Mucin-type O-glycosylation in Mesocestoides vogae (syn. corti)

Protein glycosylation is an important post-translational modification underlying host-parasite interactions, which may determine the outcome of infection. Although Mesocestoides vogae represents an important model for investigating the various aspects of cestode biology, virtually no information is available about the structure and synthesis of glycans in this parasite. In this work, focused on the initiation pathway of mucin-type O-glycosylation in M. vogae, we characterized O-glycoproteins bearing the simple mucin-type cancer-associated Tn and sialyl-Tn antigens, and the expression and activity of ppGalNAc-T, the key enzyme responsible for the first step of mucin-type O-glycosylation. Using immunohistochemistry, Tn and sialyl-Tn antigens were detected mainly in the tegument (microtriches) and in parenchymal cells. Tn expression was also observed in lateral nerve cords. Both Tn and sialyl-Tn antigens were detected in in vitro cultured parasites. Based on their electrophoretic mobility, Tn- and sialyl-Tn-bearing glycoproteins from M. vogae were separated into several components of 22 to 60 kDa. The observation that Tn and sialyl-Tn glycoproteins remained in the 0.6N perchloric acid-soluble fraction suggested that they could be good candidates for characterizing mucin-type glycosylation in this parasite. O-glycoproteins were purified and initially characterized using a proteomic approach. Immunohistochemical analysis of the tissue distribution of ppGalNAc-T revealed that this enzyme is expressed in the sub-tegumental region and in the parenchyma of the parasite. In M. vogae cultured in vitro, ppGalNAc-T was mainly detected in the suckers. Using a panel of 8 acceptor substrate synthetic peptides, we found that M. vogae ppGalNAc-T preferentially glycosylate threonine residues, the best substrates being peptides derived from human mucin MUC1 and from Trypanosoma cruzi mucin. These results suggest that M. vogae might represent a useful model to study O-glycosylation, and provide new research avenues for future studies on the glycopathobiology of helminth parasites.     

Floral anatomy,embryology, seed,and fruit development in Cephalanthus (Naucleeae-Rubiaceae), with emphasis on C. glabratus

Information on the reproductive anatomy in genera of the tribe Naucleeae, particularly Cephalanthus, is scarce and fragmented. Of the six species in the genus, only the mature megagamethophyte of Cephalanthus occidentalis has been described. This study aims to provide information on embryological aspects in flowers of C. glabratus and to analyze the morphology and anatomy of the flowers, fruit, and seed in the six species of the genus. Cephalanthus glabratus have imperfect flowers: pistillate (PF) and staminate (SF). In the PF, the ovules are functional, while in the SF, they atrophy during the formation of the embryo sac. The mature ovule has a single integument, corresponds to the Phyllis type and the embryo sac is a Polygonum type, forming only in the PF. The presence of pollenkitt and secondary presentation of pollen were observed in the SF, as well as in the pollen formation previously described, whereas in the PF, they are absent, due to the collapse of the pollen grains inside the indehiscent anthers. The analysis of the ontogeny of the ovular excrescence in C. glabratus determined its funicular origin, calling it an aril. Its development is a pre-anthesis event, initiated during megasporogenesis. In seeds, the aril is a fleshy, white appendage which almost completely envelops the seeds of Cephalanthus, except for Cephalanthus natalensis where it is noticeably more reduced. Studies of the fruit in Cephalanthus species indicate that the infructescence is a dry schizocarp which separates into uni-seminated mericarps, except in C. natalensis that has fleshy indehiscent fruit.

     

Contribution of endoplasmic reticulum stress,MAPK and PI3K/Akt pathways to the apoptotic death induced by a penicillin derivative in melanoma cells

We have previously examined the in vitro and in vivo antitumor action of TAP7f, a synthetic triazolylpeptidyl penicillin, on murine melanoma cells. In this work, we explored the signal transduction pathways modulated by TAP7f in murine B16-F0 and human A375 melanoma cells, and the contribution of some intracellular signals to the apoptotic cell death. TAP7f decreased ERK1/2 phosphorylation and increased phospho-p38, phospho-JNK and phospho-Akt levels. ERK1/2 blockage suppressed cell growth, while inhibition of p38, JNK and PI3K-I pathways reduced the antitumor effect of TAP7f. Pharmacological inhibition of p38 and JNK, or blockage of PI3K-I/Akt cascade with a dominant negative PI3K-I mutant diminished Bax expression levels and PARP-1 cleavage, indicating the involvement of these pathways in apoptosis. PI3K-I/Akt inhibition also favored an autophagic response, as evidenced by the higher expression levels of Beclin-1 and LC3-II detected in transfected cells exposed to TAP7f. However, although PI3K-I/Akt blockage promoted an autophagic survival response, this mechanism appears not to be critical for TAP7f antitumor action. It was also shown that TAP7f induced ER stress by enhancing the expression of ER stress-related genes and proteins. Downregulation of CHOP protein with specific siRNA increased cell growth and decreased cleavage of PARP-1, supporting its role in apoptosis. Furthermore, it was found that activation of p38, JNK and Akt occurred downstream ER perturbation. In summary, our results showed that TAP7f triggers an apoptotic cell death in melanoma cells through induction of ER stress and activation of p38, JNK and PI3K-I/Akt pathways.

