First Report of the Root Knot Nematode, Meloidogyne inornata, on Common Bean in Paraná State, Brazil.

Common bean (Phaseolus vulgaris F.) is one of the most important crops in Paraná State, which is responsible for almost 10% of the Brazilian production (4). Root knot nematodes, Meloidogyne spp., are common parasites of this crop worldwide, but damage caused by Meloidogyne inornata has not been reported. During a survey of nematode species present on common bean fields in Paraná State, Brazil, galled root samples of cultivars Tuiuiú and Eldorado were submitted, in June 2012, in the Nematology Laboratory from IAPAR, collected in the municipalities of Araucária (25°35'34″S, 49°24'36″W) and Santana do Itararé (23°45'18″S, 49°37'44″W). Plants did not exhibit any above-ground symptoms. The specimens were identified through perineal patterns and esterase phenotypes of 20 adult females extracted from dissected roots (2,3). The population densities observed in the samples were 140 and 700 J2 and eggs per gram of roots, respectively, for both samples. Characteristics were consistent with those described for M. inornata. For example, perineal patterns of M. inornata showed a high dorsal arch, with smooth to wavy striae, similar to those of M. incognita; but no punctate markings between anus and tail terminus were observed. However, from the esterase electrophoresis we obtained the I3 (Rm = 0.83, 1.15, and 1.32) phenotype, typical of M. inornata, a species-specific phenotype used to differentiate this species from M. incognita (1). Moreover, the excretory pore of adult females was located 32.1 (± 5.4) µm from the anterior end, consistent with the M. inornata description (25 to 53 µm) (1). To the best of our knowledge, this is the first report of M. inornata parasitizing common bean roots. This finding has great importance for Brazilian agriculture, since this nematode may damage common bean plants and become an additional problem for this crop. Additional work is necessary in order to elucidate the losses caused by M. inornata on common bean. References: (1) R. M. D. G. Carneiro et al. Nematology 10:123, 2008. (2) P. R. Esbenshade and A. C. Triantaphyllou J. Nematol. 22:10, 1990. (3) K. M. Hartman and J. N. Sasser. Page 115 in: An Advanced Treatise on Meloidogyne, Volume II Methodology. K. R. Barker et al., eds. Raleigh: North Carolina State University Graphics, 1985. (4) MAPA. Feijão, Ministério da Agricultura, Brasil. Retrieved from http://www.agricultura.gov.br/vegetal/culturas/feijao September 05, 2012.

Transmission blocking immunity in Plasmodium vivax malaria: antibodies raised against a peptide block parasite development in the mosquito vector.

One approach towards the development of a vaccine against malaria is to immunize against the parasite sexual stages that mediate transmission of the parasite from man to mosquito. Antibodies against these stages, ingested with the blood meal, inhibit the parasite development in the mosquito vector, constituting "transmission blocking immunity." Most epitopes involved in transmission-blocking immunity depend on the tertiary conformational structure of surface antigens. However, one of the transmission-blocking monoclonal antibodies we have raised against Plasmodium vivax reacts with a linear epitope on both asexual stages and gametes. This monoclonal antibody (A12) is capable of totally blocking development of the parasite in the mosquito host when tested in membrane feeding assays with gametocytes from P. vivax-infected patients. Immune screening of a P. vivax lambda gt11 genomic expression library with A12 led to the isolation of a clone to which was mapped the six-amino acid epitope recognized by A12. Antisera raised in mice against a 12-mer synthetic peptide containing this epitope coupled to bovine serum albumin not only had high titers of antipeptide antibodies as measured by enzyme-linked immunosorbent assay, but in addition recognized the same 24- and 57-kD parasite components as A12 on Western blots and reacted with the parasite by immunofluorescence. When tested in membrane feeding assays, these antibodies have significant suppressive effects on parasite development in the mosquito.

Fungal infections in recipients of hematopoietic stem cell transplants: results of the SEIFEM B-2004 study--Sorveglianza Epidemiologica Infezioni Fungine Nelle Emopatie Maligne.

