The Compatibility Between Biomphalaria glabrata Snails and Schistosoma mansoni: An Increasingly Complex Puzzle.

This review reexamines the results obtained in recent decades regarding the compatibility polymorphism between the snail, Biomphalaria glabrata, and the pathogen, Schistosoma mansoni, which is one of the agents responsible for human schistosomiasis. Some results point to the snail's resistance as explaining the incompatibility, while others support a "matching hypothesis" between the snail's immune receptors and the schistosome's antigens. We propose here that the two hypotheses are not exclusive, and that the compatible/incompatible status of a particular host/parasite couple probably reflects the balance of multiple molecular determinants that support one hypothesis or the other. Because these genes are involved in a coevolutionary arms race, we also propose that the underlying mechanisms can vary. Finally, some recent results show that environmental factors could influence compatibility. Together, these results make the compatibility between B. glabrata and S. mansoni an increasingly complex puzzle. We need to develop more integrative approaches in order to find targets that could potentially be manipulated to control the transmission of schistosomiasis.

Transcriptional changes in Crassostrea gigas oyster spat following a parental exposure to the herbicide diuron.

The Pacific oyster Crassostrea gigas is the main oyster species produced in the world, and a key coastal economic resource in France. High mortalities affect Pacific oysters since 2008 in France and Europe. Their origins have been attributed to a combination of biotic and abiotic factors, underlining the importance of environment quality. The impact of water pollution has been pointed out and one of the pollutants, the genotoxic herbicide diuron, occurs at high concentrations all along the French coasts. Previous work has revealed that a parental exposure to diuron had a strong impact on hatching rates and offspring development even if spats were not exposed to diuron themselves. In this study, we explored for the first time the transcriptional changes occurring in oyster spats (non exposed) originating from genitors exposed to an environmentally relevant concentration of diuron during gametogenesis using the RNAseq methodology. We identified a transcriptomic remodeling revealing an effect of the herbicide. Different molecular pathways involved in energy production, translation and cell proliferation are particularly disturbed. This analysis revealed modulated candidate genes putatively involved in response to oxidative stress and mitochondrial damage in offspring of genitors exposed to diuron. Complementary measures of the activity of enzymes involved in these latter processes corroborate the results obtained at the transcriptomic level. In addition, our results suggested an increase in energy production and mitotic activity in 5-month-spats from diuron-exposed genitors. These results could correspond to a "catch-up growth" phenomenon allowing the spats from diuron-exposed genitors, which displayed a growth delay at 3 months, to gain a normal size when they reach the age of 6 months. These results indicate that exposure to a concentration of diuron that is frequently encountered in the field during the oyster's gametogenesis stage can impact the next generation and may result in fitness disturbance.

Recent advances in Schistosoma genomics.

Schistosome research has entered the genomic era with the publications reporting the Schistosoma mansoni and Schistosoma japonicum genomes. Schistosome genomics is motivated by the need for new control tools. However, much can also be learned about the biology of Schistosoma, which is a tractable experimental model. In this article, we review the recent achievements in the field of schistosome research and discuss future perspectives on genomics and how it can be integrated in a usable format, on the genetic mapping and how it has improved the genome assembly and provided new research approaches, on how epigenetics provides interesting insights into the biology of the species and on new functional genomics tools that will contribute to the understanding of the function of genes, many of which are parasite- or taxon specific.

Vertebrate host protective immunity drives genetic diversity and antigenic polymorphism in Schistosoma mansoni.

Schistosomes are gonochoric blood parasites with a complex life cycle responsible for a disease of considerable medical and veterinary importance in tropical and subtropical regions. Understanding the evolution of schistosome genetic diversity is clearly of fundamental importance to interpreting schistosomiasis epidemiology and disease transmission patterns of this parasite. In this article, we investigated the putative role of the host immune system in the selection of male genetic diversity. We demonstrated the link between genetic dissimilarity and the protective effect among male worms. We then compared the proteomes of three male clones with different genotypes and differing by their capacity to protect against reinfection. The identified differences correspond mainly to antigens known or supposed to be involved in the induction of protective immunity. These results underline the role played by host immune system in the selection of schistosome genetic diversity that is linked to antigenic diversity. We discuss the evolutionary consequences in the context of schistosome infection.

Schistosoma mansoni: developmental arrest of miracidia treated with histone deacetylase inhibitors.