     

Byssal hairs in the invasive Asian freshwater bivalve Limnoperna fortunei (Mytilidae) in the Paraná River system with comments on this species in South America

We recorded, for the first time, byssal hairs in the Asian freshwater bivalve Limnoperna fortunei from the Paraná River system. We analysed the presence of hairs and their distribution on the shell in relation to habitat and shell size in 12 sites. Hairs were present in lentic habitats associated with macrophytes or organic matter, but were absent in lotic environments. The proportion of mussels with hairs was negatively correlated with current velocity. Hairs were more frequent and abundant in larger mussels. In general, the hairs are a similar length over the bivalve and almost entirely cover the shells in≥60% in lentic habitats. The projections allow L. fortunei to be camouflaged among the roots of macrophytes or coarse organic matter, assisting in avoiding visual predators. The clear-cut separation of L. fortunei populations into two different groups could be associated with phenotypic plasticity in this species.     

Presence and histopathological effects of the Parvatrema sp. (Digenea, Gymnophallidae) in the stout razor clam Tagelus plebeius (Bivalvia, Psammobiidae)

The stout razor clam Tagelus plebeius (Bivalvia, Psammobiidae) has a wide geographic distribution range, including the Brazilian coasts from the northeast (Alagoas) to the south (Santa Catarina). In March 2008, an episode of mass T. plebeius mortality (70%) occurred in an intertidal bed at The Pontal da Daniela, State of Santa Catarina, Brazil. We report here high prevalences (to 100%) of the trematode parasite Parvatrema sp. Cable, 1953 (Digenea, Gymnophallidae) infecting T. plebeius at high intensities. We describe the gymnophalid, echinostomatid and unidentified metacercariae parasites infecting the clam and the host reactions elicited by them. The use of special diagnostic techniques such as Ray’s fluid thioglycollate medium (RFTM) and PCR assays to detect Perkinsus sp. pathogens, hemolymph cytology, and histopathological examinations did not show Perkinsus sp. infections, microcell infections, or neoplastic conditions. However, neither infections or pathology caused by trematode parasites; nor any other pathological condition could be uniquely correlated with the mortality event. A coincident flash flood might have contributed to cause the mortality episode. This is the first report of the Parvatrema sp. metacercariae larvae infecting the stout razor clam T. plebeius from Brazil.     

Southernmost finding of Lymnaea viatrix orbigny, 1835 (Pulmonata: Lymnaeidae), intermediate host of Fasciola hepatica (Linnaeus, 1758) (Trematoda: Digenea), in urban and rural areas of Patagonia, Argentina

We report the first finding of Lymnaea viatrix south of parallel 41 masculine S, in rural and urban areas from Argentina. Ninety snails were collected during year 2000, from a concrete pond at a Public Square in El Bolsón Village, Río Negro province, and 811 snails in November 1999, and during 2000 from waterbodies within a farm at Cholila locality, Chubut province. Fasciola hepatica infection was detected in 0.9% snails from the rural area. We discuss the potential risk of L. viatrix to public health in urban areas and its epidemiological importance in rural areas of the Andean Patagonian region.     

Telomere length in healthy newborns is not affected by adverse intrauterine environments

Different intrauterine exposures are associated with different metabolic profiles leading to growth and development characteristics in children and also relate to health and disease patterns in adult life. The objective of this work was to evaluate the impact of four different intrauterine environments on the telomere length of newborns. This is a longitudinal observational study using a convenience sample of 222 mothers and their term newborns (>37 weeks of gestational age) from hospitals in Porto Alegre, Rio Grande do Sul (Brazil), from September 2011 to January 2016. Sample was divided into four groups: pregnant women with Gestational Diabetes Mellitus (DM) (n=38), smoking pregnant women (TOBACCO) (n=52), mothers with small-for-gestational age (SGA) children due to idiopathic intrauterine growth restriction (n=33), and a control group (n=99). Maternal and newborn genomic DNA were obtained from epithelial mucosal cells. Telomere length was assessed by qPCR, with the calculation of the telomere and single copy gene (T/S ratio). In this sample, there was no significant difference in telomere length between groups (p>0.05). There was also no association between childbirth weight and telomere length in children (p>0.05). For term newborns different intrauterine environments seems not to influence telomere length at birth.     