BACKGROUND: The purpose of our study was to evaluate the incidence and outcome of invasive fungal infection (IFI) among patients who underwent autologous or allogeneic hematopoietic stem cell transplantation (HSCT) at 11 Italian transplantation centers. METHODS: This cohort-retrospective study, conducted during 1999-2003, involved HSCT patients admitted to 11 tertiary care centers or university hospitals in Italy, who developed IFIs (proven or probable). RESULTS: Among 3228 patients who underwent HSCT (1249 allogeneic HSCT recipients and 1979 autologous HSCT recipients), IFI occurred in 121 patients (overall incidence, 3.7%). Ninety-one episodes (2.8% of all patients) were due to molds, and 30 (0.9%) were due to yeasts. Ninety-eight episodes (7.8%) occurred among the 1249 allogeneic HSCT recipients, and 23 (1.2%) occurred among the 1979 autologous HSCT recipients. The most frequent etiological agents were Aspergillus species (86 episodes) and Candida species (30 episodes). The overall mortality rate was 5.7% among allogeneic HSCT recipients and 0.4% among autologous HSCT recipients, whereas the attributable mortality rate registered in our population was 65.3% (72.4% for allogeneic HSCT recipients and 34.7% for autologous HSCT recipients). Etiology influenced the patients' outcomes: the attributable mortality rate for aspergillosis was 72.1% (77.2% and 14.3% for allogeneic and autologous HSCT recipients, respectively), and the rate for Candida IFI was 50% (57.1% and 43.8% for allogeneic and autologous HSCT recipients, respectively). CONCLUSIONS: IFI represents a common complication for allogeneic HSCT recipients. Aspergillus species is the most frequently detected agent in these patients, and aspergillosis is characterized by a high mortality rate. Conversely, autologous HSCT recipients rarely develop aspergillosis, and the attributable mortality rate is markedly lower. Candidemia was observed less often than aspergillosis among both allogeneic and autologous HSCT recipients; furthermore, there was no difference in either the incidence of or the attributable mortality rate for candidemia among recipients of the 2 transplant types.

Expressed var genes are found in Plasmodium falciparum subtelomeric regions.

The antigenic variation and cytoadherence of Plasmodium falciparum-infected erythrocytes are modulated by a family of variant surface proteins encoded by the var multigene family. The var genes occur on multiple chromosomes, often in clusters, and 50 to 150 genes are estimated to be present in the haploid parasite genome. Transcripts from var genes have been previously mapped to internal chromosome positions, but the generality of such assignments and the expression sites and mechanisms that control switches of var gene expression are still in early stages of investigation. Here we describe investigations of closely related var genes that occur in association with repetitive elements near the telomeres of P. falciparum chromosomes. DNA sequence analysis of one of these genes (FCR3-varT11-1) shows the characteristic two-exon structure encoding expected var features, including three variable Duffy binding-like (DBL) domains, a transmembrane sequence, and a carboxy-terminal segment thought to anchor the protein product in knobs at the surface of the parasitized erythrocyte. FCR3-varT11-1 cross-hybridizes with var genes located close to the telomeres of many other P. falciparum chromosomes, including a transcribed gene (FCR3-varT3-1) in chromosome 3 of the P. falciparum FCR3 line. The relatively high level transcription from this gene shows that the polymorphic chromosome ends of P. falciparum, which have been proposed to be transcriptionally silent, can be active expression sites for var genes. The pattern of the FCR3-varT11-1 and FCR3-varT3-1 genes are variable between different P. falciparum lines, presumably due to DNA rearrangements. Thus, recombination events in subtelomeric DNA may have a role in the expression of novel var forms.

Diurnal preference, mood and the response to morning light in relation to polymorphisms in the human clock gene PER3.

