+-dependent inorganic phosphate transporter in breast cancer cells: Possible functions in the tumor microenvironment

Tumor microenvironment has a high concentration of inorganic phosphate (Pi), which is actually a marker for tumor progression. Regarding Pi another class of transporter has been recently studied, an H⁺-dependent Pi transporter, that is stimulated at acidic pH in Caco2BBE human intestinal cells. In this study, we characterized the H⁺-dependent Pi transport in breast cancer cell (MDA-MB-231) and around the cancer tissue. MDA-MB-231 cell line presented higher levels of H⁺-dependent Pi transport as compared to other breast cell lines, such as MCF-10A, MCF-7 and T47-D. The Pi transport was linear as a function of time and exhibited a Michaelis-Menten kinetic of K_m = 1.387 ± 0.1674 mM Pi and V_max = 198.6 ± 10.23 Pi × h^?1 × mg protein^?1 hence reflecting a low affinity Pi transport. H⁺-dependent Pi uptake was higher at acidic pH. FCCP, Bafilomycin A1 and SCH28080, which deregulate the intracellular levels of protons, inhibited the H⁺-dependent Pi transport. No effect on pHi was observed in the absence of inorganic phosphate. PAA, an H⁺-dependent Pi transport inhibitor, reduced the Pi transport activity, cell proliferation, adhesion, and migration. Arsenate, a structural analog of Pi, inhibited the Pi transport. At high Pi conditions, the H⁺-dependent Pi transport was five-fold higher than the Na⁺-dependent Pi transport, thus reflecting a low affinity Pi transport. The occurrence of an H⁺-dependent Pi transporter in tumor cells may endow them with an alternative path for Pi uptake in situations in which Na⁺-dependent Pi transport is saturated within the tumor microenvironment, thus regulating the energetically expensive tumor processes.     

Evolution of infection in mice inoculated by the oral route with different developmental forms of Trypanosoma cruzi I and II

Oral infection has become the most important transmission mechanism of Chagas disease in Brazil. For this study, the development of Trypanosoma cruzi infection in mice, induced by the oral and intraperitoneal (IP) routes, was compared. Four groups of Swiss mice were used to evaluate the influence of parasite genetics, number of parasites, inoculation volume and developmental stages on the development of the orally induced infection: 1 – blood trypomastigotes (BT) via oral; 2 – BT via IP; 3 – culture metacyclic trypomastigotes (MT) via oral; and 4 – culture MT via IP. Animals inoculated orally showed levels of parasitemia, as well as infectivity and mortality rates, lower than animals inoculated via IP, regardless of DTU (discrete typing unit) and inoculum. Animals infected with TcII showed higher levels of these parameters than did animals infected with TcI. The larger volume of inoculum showed a greater capacity to cause an infection when administered via the oral route. BT infection was more virulent than culture MT infection for both routes (oral and IP). However, mice inoculated orally with BT showed lower levels than via IP, while mice inoculated orally with culture MT showed similar levels of infection to those inoculated via IP. Mice inoculated with culture MT showed more histopathological changes than those inoculated with BT, regardless of the inoculation route. These results indicate that this alternative experimental model is useful for evaluating infection by T. cruzi isolates with subpatent parasitemia and low virulence, such as those belonging to the TcI and TcIV DTUs, which are prevalent in outbreaks of orally transmitted Chagas disease.     

Serological diagnosis and prognostic of tegumentary and visceral leishmaniasis using a conserved Leishmania hypothetical protein

New candidates for serological markers against leishmaniasis are required to be identified, since the presence of high titers of anti-Leishmania antibodies remain detected in sera of treated and cured patients, when current antigens have being employed. In this study, the diagnostic performance of a conserved Leishmania hypothetical protein was evaluated against a human and canine serological panel. The serological follow-up of the patients was also evaluated, using this recombinant antigen (rLiHyS) in ELISA assays. In the results, high sensitivity and specificity values were found when rLiHyS was used in the serological tests, while when the recombinant A2 (rA2) protein or an antigenic Leishmania preparation were used as controls, low sensitivity and specificity were found. Regarding the serological follow-up of the patients, significant reductions in the anti-rLiHyS antibody levels were found and, one year after the treatments, the anti-protein IgG production was similar to this found in the non-infected groups, reflecting a drop of the anti-rLiHyS antibody production. In conclusion, the present study shows for the first time a new recombinant antigen used to identify tegumentary and visceral leishmaniasis, as well as being able to serologically distinguish treated and cured patients from those developing active disease.     

