Trypanosoma cruzi: genetic group with peculiar biochemical and biological behavior

Thirty-two Trypanosoma cruzi strains, isolated from chronic chagasic patients in the northwest of the state of Paraná (Brazil), were analyzed using molecular, biochemical and biological characteristics. Genotypic analysis using randomly amplified polymorphic DNA and simple sequence repeat-anchored polymerase chain reaction amplified profiles showed a large, genetically well-correlated group that contained the majority of the strains and a divergent group that included the PR-150 strain. For glycoconjugate composition, the PR-150 strain was different from the other strains considering the absence or presence of specific bands in aqueous or detergent phases. This strain was also totally different from the others in one out of the six parameters related to in vitro and in vivo biological behavior. We highlight the fact that the PR-150 was totally resistant to benznidazole. For the other biological parameters this strain was not totally distinct from the others, but it showed a peculiar behavior.

Saponins from Swartzia langsdorffii: biological activities

The presence of saponins and the molluscicidal activity of the roots, leaves, seeds and fruits of Swartzia langsdorffii Raddi (Leguminosae) against Biomphalaria glabrata adults and eggs were investigated. The roots, seeds and fruits were macerated in 95% ethanol. These extracts exerted a significant molluscicidal activity against B. glabrata, up to a dilution of 100 mg/l. Four mixtures (A2, B2, C and D) of triterpenoid oleanane type saponins were chromatographically isolated from the seed and fruit extracts. Two known saponins (1 and 2) were identified as beta-D-glucopyranosyl-[alpha-L-rhamnopyranosyl-(1->3)- beta-D-glucuronopyranosyl-(1->3)]-3beta-hydroxyolean-12-ene-28 -oate, and beta-D-glucopyranosyl-(1->3)-beta-D-glucuronopyranosyl-(1 ->3)]-3beta-hydroxyolean-12-ene-28-oate, respectively. These two saponins were present in all the mixtures, together with other triterpenoid oleane type saponins, which were shown to be less polar, by reversed-phase HPLC. The saponin identifications were based on spectral evidence, including ¹H-¹H two-dimensional correlation spectroscopy, nuclear Overhauser and exchange spectroscopy, heteronuclear multiple quantum coherence, and heteronuclear multiple-bond connectivity experiments. The toxicity of S. langsdorffii saponins to non-target organisms was prescreened by the brine shrimp lethality test.

Biological effects of extracts obtained from Stryphnodendron adstringens on Herpetomonas samuelpessoai

We report the effect of Stryphnodendron adstringens on the trypanosomatid Herpetomonas samuelpessoai. The parasites were grown at 28ºC in a chemically defined medium containing crude extract and fractions at concentrations from 100 to 5000 µg/ml obtained from S. adstringens. Concentrations of 500, 1000, 2500, and 5000 µg/ml both crude extract and semi-purified fraction progressively inhibited the protozoans' growth. At a concentration of 100 µg/ml, crude extract or a semi-purified (F3) fraction did not affect the growth of the protozoans. The F3-9 - F3-12 sub-fractions, at a concentration of 1000 µg/ml, also showed increased inhibitory activity on H. samuelpessoai. The IC50 of the crude extract and the F3 fraction were 538 and 634 µg/ml, respectively. Ultrastructural and enzymatic alterations in the trypanosomatids were also evaluated. H. samuelpessoai cultivated in the presence of IC50 crude extract showed considerable ultrastructural alterations, such as marked mitochondrial swelling with a large number of cristae and evident Golgi complex vesiculation, as observed by transmission electron microscopy. Cells exposed to 538 µg/ml of crude extract at 28ºC for 72 h, showed decreased activity of the enzyme succinate cytochrome c reductase, a typical mitochondrion marker, as compared to untreated cells

Fluorescence imaging reveals the nuclear behavior of peroxisome proliferator-activated receptor/retinoid X receptor heterodimers in the absence and presence of ligand.