PER3 gene polymorphisms have been associated with differences in human sleep-wake phenotypes, and sensitivity to light. The aims of this study were to assess: i) the frequency of allelic variants at two PER3 polymorphic sites (rs57875989 length polymorphism: PER3 4, PER3 5; rs228697 SNP: PER3 C, PER3 G) in relation to sleep-wake timing; ii) the effect of morning light on behavioural/circadian variables in PER3 4 /PER3 4 and PER3 5 /PER3 5 homozygotes. 786 Caucasian subjects living in Northern Italy donated buccal DNA and completed diurnal preference, sleep quality/timing and sleepiness/mood questionnaires. 19 PER3 4 /PER3 4 and 11 PER3 5 /PER3 5 homozygotes underwent morning light administration, whilst monitoring sleep-wake patterns and the urinary 6-sulphatoxymelatonin (aMT6s) rhythm. No significant relationship was observed between the length polymorphism and diurnal preference. By contrast, a significant association was observed between the PER3 G variant and morningness (OR = 2.10), and between the PER3 G-PER3 4 haplotype and morningness (OR = 2.19), for which a mechanistic hypothesis is suggested. No significant differences were observed in sleep timing/aMT6s rhythms between PER3 5 /PER3 5 and PER3 4 /PER3 4 subjects at baseline. After light administration, PER3 4 /PER3 4 subjects advanced their aMT6s acrophase (p < 0.05), and showed a trend of advanced sleep-wake timing. In conclusion, significant associations were observed between PER3 polymorphic variants/their combinations and both diurnal preference and the response to light.

Maternal immune activation results in complex microglial transcriptome signature in the adult offspring that is reversed by minocycline treatment.

Maternal immune activation (MIA) during pregnancy has been linked to an increased risk of developing psychiatric pathologies in later life. This link may be bridged by a defective microglial phenotype in the offspring induced by MIA, as microglia have key roles in the development and maintenance of neuronal signaling in the central nervous system. The beneficial effects of the immunomodulatory treatment with minocycline on schizophrenic patients are consistent with this hypothesis. Using the MIA mouse model, we found an altered microglial transcriptome and phagocytic function in the adult offspring accompanied by behavioral abnormalities. The changes in microglial phagocytosis on a functional and transcriptional level were similar to those observed in a mouse model of Alzheimer's disease hinting to a related microglial phenotype in neurodegenerative and psychiatric disorders. Minocycline treatment of adult MIA offspring reverted completely the transcriptional, functional and behavioral deficits, highlighting the potential benefits of therapeutic targeting of microglia in psychiatric disorders.

Microcystin diversity in a Planktothrix rubescens population from Lake Albano (Central Italy).

The cyanobacterium Planktothrix rubescens Anagnostidis & Komarek (previously Oscillatoria rubescens DC ex Gomont) is present in several Italian lakes and it is known to produce cyanotoxins. The dynamics and toxin production of P. rubescens population in Lake Albano, a volcanic crater lake in Central Italy, has been studied for 5 years (January 2001-April 2005). Winter-spring superficial blooms with frequent scums were observed every year. Total microcystin (MC) levels were measured from April 2004 to October 2005 by liquid chromatography-tandem mass spectrometry. MC levels up to 14.2mug/l were measured, with high concentrations found in summer at a 20-25m depth. The intracellular toxin content varied between 1.5 (surface, January 2004) and 0.21pg/cell (surface, May 2004). Six different MCs were detected, the most abundant being two desmethyl-MC-RR isomers. Of the 13 water wells monitored in the Lake Albano area, two of them showed MC contamination during winter, confirming the ability of these toxins to migrate through groundwater towards public water sources. These results highlight the need for further studies on the mobility and fate of these pervasive cyanobacterial toxins.

ELISA and LC-MS/MS methods for determining cyanobacterial toxins in blue-green algae food supplements.

The use of natural products as a diet supplement is increasing worldwide but sometimes is not followed by adequate sanitary controls and analyses. Twenty samples of pills and capsules of lyophilised cyanobacteria (blue-green algae), commercialised in Italy as dietary supplements, were found positive at the Vibrio fischeri bioassay. Further analyses with ELISA and LC-MS/MS methods revealed the presence of four microcystin (MC) analogues, MC-LR, -YR, -LA, -RR and two demethylated forms of MC-RR. The highest total microcystin content was 4.5 and 1.4 microg g-1 in pills and capsules, respectively. The ELISA measurements, compared to the LC-MS/MS analyses, showed significantly lower concentrations of microcystins in pills, this confirming a possible ELISA underestimate of mixed microcystins, due to different sensitivities for some toxic analogues.