Bacterial and plant natriuretic peptides improve plant defence responses against pathogens

Plant natriuretic peptides (PNPs) have been implicated in the regulation of ions and water homeostasis, and their participation in the plant immune response has also been proposed. Xanthomonas citri ssp. citri contains a gene encoding a PNP‐like protein (XacPNP) which has no homologues in other bacteria. XacPNP mimics its Arabidopsis thaliana homologue AtPNP‐A by modifying host responses to create favourable conditions for pathogen survival. However, the ability of XacPNP to induce plant defence responses has not been investigated. In order to study further the role of XacPNP in vivo, A. thaliana lines over‐expressing XacPNP, lines over‐expressing AtPNP‐A and AtPNP‐A‐deficient plants were generated. Plants over‐expressing XacPNP or AtPNP‐A showed larger stomatal aperture and were more resistant to saline or oxidative stress than were PNP‐deficient lines. In order to study further the role of PNP in biotic stress responses, A. thaliana leaves were infiltrated with pure recombinant XacPNP, and showed enhanced expression of genes related to the defence response and a higher resistance to pathogen infections. Moreover, AtPNP‐A expression increased in A. thaliana on Pseudomonas syringae pv. tomato (Pst) infection. This evidence led us to analyse the responses of the transgenic plants to pathogens. Plants over‐expressing XacPNP or AtPNP‐A were more resistant to Pst infection than control plants, whereas PNP‐deficient plants were more susceptible and showed a stronger hypersensitive response when challenged with non‐host bacteria. Therefore, XacPNP, acquired by horizontal gene transfer, is able to mimic PNP functions, even with an increase in plant defence responses.     

Impaired Replication Capacity of Acute/Early Viruses in Persons Who Become HIV Controllers

Human immunodeficiency virus type 1 (HIV-1) controllers maintain viremia at <2,000 RNA copies/ml without antiretroviral therapy. Viruses from controllers with chronic infection were shown to exhibit impaired replication capacities, in part associated with escape mutations from cytotoxic-T-lymphocyte (CTL) responses. In contrast, little is known about viruses during acute/early infection in individuals who subsequently become HIV controllers. Here, we examine the viral replication capacities, HLA types, and virus sequences from 18 HIV-1 controllers identified during primary infection. gag-protease chimeric viruses constructed using the earliest postinfection samples displayed significantly lower replication capacities than isolates from persons who failed to control viremia (P = 0.0003). Protective HLA class I alleles were not enriched in these early HIV controllers, but viral sequencing revealed a significantly higher prevalence of drug resistance mutations associated with impaired viral fitness in controllers than in noncontrollers (6/15 [40.0%] versus 10/80 [12.5%], P = 0.018). Moreover, of two HLA-B57-positive (B57⁺) controllers identified, both harbored, at the earliest time point tested, signature escape mutations within Gag that likewise impair viral replication capacity. Only five controllers did not express “protective” alleles or harbor viruses with drug resistance mutations; intriguingly, two of them displayed typical B57 signature mutations (T242N), suggesting the acquisition of attenuated viruses from B57⁺ donors. These data indicate that acute/early stage viruses from persons who become controllers have evidence of reduced replication capacity during the initial stages of infection which is likely associated with transmitted or acquired CTL escape mutations or transmitted drug resistance mutations. These data suggest that viral dynamics during acute infection have a major impact on HIV disease outcome.Human immunodeficiency virus type I (HIV-1)-infected individuals who control viremia spontaneously without antiviral therapy have been termed HIV controllers (3, 18, 21, 48, 52). Unraveling the mechanisms associated with this phenotype should provide important insights regarding HIV pathogenesis and could contribute to vaccine development.Host and viral genetics, as well as host innate and adaptive immune responses, influence the rate of disease progression in HIV-1 infection (reviewed in reference 18). Several studies have reported the correlation between in vitro HIV replication capacity and level of plasma virus loads or disease progression in individuals with chronic infection (6, 13, 35, 45, 50, 55). Studies of HIV-1 elite controllers (EC), who control viremia to below the limit of detection in commercial assays, have revealed the presence of replication-competent viruses in these individuals (7), although these viruses appear to be less fit based on studies of envelope (35) and Gag-protease (45). This fitness defect in the chronic phase of infection is due at least in part to fitness-impairing mutations induced by cytotoxic-T-lymphocyte (CTL) responses restricted by “protective” HLA class I alleles (46).In contrast, little is known about viruses obtained from the acute/early phase of infection in persons who subsequently become HIV-1 controllers, largely due to the difficulty in enrolling such people during the acute/early phase of infection. The characterization of acute/early-phase viruses in individuals who subsequently achieve low set-point virus loads is of paramount importance to our understanding of the mechanisms of HIV-1 control.In the present study, we analyzed acute/early-phase plasma HIV RNA sequences from 18 untreated individuals who were diagnosed during the acute/early phase and subsequently became controllers (<2,000 RNA copies/ml). We compared these to sequences from a group of HIV-1 noncontrollers enrolled similarly during acute/early infection. We also generated chimeric viruses carrying patient-derived gag-protease sequences from acute/early-phase infection and compared the viral replication capacities of the chimeric viruses from controllers and from noncontrollers.We observed that the chimeric viruses derived from controllers have significantly reduced replicative capacities compared to those from noncontrollers. Moreover, we observed that at least 80% of these individuals who go on to become controllers featured transmission of attenuated drug-resistant viruses, transmission of HLA-B57-restricted CTL escape variants to HLA-mismatched recipients, selection of attenuated CTL escape variants in HLA-B57-positive (B57⁺) recipients, or combinations of these factors. Taken together, these results indicate that the initial viral dynamics have a major influence on the subsequent course of disease.