In a global approach combining fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and fluorescence resonance energy transfer (FRET), we address the behavior in living cells of the peroxisome proliferator-activated receptors (PPARs), a family of nuclear receptors involved in lipid and glucose metabolism, inflammation control, and wound healing. We first demonstrate that unlike several other nuclear receptors, PPARs do not form speckles upon ligand activation. The subnuclear structures that may be observed under some experimental conditions result from overexpression of the protein and our immunolabeling experiments suggest that these structures are subjected to degradation by the proteasome. Interestingly and in contrast to a general assumption, PPARs readily heterodimerize with retinoid X receptor (RXR) in the absence of ligand in living cells. PPAR diffusion coefficients indicate that all the receptors are engaged in complexes of very high molecular masses and/or interact with relatively immobile nuclear components. PPARs are not immobilized by ligand binding. However, they exhibit a ligand-induced reduction of mobility, probably due to enhanced interactions with cofactors and/or chromatin. Our study draws attention to the limitations and pitfalls of fluorescent chimera imaging and demonstrates the usefulness of the combination of FCS, FRAP, and FRET to assess the behavior of nuclear receptors and their mode of action in living cells.

A cell biological view of the siderophore pyochelin iron uptake pathway in Pseudomonas aeruginosa.

Pyochelin (PCH) is a siderophore produced and secreted by Pseudomonas aeruginosa for iron capture. Using (55) Fe uptake and binding assays, we showed that PCH-Fe uptake in P. aeruginosa involves, in addition to the highly studied outer membrane transporter FptA, the inner membrane permease FptX, which recognizes PCH-(55) Fe with an affinity of 0.6 ± 0.2 nM and transports the ferri-siderophore complex from the periplasm into the cytoplasm: fptX deletion inhibited (55) Fe accumulation in the bacterial cytoplasm. Chromosomal replacement was used to generate P. aeruginosa strains producing fluorescent fusions with FptX, PchR (an AraC regulator), PchA (the first enzyme involved in the PCH biosynthesis) and PchE (a non-ribosomic peptide-synthetase involved in a further step). Fluorescence imaging and cellular fractionation showed a uniform repartition of FptX in the inner membrane. PchA and PchE were found in the cytoplasm, associated to the inner membrane all over the bacteria and also concentrated at the bacterial poles. PchE clustering at the bacterial poles was dependent on PchA expression, but on the opposite PchA clustering and membrane association was PchE-independent. PchA and PchE cellular organization suggests the existence of a siderosome for PCH biosynthesis as previously proposed for pyoverdine biosynthesis (another siderophore produced by P. aeruginosa).

Genetic and biological characterization of a densovirus isolate that affects dengue virus infection

Brevidensoviruses have an encapsidated, single-stranded DNA genome that predominantly has a negative polarity. In recent years, they have received particular attention due to their potential role in the biological control of pathogenic arboviruses and to their unnoticed presence in cell cultures as contaminants. In addition, brevidensoviruses may also be useful as viral vectors. This study describes the first genetic and biological characterization of a mosquito densovirus that was isolated in Brazil; moreover, we examined the phylogenetic relationship between this isolate and the other brevidensoviruses. We further demonstrate that this densovirus has the potential to be used to biologically control dengue virus (DENV) infection with in vitro co-infection experiments. The present study provides evidence that this densovirus isolate is a fast-spreading virus that affects cell growth and DENV infection.

Some biological data on cetaceans populations present in the western coasts of Ireland

Ireland’s waters constitute one of the richest habitats for cetaceans in Europe. Marine mammals, particularly cetaceans, are known to be definitive hosts of digestive parasites from the Fm.Anisakidae. The main aim of this study is to collect and compile all the information available out there regarding parasites of the Fm. Anisakidae and their definitive hosts. Secondary objectives are to relate the presence of cetacean species with the presence of parasites of the Fm. Anisakidae and to determine whether this greater number of cetaceans relates to a greater level of parasitism. Prevalence and burdens of anisakids in definitive hosts vary widely with host species, geographic location, and season. Results from several post-mortem exams are given. However, they cannot be compared due to differences in collecting techniques. Anisakis simplex is the most commonly and widespread parasite found in the majority of the samples and in a majornumber of hosts, which include harbour porpoise, short-beaked common dolphin and bottlenose dolphin. Studies on harbour porpoise obtained prevalences of Anisakis spp. of 46% (n=26) and of 100% (n= 12). Another study in common dolphin reported a prevalence of 68% (n=25). Several reasons could influence the variations in the presence of Anisakis. Studies on commerciallyexploited fish have reported prevalences of Anisakis simplex ranging from 65-100% in wildAtlantic salmon and from 42-53.4% in Atlantic cod

