Plasmodium vivax Duffy binding protein: baseline antibody responses and parasite polymorphisms in a well-consolidated settlement of the Amazon Region

Objective To investigate risk factors associated with the acquisition of antibodies against Plasmodium vivax Duffy binding protein (PvDBP) a leading malaria vaccine candidate in a well-consolidated agricultural settlement of the Brazilian Amazon Region and to determine the sequence diversity of the PvDBP ligand domain (DBPII) within the local malaria parasite population. Methods Demographic, epidemiological and clinical data were collected from 541 volunteers using a structured questionnaire. Malaria parasites were detected by conventional microscopy and PCR, and blood collection was used for antibody assays and molecular characterisation of DBPII. Results The frequency of malaria infection was 7% (6% for P. vivax and 1% for P. falciparum), with malaria cases clustered near mosquito breeding sites. Nearly 50% of settlers had anti-PvDBP IgG antibodies, as detected by enzyme-linked immunosorbent assay (ELISA) with subjects age being the only strong predictor of seropositivity to PvDBP. Unexpectedly, low levels of DBPII diversity were found within the local malaria parasites, suggesting the existence of low gene flow between P. vivax populations, probably due to the relative isolation of the studied settlement. Conclusion The recognition of PvDBP by a significant proportion of the community, associated with low levels of DBPII diversity among local P. vivax, reinforces the variety of malaria transmission patterns in communities from frontier settlements. Such studies should provide baseline information for antimalarial vaccines now in development.

Distribution and molecular characterization of hepatitis C virus (HCV) genotypes in patients with chronic infection from Pernambuco State, Brazil

Hepatitis C virus (HCV) is a public health problem throughout the world and 3% of the world population is infected with this virus. It is estimated that 3-4 millions individuals are being infected every year. It has been estimated that around 1.5% of Brazilian population is anti-HCV positive and the Northeast region showed the highest prevalence in Brazil. The aim of this study was to characterize HCV genotypes circulating in Pernambuco State (PE), Brazil, located in the Northeast region of the country. This study included 85 anti-HCV positive patients followed up between 2004 and 2011. For genotyping, a 380bp fragment of HCV RNA in the NS5B region was amplified by nested PCR. Phylogenetic analysis was conducted using Bayesian Markov chain Monte Carlo simulation (MCMC) using BEAST v.1.5.3. From 85 samples, 63 (74.1%) positive to NS5B fragment were successfully sequenced. Subtype 1b was the most prevalent in this population (42-66.7%), followed by 3a (16-25.4%), 1a (4-6.3%) and 2b (1-1.6%). Twelve (63.1%) and seven (36.9%) patients with HCV and schistosomiasis were infected with subtypes 1b and 3a, respectively. Brazil is a large country with many different population backgrounds; a large variation in the frequencies of HCV genotypes is predictable throughout its territory. This study reports HCV genotypes from Pernambuco State where subtype 1b was found to be the most prevalent. Phylogenetic analysis suggests the presence of the different HCV strains circulating within this population. (C) 2012 Elsevier B.V. All rights reserved.

Complete Sequence of Broad-Host-Range Plasmid pRIO-5 Harboring the Extended-Spectrum-beta-Lactamase Gene bla(BES-1)

Broad-host-range plasmid pRIO-5, harboring the extended-spectrum beta-lactamase bla(BES-1) gene in Serratia marcescens, was fully sequenced. Analysis of the 12,957-bp sequence of this IncP6-type plasmid revealed that the bla(BES-1) gene was associated with two copies of the insertion sequence IS26. The promoter responsible for the bla(BES-1) expression was hybrid, made of a - 35 box located inside the inverted repeat of IS26 and a - 10 box inside a remnant of an insertion sequence.

ORAOV1 is amplified in oral squamous cell carcinoma

BACKGROUND: Oral cancer overexpressed 1 (ORAOV1) was found as a candidate oncogene in the 11q13 chromosomal region, based on its amplification and overexpression in oral cancer cell lines. Because gene amplification often leads to increased levels of gene expression, we aimed to verify the relationship between ORAOV1 gene status and mRNA expression primarily in oral squamous cell carcinoma (OSCC) by quantitative assay, correlating with clinical and pathological characteristics in patients. METHODS: Levels of ORAOV1 amplification and expression were evaluated by qPCR and RT-qPCR in OSCC cell lines and in tumor and non-tumoral surgical margins from 33 patients with OSCC. All subjects were smokers and habitual alcohol drinkers, mostly men above 40 years of age and with a single primary tumor. RESULTS: ORAOV1 exhibited increased gene expression levels as well as higher copy number in three OSCC cell lines with 11q13 amplified chromosomal region when compared with the OSCC cell line without the amplification (one-way ANOVA, P < 0.05). Weak correlation between ORAOV1 mRNA levels and DNA copy number was seen in tumor samples (Spearman, P = 0.07). Although ORAOV1 was amplified in tumor (Wilcoxon, P < 0.01), high levels of transcripts in margin did not reveal differences in comparison with tumor (Wilcoxon, P = 0.85). Aggressiveness and survival rate did not demonstrate statistical difference for both events in OSCC. CONCLUSION: The overexpression of ORAOV1 in non-tumoral margin samples can occur in the absence of amplification. The weak correlation between ORAOV1 amplification and expression in OSSC suggests that ORAOV1 expression can be regulated by mechanisms other than gene amplification. J Oral Pathol Med (2012) 41: 5460