In the present study, we examined the effect of the histone deacetylase (HDAC) inhibitors trichostatin A (TSA), valproic acid (VA) and sodium-butyrate on the metamorphosis of larvae of the human blood-fluke Schistosoma mansoni from the free-swimming miracidia into the intramolluskal sporocyst. We show that HDAC inhibitors block transformation in concentration dependant manner. TSA reversibly blocks this developmental process: only 13+/-11% of TSA treated miracidia transform into sporocysts in-vitro, compared to 92+/-3% in the mock-treated control. Other enzyme inhibitors such as cycloheximide or hydroxyurea had no effect on metamorphosis. For treatment of up to 4 h, the effect of TSA was completely reversible. Our data indicates that HDAC activity is necessary for the transformation of S. mansoni miracidia during infection of the snail host.

The compatibiuty polymorphism in invertebrate host/trematodes interactions: research of molecular determinants.

The co-evolutionary dynamics that exist in many host-parasite interactions sometimes leads to compatibility polymorphism. This phenomenon is well documented in mollusc/trematodes interactions but its molecular base is unknown. In order to identify key molecules involved in this phenomenon, we developed several molecular approaches comparing compatible or incompatible strains of mollusc or parasite. These comparisons led to the identification of numerous candidate genes listed and discussed (some of them) in the present review.

Bisulfite genomic sequencing: systematic investigation of critical experimental parameters.

Bisulfite genomic sequencing is the method of choice for the generation of methylation maps with single-base resolution. The method is based on the selective deamination of cytosine to uracil by treatment with bisulfite and the sequencing of subsequently generated PCR products. In contrast to cytosine, 5-methylcytosine does not react with bisulfite and can therefore be distinguished. In order to investigate the potential for optimization of the method and to determine the critical experimental parameters, we determined the influence of incubation time and incubation temperature on the deamination efficiency and measured the degree of DNA degradation during the bisulfite treatment. We found that maximum conversion rates of cytosine occurred at 55 degrees C (4-18 h) and 95 degrees C (1 h). Under these conditions at least 84-96% of the DNA is degraded. To study the impact of primer selection, homologous DNA templates were constructed possessing cytosine-containing and cytosine-free primer binding sites, respectively. The recognition rates for cytosine (>/=97%) and 5-methylcytosine (>/=94%) were found to be identical for both templates.

MethDB--a public database for DNA methylation data.

Methylation of cytosine in the 5 position of the pyrimidine ring is a major modification of the DNA in most organisms. In eukaryotes, the distribution and number of 5-methylcytosines (5mC) along the DNA is heritable but can also change with the developmental state of the cell and as a response to modifications of the environment. While DNA methylation probably has a number of functions, scientific interest has recently focused on the gene silencing effect methylation can have in eukaryotic cells. In particular, the discovery of changes in the methylation level during cancer development has increased the interest in this field. In the past, a vast amount of data has been generated with different levels of resolution ranging from 5mC content of total DNA to the methylation status of single nucleotides. We present here a database for DNA methylation data that attempts to unify these results in a common resource. The database is accessible via WWW (http://www.methdb.de). It stores information about the origin of the investigated sample and the experimental procedure, and contains the DNA methylation data. Query masks allow for searching for 5mC content, species, tissue, gene, sex, phenotype, sequence ID and DNA type. The output lists all available information including the relative gene expression level. DNA methylation patterns and methylation profiles are shown both as a graphical representation and as G/A/T/C/5mC-sequences or tables with sequence positions and methylation levels, respectively.

Large-scale methylation analysis of human genomic DNA reveals tissue-specific differences between the methylation profiles of genes and pseudogenes.

Cytosine in CpG dinucleotides is frequently found to be methylated in the DNA of higher eukaryotes and differential methylation has been proposed to be a key element in the organization of gene expression in man. To address this question systematically, we used bisulfite genomic sequencing to study the methylation patterns of three X-linked genes and one autosomal pseudogene in two adult individuals and across nine different tissues. Two of the genes, SLC6A8 and MSSK1, are tissue-specifically expressed. CDM is expressed ubiquitously. The pseudogene, psi SLC6A8, is exclusively expressed in the testis. The promoter regions of the SLC6A8, MSSK1 and CDM genes were found to be essentially unmethylated in all tissues, regardless of their relative expression level. In contrast, the pseudogene psi SLC6A8 shows high methylation of the CpG islands in all somatic tissues but complete demethylation in testis. Methylation profiles in different tissues are similar in shape but not identical. The data for the two investigated individuals suggest that methylation profiles of individual genes are tissue specific. Taken together, our findings support a model in which the bodies of the genes are predominantly methylated and thus insulated from the interaction with DNA-binding proteins. Only unmethylated promoter regions are accessible for binding and interaction. Based on this model we propose to use DNA methylation studies in conjunction with large-scale sequencing approaches as a tool for the prediction of cis-acting genomic regions, for the identification of cryptic and potentially active CpG islands and for the preliminary distinction of genes and pseudogenes.