When do we dare to stop biological or immunomodulatory therapy for Crohn's disease? Results of a multidisciplinary European expert panel.

BACKGROUND: Safety and economic issues have increasingly raised concerns about the long term use of immunomodulators or biologics as maintenance therapies for Crohn's disease (CD). Despite emerging evidence suggesting that stopping therapy might be an option for low risk patients, criteria identifying target groups for this strategy are missing, and there is a lack of recommendations regarding this question. METHODS: Multidisciplinary European expert panel (EPACT-II Update) rated the appropriateness of stopping therapy in CD patients in remission. We used the RAND/UCLA Appropriateness Method, and included the following variables: presence of clinical and/or endoscopic remission, CRP level, fecal calprotectin level, prior surgery for CD, and duration of remission (1, 2 or 4 years). RESULTS: Before considering withdrawing therapy, the prerequisites of a C-reactive protein (CRP) and fecal calprotectin measurement were rated as "appropriate" by the panellists, whereas a radiological evaluation was considered as being of "uncertain" appropriateness. Ileo-colonoscopy was considered appropriate 1 year after surgery or after 4 years in the absence of prior surgery. Stopping azathioprine, 6-mercaptopurine or methotrexate mono-therapy was judged appropriate after 4 years of clinical remission. Withdrawing anti-TNF mono-therapy was judged appropriate after 2 years in case of clinical and endoscopic remission, and after 4 years of clinical remission. In case of combined therapy, anti-TNF withdrawal, while continuing the immunomodulator, was considered appropriate after two years of clinical remission. CONCLUSION: A multidisciplinary European expert panel proposed for the first time treatment stopping rules for patients in clinical and/or endoscopic remission, with normal CRP and fecal calprotectin levels.

Characterization of a protein of the plastid inner envelope having homology to animal inorganic phosphate, chloride and organic-anion transporters.

A protein from Arabidopsis thaliana (L.) Heynh. showing homology to animal proteins of the NaPi-1 family, involved in the transport of inorganic phosphate, chloride, glutamate and sialic acid, has been characterized. This protein, named ANTR2 (for anion transporters) was shown by chloroplast subfractionation to be localized to the plastid inner envelope in both A. thaliana and Spinacia oleracea (L.). Immunolocalization revealed that ANTR2 was expressed in the leaf mesophyll cells as well as in the developing embryo at the upturned-U stage. Five additional homologues of ANTR2 are found in the Arabidopsis genome, of which one was shown by green fluorescent protein (GFP) fusion to be also located in the chloroplast. All ANTR proteins share homology to the animal NaPi-1 family, as well as to other organic-anion transporters that are members of the Anion:Cation Symporter (ACS) family, and share the main features of transporters from this family, including the presence of 12 putative transmembrane domains and of a 7-amino acid motif in the fourth putative transmembrane domain. ANTR2 thus represent a novel protein of the plastid inner envelope that is likely to be involved in anion transport.

DRB1*03:01 haplotypes: differential contribution to multiple sclerosis risk and specific association with the presence of intrathecal IgM bands.