Mapping elements distribution in carapace of Caretta caretta: A strategy for biomonitoring contamination in sea turtles?

This study analyzed the carapace distribution of Ca, Cd, Cr, Cu, Mg, Mn, Pb, Sb, U, V and Zn by GF-AAS and ICP-AES in one specimen of Caretta caretta from Mediterranean Sea. Calcium, Mg, Mn, Pb, U, Zn were mainly distributed in the central area while Cd, Cr, Cu, Sb, V in lateral areas. Cadmium, Cr, Mg, Mn, Sb, U and V were different between lateral areas. The different distribution may be related to several exposures during lifetime and/or the shell ossification during growth. Carapace may be a suitable matrix for metal biomonitoring, however, further studies are required to confirm these findings.

The Pf332 gene of Plasmodium falciparum codes for a giant protein that is translocated from the parasite to the membrane of infected erythrocytes.

We studied the gene structure of the Plasmodium falciparum antigen 332 (Ag332). The gene size was estimated to be approx. 20 kb based on the large size of both the transcript found in mature asexual blood stage parasites and mung bean nuclease fragment generated from genomic DNA. Sequence analysis of genomic and cDNA clones representing different regions of the Pf332 locus showed that the gene product contains a large number of highly degenerated glutamic acid (Glu)-rich repeats (32% Glu). The gene shows dramatic restriction fragment length polymorphism in various P. falciparum isolates and was mapped to the subtelomeric region of chromosome 11. The recombinant 332 fusion protein reacts strongly with the human monoclonal antibody (mAb) 33G2, which is able to inhibit the cytoadherence of parasitized red blood cells on the melanoma cell line C32 and merozoite invasion in in vitro assays. The epitope recognized by this mAb is found frequently in the reported sequence. Ag332 monospecific antibodies were obtained by immunization of mice with a recombinant fusion protein. These antibodies react with a large parasite molecule with an apparent molecular size of 2500 kDa of trophozoite and schizont-infected erythrocytes on Western blot and by immunoprecipitation analysis. Immunofluorescence studies using a confocal microscope showed that Ag332 is exported from the parasite to the infected red blood cell membrane within large vesicle-like structures of about 1 micron diameter.

Genetic diversity in the major merozoite surface antigen of Plasmodium falciparum: high prevalence of a third polymorphic form detected in strains derived from malaria patients.

We studied the diversity of the polymorphic 195-kDa antigen (p190) of Plasmodium from infected individuals. Genomic parasite DNA was extracted from the blood of 30 donors from different endemic areas of Brazil. The 5' region, encoding the polymorphic N-terminal part of p190 was analysed following polymerase chain reaction (PCR). Multiple infections of genetically distinct parasites could be detected within infected malaria patients. Sequence analysis and oligodeoxyribonucleotide typing of the PCR products demonstrated the prevalence of a third polymorphic form of p190.

Cloning and characterisation of a gene from Plasmodium vivax and P. knowlesi: homology with valine-tRNA synthetase.

We have previously described a lambdagt11 clone detected by immune screening with a monoclonal antibody (mAb) A12. This mAb is capable of completely blocking Plasmodium vivax transmission in the mosquito vector. An epitope recognised by A12 was mapped to six amino acids (aa) within the translated sequence of this clone. Here, we describe the complete sequence of the gene within which we mapped this epitope. Surprisingly, the translated sequence of the full-length open reading frame shows homology with that of valine-tRNA synthetases (Val-tRS) from other organisms. DNA cross-hybridisation with several of these species was observed by Southern blot. In addition, the corresponding gene has been obtained from the closely related simian malaria parasite, P. knowlesi. The two aa sequences show 66% identity and yet are very divergent from other Val-tRS sequences, apart from conserved blocks related to functional activity. Multiple sequence alignments reflect this dichotomy, as do predicted differences in antigenicity.

Parasite antigens expressed in Escherichia coli. A refined approach for epidemiological analysis.