MethTools--a toolbox to visualize and analyze DNA methylation data.

The Bisulfite Genomic Sequencing technique has found wide acceptance for the generation of DNA-methylation maps with single-base resolution. The method is based on the selective deamination of cytosine to uracil (and subsequent conversion to thymine via PCR), whereas 5-methylcytosine residues remain unchanged. Methylation maps are created by the comparison of bisulfite converted sequences with the untreated genomic sequence. 'MethTools' is a collection of software tools that replaces the time-consuming manual comparison process, generates graphical outputs of methylation patterns and methyl-ation density, estimates the systematic error of the experiment and searches for conserved methylated nucleotide patterns. The programs are written in Perl 5 and C, and the source code can be downloaded. All tools run independently but the programs are interfaced. Thus, a script can perform the entire analysis procedure automatically. In addition, a web-based remote analysis service is offered. Both the source code and the remote analysis are available at http://genome.imb-jena.de/methtools/

Carpel, a new Arabidopsis epi-mutant of the SUPERMAN gene: phenotypic analysis and DNA methylation status.

The carpel (car) mutation affects the morphology of reproductive organs in Arabidopsis thaliana. car flowers have an increased number of carpels, on average 2.7 +/- 0.8 instead of two in the wild type. Through allelism test with fon1-3 and analysis of the methylation state of the SUPERMAN (SUP) gene in car mutants, we show that car is an epi-mutation of SUP. The methylation pattern of car is clearly distinct from that of fon1-3, another epi-mutation of the SUP gene. Methylation was found predominantly in Cp(A/T)p(A/G) triplets and in CpG pairs. We suggest that the extensive SUP methylation in car has arisen from an abundant methylation of a single CpG site that was already present in abscisic acid-insensitive (abi3-4) mutants, from which car was segregating.

Phylogenetic identification of the symbiotic hypermastigote Trichonympha agilis in the hindgut of the termite Reticulitermes speratus based on small-subunit rRNA sequence.

The phylogeny of a symbiotic hypermastigote Trichonympha agilis (class Parabasalia; order Hypermastigida) in the hindgut of the lower termite Reticulitermes speratus was examined by a strategy that does not rely on cultivation. From mixed-population DNA obtained from the termite gut, small subunit (16S-like) ribosomal RNA sequences were directly amplified by the polymerase chain reaction method using primers specific for eukaryotes. Comparative sequence analysis of the clones revealed two kinds of sequences, one from the termite itself and the other from a symbiotic protist. A fluorescent-labeled oligonucleotide probe for the latter sequence was designed and used in whole-cell hybridization experiments to provide direct visual evidence that the sequence originated from a larger hypermastigote in the termite hindgut, Trichonympha agilis. According to the phylogenetic trees constructed, the hypermastigote represented one of the deepest branches of eukaryotes. The hypermastigote along with members of the order Trichomonadida formed a monophyletic lineage, indicating that this hypermastigote and trichomonads shared a recent common ancestry.

Bovine adrenodoxin--a mitochondrial iron-sulphur protein--binds to chaperonin GroEL.

The interaction of bovine adrenodoxin with the chaperonin GroEL was investigated using sucrose density centrifugation and analytical ultracentrifugation. It could be clearly established that denatured mature adrenodoxin comigrated in a sucrose density gradient with the GroEL oligomer, indicating that a complex had been formed. Up to 2 moles of adrenodoxin/mol GroEL can be bound. From the partial concentrations, association constants of 4.3 x 10(5) M-1 for the first adrenodoxin molecule and of 1.08 x 10(5) M-1 for the second molecule to the complex, respectively, were calculated. Upon addition of the cochaperonin GroES and Mg-ATP to the adrenodoxin-GroEL complex, adrenodoxin was released, indicating a specific binding between GroEL and adrenodoxin.

Immunocytochemical localization of heterologously expressed adrenodoxin and its electron acceptor cytochrome P45011B1 in Escherichia coli.

Adrenal steroid hydroxylase P45011B1 and its electron donor adrenodoxin were localized in the cortex of bovine adrenals by immunogold-silver staining. In order to test recently developed heterologous expression systems for both enzymes to enable structure-function studies, immunocytochemical marker methods were applied. Adrenodoxin, the ferredoxin of the adrenal gland, was successfully expressed and for the first time localized in Escherichia coli. By use of ultrathin cryosections and the protein A-gold technique, adrenodoxin was detectable in large amounts in the cytoplasm of the bacterial cells, and, following the insertion of the outer membrane protein A leader sequence of E. coli, also in the periplasmic space. A fusion protein between mature adrenodoxin and human P45011B1 was constructed and clearly localized in E. coli by antibodies against both proteins.