BACKGROUND Multiple sclerosis (MS) is a multifactorial disease with a genetic basis. The strongest associations with the disease lie in the Human Leukocyte Antigen (HLA) region. However, except for the DRB1*15:01 allele, the main risk factor associated to MS so far, no consistent effect has been described for any other variant. One example is HLA-DRB1*03:01, with a heterogeneous effect across populations and studies. We postulate that those discrepancies could be due to differences in the diverse haplotypes bearing that allele. Thus, we aimed at studying the association of DRB1*03:01 with MS susceptibility considering this allele globally and stratified by haplotypes. We also evaluated the association with the presence of oligoclonal IgM bands against myelin lipids (OCMB) in cerebrospinal fluid. METHODS Genotyping of HLA-B, -DRB1 and -DQA1 was performed in 1068 MS patients and 624 ethnically matched healthy controls. One hundred and thirty-nine MS patients were classified according to the presence (M+, 58 patients)/absence (M-, 81 patients) of OCMB. Comparisons between groups (MS patients vs. controls and M+ vs. M-) were performed with the chi-square test or the Fisher exact test. RESULTS Association of DRB1*03:01 with MS susceptibility was observed but with different haplotypic contribution, being the ancestral haplotype (AH) 18.2 the one causing the highest risk. Comparisons between M+, M- and controls showed that the AH 18.2 was affecting only M+ individuals, conferring a risk similar to that caused by DRB1*15:01. CONCLUSIONS The diverse DRB1*03:01-containing haplotypes contribute with different risk to MS susceptibility. The AH 18.2 causes the highest risk and affects only to individuals showing OCMB.

Genetic variability in G2 and F2 region between biological clones of human respiratory syncytial virus with or without host immune selection pressure

Human respiratory syncytial virus (HRSV) is an important respiratory pathogens among children between zero-five years old. Host immunity and viral genetic variability are important factors that can make vaccine production difficult. In this work, differences between biological clones of HRSV were detected in clinical samples in the absence and presence of serum collected from children in the convalescent phase of the illness and from their biological mothers. Viral clones were selected by plaque assay in the absence and presence of serum and nucleotide sequences of the G2 and F2 genes of HRSV biological clones were compared. One non-synonymous mutation was found in the F gene (Ile5Asn) in one clone of an HRSV-B sample and one non-synonymous mutation was found in the G gene (Ser291Pro) in four clones of the same HRSV-B sample. Only one of these clones was obtained after treatment with the child's serum. In addition, some synonymous mutations were determined in two clones of the HRSV-A samples. In conclusion, it is possible that minor sequences could be selected by host antibodies contributing to the HRSV evolutionary process, hampering the development of an effective vaccine, since we verify the same codon alteration in absence and presence of human sera in individual clones of BR-85 sample.

Presence of an oligodendroglioma-like component in newly diagnosed glioblastoma identifies a pathogenetically heterogeneous subgroup and lacks prognostic value: central pathology review of the EORTC_26981/NCIC_CE.3 trial.

Glioblastoma (GBM) is a morphologically heterogeneous tumor type with a median survival of only 15 months in clinical trial populations. However, survival varies greatly among patients. As part of a central pathology review, we addressed the question if patients with GBM displaying distinct morphologic features respond differently to combined chemo-radiotherapy with temozolomide. Morphologic features were systematically recorded for 360 cases with particular focus on the presence of an oligodendroglioma-like component and respective correlations with outcome and relevant molecular markers. GBM with an oligodendroglioma-like component (GBM-O) represented 15% of all confirmed GBM (52/339) and was not associated with a more favorable outcome. GBM-O encompassed a pathogenetically heterogeneous group, significantly enriched for IDH1 mutations (19 vs. 3%, p = 0.003) and EGFR amplifications (71 vs. 48%, p = 0.04) compared with other GBM, while co-deletion of 1p/19q was found in only one case and the MGMT methylation frequency was alike (47 vs. 46%). Expression profiles classified most of the GBM-O into two subtypes, 36% (5/14 evaluable) as proneural and 43% as classical GBM. The detection of pseudo-palisading necrosis (PPN) was associated with benefit from chemotherapy (p = 0.0002), while no such effect was present in the absence of PPN (p = 0.86). In the adjusted interaction model including clinical prognostic factors and MGMT status, PPN was borderline nonsignificant (p = 0.063). Taken together, recognition of an oligodendroglioma-like component in an otherwise classic GBM identifies a pathogenetically mixed group without prognostic significance. However, the presence of PPN may indicate biological features of clinical relevance for further improvement of therapy.