A simple method is described to generate carrier-free recombinant antigens following their expression in Escherichia coli. A plasmid, called pMSgt11, has been constructed such that the cleavage site for the protease factor Xa separates the recombinant antigen from an enzymatically active beta-galactosidase. Thus, rapid purification of the active beta-galactosidase recombinant protein, followed by digestion with factor Xa, releases the antigen of interest. The pMSgt11 plasmid is compatible with the phage expression vector, lambda gt11 and the feasibility of applying this system has been demonstrated using malarial recombinant antigens. Inserts from lambda gt11 recombinant Plasmodium falciparum clones have been recloned into the EcoRI site of pMSgt11 and the expressed soluble fusion proteins have been purified from crude extracts using a one step affinity chromatography. After protease digestion, the fusion protein cleavage products were analysed by immunoblot with a panel of different human immune sera. We were able to successfully demonstrate specific antibody titers to the parasite-derived carrier-free antigen, without interference from anti-Escherichia coli-specific antibodies. The general application of this approach to epidemiological analysis is discussed.

In vitro phagocytosis inhibition assay for the screening of potential candidate antigens for sub-unit vaccines against the asexual blood stage of Plasmodium falciparum.

We have previously established a direct correlation between immune protection against the asexual blood stage Plasmodium falciparum infection and the presence of opsonizing antibodies promoting phagocytosis of parasitized red blood cells. In the present communication we describe an in vitro assay for measuring phagocytosis inhibition (PIA) specific for P. falciparum-infected erythrocytes. The phagocytosis inhibition assay is a simple procedure for screening potential candidates for sub-unit vaccines against P. falciparum based on the correlation between opsonizing antibodies and immunoprotection. The assay was used to analyse 18 recombinant molecules, corresponding to 11 distinct antigens of P. falciparum. Pre-incubation and selective antibody depletion experiments demonstrate the antigen-antibody specificity of the PIA. The presence of epitopes participating as targets of opsonic antibodies were demonstrated in six distinct polypeptide antigens.

Plaque antibody selection: rapid immunological analysis of a large number of recombinant phage clones positive to sera raised against Plasmodium falciparum antigens.

A library of Plasmodium falciparum genomic DNA on the lambda gt11 phage vector was screened for clones positive to a rabbit serum raised against a purified fraction of P. falciparum proteins and a pool of sera from malaria patients. The positive clones were characterized with antibodies purified by the plaque antibody selection technique. This technique consist of purifying specific antibodies on a nitrocellulose filter blotted directly on a lawn of plaques of an antigen-producing phage clone. The purified antibodies are then used as a probe in a Western blot of parasite protein extract, for preliminary characterization of the clones. Using this method, two different clones coding for P. falciparum antigens were identified with the rabbit serum and about 20 with the human sera. This method can be of general use, i.e. it is not limited to parasite systems, and facilitates the immunological analysis and identification of a large number of clones.

Characterisation of PfSec61, a Plasmodium falciparum homologue of a component of the translocation machinery at the endoplasmic reticulum membrane of eukaryotic cells.

Plasmodium falciparum secretes several proteins that cause changes in the erythrocyte membrane enabling it to survive within red blood cells. Little is known of the mechanisms involved in the secretion and targeting of parasite polypeptides to the various cell compartments. The P. falciparum gene homologous to the mammalian Sec61alpha, gene, which encodes a component of the translocation pore in the endoplasmic reticulum of eukaryotic cells, was characterised to investigate the translocation process in the parasite. PfSec61 is present as a unique copy in the parasite genome and was mapped to chromosome 13. It encodes a 40 kDa polypeptide, as shown by immunoblotting and immunoprecipitation of [35S]methionine metabolically-labelled parasite extracts. The deduced amino acid sequence of PfSec61 is 87% similar to the mammalian polypeptide, and the two proteins give similar hydropathy plots. These results strongly suggest that PfSec61 has the same topological orientation and functional role as Sec61alpha. Anti-PfSec61 antibodies were used to investigate the cellular location and kinetics of expression of the polypeptide in the parasite. Immunofluorescence confocal microscopy showed that PfSec61 was located in the parasite cytoplasm, close to the nucleus, in a position consistent with its being in the endoplasmic reticulum.