Nonlinear transformation of the resting electrocardiogram in the diagnosis of coronary artery disease.

Despite the common use of the standard 12-lead ECG, its reliability as an indicator of coronary artery disease (CAD) is poor. The normal ECG is falsely negative in more than 50% of angiographically proven CAD. The waveform of the standard ECG, however, can be transformed mathematically by nonlinear signal transformation to enhance its interpretation by computer. Using such a process it is possible that abnormalities can be identified in "normal" ECGs that can be correlated with CAD, thus identifying high-risk patients. A computer template that represents grouped data of normal ECGs for patients who also have normal coronary angiography was developed. Unblinded, preliminary testing of the template on normal ECGs of 107 white patients who had normal or abnormal coronary angiograms was performed. The process identified presence or absence of CAD with 82% specificity and 71% sensitivity for 53 women, and with 82% specificity and 86% sensitivity for 54 men. These preliminary results are promising, but further refinement of the templates is required and blinded studies with larger numbers and varieties of patients are needed.

Diagnosis of complex acid-base disorders: physician performance versus the microcomputer.

Patients with acid-base disturbances that are often complex frequently present to the emergency department. The sometimes hectic nature of the ED can preclude the appropriate quantitative analysis required by these disorders, especially when mixed disturbances are present. A computer program using generally accepted acid-base and electrolyte formulae was developed for use on the Apple II+ or IBM-PC microcomputer. Each of a series of 35 acid-base disturbances incorporating single, double, and triple disorders was correctly identified by the computer in less than 45 seconds. Problem sets based on the same 35 disturbances were presented to 21 physician-subjects at various levels of training from the emergency medicine, internal medicine, pediatrics, surgery, and family practice specialties. Although the physicians were given unlimited time and the necessary formulae to reach a diagnosis, they were requested to perform their analyses in the same fashion used in the ED. Although times varied widely, no physician spent more than five minutes on any problem. The physician correct response rates were 86%, 49%, and 17% for single, double, and triple disorders, respectively. The primary disorder correct response rate was 89% for double disorders and 94% for triple disorders. The primary and secondary disorder correct response rate was 58% for triple disorders. The data suggest that the microcomputer may be beneficial in the rapid assessment of complex disorders.

Renal artery obstruction.

A 57-year-old woman with an extensive cardiac history presented complaining of left flank pain. An intravenous pyelogram performed for the presumptive diagnosis of renal calculus showed poor function of the left kidney. Angiography demonstrated a 95% obstructing embolus in the left renal artery, which was removed surgically. This case illustrates some of the pitfalls in the diagnosis of renal artery obstruction and the need for a high index of suspicion. The intrarenal infusion of thrombolytic agents such as streptokinase may become the treatment of choice despite the success of surgical embolectomy. The diagnosis, laboratory analysis, and treatment of renal artery obstruction is discussed.

Pancreatic injury associated with interposed abdominal compressions in pediatric cardiopulmonary resuscitation.

A case in which conventional CPR was augmented with interposed abdominal compressions on a child is reported. Animal studies and electrical models of this new form of CPR have demonstrated improved hemodynamics without instance of intra-abdominal injury. In this case, intraperitoneal visceral injury was noticed in the form of blood within the stomach and small intestine and parenchymal hemorrhage within the pancreas. Similar pancreatic injury has not been reported with conventional pediatric CPR, and caution may have to be exercised if this form of CPR with interposed abdominal compressions is to be used on children.

Doppler ultrasound monitoring of systemic blood flow during CPR.

During cardiopulmonary resuscitation in 12 patients, Doppler ultrasound monitoring of radial arterial flow provided an audible, instantaneous flow sound to which the resuscitation team referred, along with the monitor electrocardiogram (EKG), in determining hemodynamic status. Incidental to the resuscitation effort, a separate analog flow signal and the monitor EKG were simultaneously recorded in eight patients. Doppler blood flow monitoring allowed evaluation of effectiveness of cardiac massage; immediate recognition of electromechanical dissociation; rapid determinations of blood pressure, often during profound hypotension, and estimates of changes in cardiac output. When the hemodynamic consequences were immediately obvious, both ineffective chest compression and pauses longer than five seconds during effective chest compression were not tolerated by those in attendance, for whom the Doppler flow signal often became the primary reference in determining the patient's cardiac status.