The role of LEKTI in fate determination of keratinocyte stem cells

SPINK5 (serine protease inhibitor Kazal-type 5) encodes the putative proteinase inhibitor LEKTI (lympho-epithelial Kazal-type related inhibitor). In skin, LEKTI expression is restricted to the stratum granulosum of the epidermis and the inner root sheath of hair follicles. Mutations that create premature termination codons in SPINK5 have been reported as the cause of Netherton syndrome (NS), a human autosomal recessive disorder characterized by congenital ichthyosis with defective cornification, a specific hair shaft defect known as trichorrexis invaginata or 'bamboo hair', and severe atopic manifestations, including atopic dermatitis and hayfever. Althought recombinant human LEKTI inhibits a battery of serine proteases including plasmin, trypsin, subtilisin A, cathepsin G, and elastase, the precise role of LEKTI in the physiopathology of NS remains unclear. Spink5−/− mice display a NS-like phenotype. Surprisingly, a psoriasis-like hyperplasia, basement membrane breakdown followed by evagination of spindle-shaped epidermal cells into the dermal compartment, and the presence of numerous sweat gland-like structures were also observed when the skin of Spink5−/− newborn mice, which die at birth, was transplanted onto the back of nude mice. Collectively, these observations suggest that LEKTI may play a role on cell proliferation and stem cell fate. Our current work aims at elucidating the mechanisms by which LEKTI impact these biological processes. Using keratinocyte stem cells obtained from NS patients, we have identified LEKTI as a regulator node in several signaling pathways involved in stem cell behavior.

Physicochemical and biological characterization of single-walled and double-walled carbon nanotubes in biological media

To study the toxicity of nanoparticles under relevant conditions, it is important to reproducibly disperse nanoparticles in biological media in in vitro and in vivo studies. Here, single-walled nanotubes (SWNTs) and double-walled nanotubes (DWNTs) were physicochemically and biologically characterized when dispersed in phosphate-buffered saline (PBS) and bovine serum albumin (BSA). BSA-SWNT/DWNT interaction resulted in a reduction of aggregation and an increase in particle stabilization. Based on the protein sequence coverage and protein binding results, DWNTs exhibited higher protein binding than SWNTs. SWNT and DWNT suspensions in the presence of BSA increased interleukin-6 (IL-6) levels and reduced tumor necrosis factor-alpha (TNF-α) levels in A549 cells as compared to corresponding samples in the absence of BSA. We next determined the effects of SWNTs and DWNTs on pulmonary protein modification using bronchoalveolar lavage fluid (BALF) as a surrogate collected form BALB/c mice. The BALF proteins bound to SWNTs (13 proteins) and DWNTs (11 proteins), suggesting that these proteins were associated with blood coagulation pathways. Lastly, we demonstrated the importance of physicochemical and biological alterations of SWNTs and DWNTs when dispersed in biological media, since protein binding may result in the misinterpretation of in vitro results and the activation of protein-regulated biological responses.

Large-scale association analyses identify new loci influencing glycemic traits and provide insight into the underlying biological pathways.

Through genome-wide association meta-analyses of up to 133,010 individuals of European ancestry without diabetes, including individuals newly genotyped using the Metabochip, we have increased the number of confirmed loci influencing glycemic traits to 53, of which 33 also increase type 2 diabetes risk (q < 0.05). Loci influencing fasting insulin concentration showed association with lipid levels and fat distribution, suggesting impact on insulin resistance. Gene-based analyses identified further biologically plausible loci, suggesting that additional loci beyond those reaching genome-wide significance are likely to represent real associations. This conclusion is supported by an excess of directionally consistent and nominally significant signals between discovery and follow-up studies. Functional analysis of these newly discovered loci will further improve our understanding of glycemic control.