Assessment of two CRISPR-Cas9 genome editing protocols for rapid generation of Trypanosoma cruzi gene knockout mutants

CRISPR/Cas9 technology has been used to edit genomes in a variety of organisms. Using the GP72 gene as a target sequence, we tested two distinct approaches to generate Trypanosoma cruzi knockout mutants using the Cas9 nuclease and in vitro transcribed single guide RNA. Highly efficient rates of disruption of GP72 were achieved either by transfecting parasites stably expressing Streptococcus pyogenes Cas9 with single guide RNA or by transfecting wild type parasites with recombinant Staphylococcus aureus Cas9 previously associated with single guide RNA. In both protocols, we used single-stranded oligonucleotides as a repair template for homologous recombination and insertion of stop codons in the target gene.     

Enzymological properties of the LPP1-encoded lipid phosphatase from Saccharomyces cerevisiae1

The product of the LPP1 gene in Saccharomyces cerevisiae is a membrane-associated enzyme that catalyzes the Mg²⁺-independent dephosphorylation of phosphatidate (PA), diacylglycerol pyrophosphate (DGPP), and lysophosphatidate (LPA). The LPP1-encoded lipid phosphatase was overexpressed 681-fold in Sf-9 insect cells and used to examine the enzymological properties of the enzyme using PA, DGPP, and LPA as substrates. The optimum pH values for PA phosphatase, DGPP phosphatase, and LPA phosphatase activities were 7.5, 7.0, and 7.0, respectively. Divalent cations (Mn²⁺, Co²⁺, and Ca²⁺), NaF, heavy metals, propranolol, phenylglyoxal, and N-ethylmaleimide inhibited the PA phosphatase, DGPP phosphatase, and LPA phosphatase activities of the enzyme. The inhibitory effects of N-ethylmaleimide and phenylglyoxal on the LPP1-encoded enzyme were novel properties when compared with other Mg²⁺-independent lipid phosphate phosphatases from S. cerevisiae and mammalian cells. The LPP1-encoded enzyme exhibited saturation kinetics with respect to the surface concentrations of PA (K_m=0.05 mol%), DGPP (K_m=0.07 mol%), and LPA (K_m=0.08 mol%). Based on specificity constants (V_max/K_m), the order of substrate preference was PA (4.2 units/mg/mol%)>DGPP (3.5 units/mg/mol%)>LPA (1.3 units/mg/mol%). DGPP (K_i=0.12 mol%) was a competitive inhibitor with respect to PA, and PA (K_i=0.12 mol%) was a competitive inhibitor with respect to DGPP. This suggested that the binding sites for these substrates were the same. The enzymological properties of the LPP1-encoded enzyme differed significantly from those of the S. cerevisiae DPP1-encoded lipid phosphatase, a related enzyme that also utilizes PA, DGPP, and LPA as substrates.     

Antiulcerogenic activity of the hydroalcoholic extract of leaves of Annona muricata Linnaeus in mice

Annona muricata Linnaeus, popularly known as “graviola” and also called soursop, is a species typical of countries with a tropical climate, and it is used in folk medicine as an anticancer, analgesic and antispasmodic agent. The aim of the present study was to validate the gastroprotective activity of the hydroalcoholic extract of the leaves of A. muricata (HEAM) and to investigate the underlying mechanisms of action for this effect. Gastric lesions were induced in mice by absolute ethanol, acidified ethanol or indomethacin. Before, the animals were pretreated with saline, omeprazole or HEAM orally at doses of 50–400 mg/kg. To determine the mechanism of action of the extract, we investigated, using specific inhibitors, the involvement of nitric oxide (NO), prostaglandins (PGEs), ATP-dependent K⁺ channels and α2-noradrenergic receptors. HEAM showed significant antiulcer activity against lesions induced by absolute ethanol, acidified ethanol or indomethacin, which was mediated by endogenous gastric prostaglandins.     

Diversity of Entamoeba spp. in African great apes and humans: an insight from Illumina MiSeq high-throughput sequencing

Understanding the complex Entamoeba communities in the mammalian intestine has been, to date, complicated by the lack of a suitable approach for molecular detection of multiple variants co-occurring in mixed infections. Here, we report on the application of a high throughput sequencing approach based on partial 18S rDNA using the Illumina MiSeq platform. We describe, to our knowledge, for the first time, the Entamoeba communities in humans, free-ranging western lowland gorillas and central chimpanzees living in the Dja Faunal Reserve in Cameroon. We detected 36 Entamoeba haplotypes belonging to six haplotype clusters, containing haplotypes possessing high and low host specificity. Most of the detected haplotypes belonged to commensal Entamoeba, however, the pathogenic species (Entamoeba histolytica and Entamoeba nuttalli) were also detected. We observed that some Entamoeba haplotypes are shared between humans and other hosts, indicating their zoonotic potential. The findings are important not only for understanding the epidemiology of amoebiasis in humans in rural African localities, but also in the context of wild great ape conservation.     