Expression and cellular localization of Trypanosoma cruzi type II DNA topoisomerase.

Topoisomerases are enzymes that participate in many cellular functions involving topological manipulation of DNA strands. There are two types of topoisomerases in the cell: (a) type I topoisomerases; and (b) type II topoisomerases (topo II). Previously we have cloned and sequenced the gene encoding Trypanosoma cruzi topo II (TcTOP2). This study group has raised an antiserum against recombinant type II DNA topoisomerase (TctopoII) to study the expression of this gene during T. cruzi differentiation and to determine the cellular location of the enzyme. Western blot analysis showed that T. cruzi TctopoII is expressed in the replicative epimastigotes but not in the infective and non-replicative trypomastigotes. However, slot blot analysis of RNAs extracted from epimastigotes and metacyclic trypomastigotes showed that the mRNA encoding the enzyme is present in both developmental stages of the parasite. Confocal laser microscopy using the antiserum raised against recombinant TctopoII showed that the enzyme is located exclusively in the nucleus of the parasite. Similar results were obtained by immunofluorescence analysis of Crithidia fasciculata. However, monoclonal antisera against the corresponding enzyme extracted from C. fasciculata recognizes a kinetoplast protein in both T. cruzi and Crithidia.

The S-antigen of Plasmodium falciparum Palo Alto represents a new S-antigen serotype.

The S-antigen from the Palo Alto isolate of Plasmodium falciparum has been characterized. The partial sequence for the gene coding for this antigen (clone 281) reveals the presence of tandem repeats of eight amino acids which defines a new S-antigen serotype. Antibodies raised against the 281 recombinant clone reacted with a 140 kDa antigen by immunoblotting with parasite extracts and culture supernatants. The 140 kDa peptide was also identified by immunoprecipitation of metabolic labelled parasites. The 281 mouse antiserum was used to localize the antigen on parasite smears by indirect immunofluorescence assay and more precisely by immunoelectron microscopy. The S-antigen is localized within the parasitophorous vacuole. Furthermore, different isolates were examined for the presence of the Palo Alto S-antigen specificity.

Pfsbp1, a Maurer's cleft Plasmodium falciparum protein, is associated with the erythrocyte skeleton.

Antibodies from hyperimmune monkey sera, selected by absorption to Plasmodium falciparum-infected erythrocytes, and elution at acidic pH, allowed us to characterize a novel parasite protein, Pfsbp1 (P. falciparum skeleton binding protein 1). Pfsbp1 is an integral membrane protein of parasite-induced membranous structures associated with the erythrocyte plasma membrane and referred to as Maurer's clefts. The carboxy-terminal domain of Pfsbp1, exposed within the cytoplasm of the host cell, interacts with a 35 kDa erythrocyte skeletal protein and might participate in the binding of the Maurer's clefts to the erythrocyte submembrane skeleton. Antibodies to the carboxy- and amino-terminal domains of Pfsbp1 labelled similar vesicular structures in the cytoplasm of Plasmodium chabaudi and Plasmodium berghei-infected murine erythrocytes, suggesting that the protein is conserved among malaria species, consistent with an important role of Maurer's cleft-like structures in the intraerythrocytic development of malaria parasites.

Plasmodium falciparum signal sequences: simply sequences or special signals?

The malaria parasite, Plasmodium falciparum, synthesises and exports several proteins inducing morphological and biochemical modifications of erythrocytes during the erythrocytic cycle. The protein trafficking machinery of the parasite is similar to that of other eukaryotic cells in several ways. However, some unusual features are also observed. The secretion of various polypeptides was inhibited when P. falciparum-infected erythrocytes were incubated with Brefeldin A. Immunoelectron microscopy studies revealed substantial morphological changes in the endoplasmic reticulum following exposure of parasitised erythrocytes to the drug. Immunofluorescence studies of Brefeldin A-treated parasites suggest that polypeptide sorting to different intracellular destinations begins at the endoplasmic reticulum. The parasite also secretes polypeptides by a Brefeldin A-insensitive route that bypasses the classical endoplasmic reticulum-Golgi complex pathway.