Detection of immune complexes and evaluation of alcoholic individuals' serological profile in the diagnosis of strongyloidiasis

Strongyloidiasis is a human parasitosis that is considered a public health problem. Early diagnosis of this infection is extremely important in immunocompromised patients (i.e. subjects with alcoholism). This study aimed to evaluate anti-Strongyloides immunoglobulin G (IgG) and immunoglobulin A (IgA), assess levels of circulating immune complexes (IC) and determine IgG avidity in serum samples from alcoholic and nonalcoholic individuals. A total of 140 blood samples were collected from male individuals (70 alcoholic and 70 nonalcoholic subjects). Serum was obtained and analysed by enzyme-linked immunosorbent assay for IgG, IgA, IC detection and avidity determination. Anti-Strongyloides IgG was detected in 55.7% of alcoholic subjects and 32.8% nonalcoholics, while IC levels showed frequencies of 38.6% and 17.1% in these groups, respectively. Anti-Strongyloides IgA was lower among alcoholics (4.3%) than nonalcoholics (34.3%). Spearman's correlation coefficient reported a positive correlation between IgG, IC and IgA in alcoholic individuals and no correlation in nonalcoholics. The median avidity index was higher in alcoholics (83.8%) than nonalcoholic subjects (73.2%). In conclusion, this study shows that alcoholic subjects produced specific antibodies against S. stercoralis regardless of the possible immunosuppression caused by chronic alcoholism. Considering that alcoholics are more susceptible to the severe forms of strongyloidiasis, the implementation of immunological methods as a complementary approach to parasitological diagnostics (i.e. detection of IgG, IC and antibody avidity) appears to be an alternative method for early diagnosis in these individuals.     

Synthesis and biological evaluation of aminothiazoles against Histoplasma capsulatum and Cryptococcus neoformans

The design and synthesis of a library of forty novel 2-aminoazole analogues as well as their evaluation as antifungal compounds against Histoplasma capsulatum and Cryptococcus neoformans is described. These structures were derived from N-[5-(1-naphthalenylmethyl)-2-thiazolyl]cyclohexanecarboxamide (41F5), a fungistatic agent previously identified through phenotypic screening (Antimicrob Agents Chemother. 2013;57:4349). Modifications to improve potency and water-solubility of 41F5 focused primarily on the 5-naphthalenyl group, the thiazole core, and the methylene linker between these two structural elements. In general, compounds with lipophilic [5+6] bicyclic ring systems, such as the 7-benzothiophenyl- and 4-indanyl groups, at the 5-position were 2–3 times more active against both fungal species as compared to 41F5. Also, introduction of a carbonyl group at the methylene linker of 41F5 resulted in a 2–3-fold increase in potency. These highly active compounds also showed generally low toxicities against murine P388D1 macrophages resulting in selectivity indices ranging from 63 to >200. Compounds that were highly active against fluconazole-sensitive C. neoformans strains had almost identical activity against fluconazole-resistant variants of this fungus indicating that 14α-demethylase is not their molecular target. Highly active compounds also retained activity against H. capsulatum phagocytosed into P388D1 macrophages.     

Synthesis and biological evaluation of new long-chain squaramides as anti-chagasic agents in the BALB/c mouse model

Chagas Disease is caused by infection with the insect-transmitted protozoan Trypanosoma cruzi and affects more than 10 million people. It is a paradigmatic example of a chronic disease without an effective treatment in Latin America where the current therapies, based on Benznidazole and Nifurtimox, are characterised by limited efficacy, toxic side-effects and frequent failures in the treatment. We present a series of new long-chain squaramides, identified based on their ¹H and ¹³C NMR spectra, and their trypanocidal activity and cytotoxicity were tested in vitro through the determination of IC₅₀ values. Compounds 4 and 7 were more active and less toxic than the reference drug Benznidazole, and these results were the basis of promoting in vivo assays, where parasitaemia levels, assignment of cure, reactivation of parasitaemia and others parameters were determined in mice treated in both the acute and chronic phases. Finally, the mechanisms of action were elucidated at metabolic and mitochondrial levels and superoxide dismutase inhibition. The experiments allowed us to select compound 7 as a promising candidate for treating Chagas Disease, where the activity, stability and low cost make long-chain squaramides appropriate molecules for the development of an affordable anti-chagasic agent versus current treatments.