Polymorphic toxin systems: Comprehensive characterization of trafficking modes, processing, mechanisms of action, immunity and ecology using comparative genomics

Background: Proteinaceous toxins are observed across all levels of inter-organismal and intra-genomic conflicts. These include recently discovered prokaryotic polymorphic toxin systems implicated in intra-specific conflicts. They are characterized by a remarkable diversity of C-terminal toxin domains generated by recombination with standalone toxin-coding cassettes. Prior analysis revealed a striking diversity of nuclease and deaminase domains among the toxin modules. We systematically investigated polymorphic toxin systems using comparative genomics, sequence and structure analysis. Results: Polymorphic toxin systems are distributed across all major bacterial lineages and are delivered by at least eight distinct secretory systems. In addition to type-II, these include type-V, VI, VII (ESX), and the poorly characterized "Photorhabdus virulence cassettes (PVC)", PrsW-dependent and MuF phage-capsid-like systems. We present evidence that trafficking of these toxins is often accompanied by autoproteolytic processing catalyzed by HINT, ZU5, PrsW, caspase-like, papain-like, and a novel metallopeptidase associated with the PVC system. We identified over 150 distinct toxin domains in these systems. These span an extraordinary catalytic spectrum to include 23 distinct clades of peptidases, numerous previously unrecognized versions of nucleases and deaminases, ADP-ribosyltransferases, ADP ribosyl cyclases, RelA/SpoT-like nucleotidyltransferases, glycosyltranferases and other enzymes predicted to modify lipids and carbohydrates, and a pore-forming toxin domain. Several of these toxin domains are shared with host-directed effectors of pathogenic bacteria. Over 90 families of immunity proteins might neutralize anywhere between a single to at least 27 distinct types of toxin domains. In some organisms multiple tandem immunity genes or immunity protein domains are organized into polyimmunity loci or polyimmunity proteins. Gene-neighborhood-analysis of polymorphic toxin systems predicts the presence of novel trafficking-related components, and also the organizational logic that allows toxin diversification through recombination. Domain architecture and protein-length analysis revealed that these toxins might be deployed as secreted factors, through directed injection, or via inter-cellular contact facilitated by filamentous structures formed by RHS/YD, filamentous hemagglutinin and other repeats. Phyletic pattern and life-style analysis indicate that polymorphic toxins and polyimmunity loci participate in cooperative behavior and facultative 'cheating' in several ecosystems such as the human oral cavity and soil. Multiple domains from these systems have also been repeatedly transferred to eukaryotes and their viruses, such as the nucleo-cytoplasmic large DNA viruses. Conclusions: Along with a comprehensive inventory of toxins and immunity proteins, we present several testable predictions regarding active sites and catalytic mechanisms of toxins, their processing and trafficking and their role in intra-specific and inter-specific interactions between bacteria. These systems provide insights regarding the emergence of key systems at different points in eukaryotic evolution, such as ADP ribosylation, interaction of myosin VI with cargo proteins, mediation of apoptosis, hyphal heteroincompatibility, hedgehog signaling, arthropod toxins, cell-cell interaction molecules like teneurins and different signaling messengers.

Insertional translocation of 15q25-q26 into 11p13 and duplication at 8p23.1 characterized by high resolution arrays in a boy with congenital malformations and aniridia

We report on a boy presenting submucous cleft palate, hydronephrosis, ventriculoseptal defect, aniridia, and developmental delay. Additional material on 11p13 was cytogenetically visible and array analyses identified a duplicated segment on 15q25-26 chromosome region; further, array analyses revealed a small deletion (49?kb) at 11p13 region involving the ELP4 gene and a duplication at 8p23.1. Results were confirmed with both molecular and molecular cytogenetics techniques. Possibilities for etiological basis of clinical phenotype are discussed. (c) 2012 Wiley Periodicals, Inc.

The young stellar cluster [DBS2003] 157 associated with the H ii region GAL 331.31-00.34

We report a study of the stellar content of the near-infrared (NIR) cluster [DBS2003] 157 embedded in the extended H ii region GAL 331.31-00.34, which is associated with the IRAS source 16085-5138. JHK photometry was carried out in order to identify potential ionizing candidates, and the follow-up NIR spectroscopy allowed the spectral classification of some sources, including two O-type stars. A combination of NIR photometry and spectroscopy data was used to obtain the distance of these two stars, with the method of spectroscopic parallax: IRS 298 (O6 V, 3.35 +/- 0.61 kpc) and IRS 339 (O9 V, 3.24 +/- 0.56 kpc). Adopting the average distance of 3.29 +/- 0.58 kpc and comparing the Lyman continuum luminosity of these stars with that required to account for the radio continuum flux of the H ii region, we conclude that these two stars are the ionizing sources of GAL 331.31-00.34. Young stellar objects (YSOs) were searched by using our NIR photometry and mid-infrared (MIR) data from the Galactic Legacy Infrared Mid-Plane Survey Extraordinaire (GLIMPSE) survey. The analysis of NIR and MIR colourcolour diagrams resulted in 47 YSO candidates. The GLIMPSE counterpart of IRAS 16085-5138, which presents IRAS colour indices compatible with an ultracompact H ii region, has been identified. The analysis of its spectral energy distribution between 2 and m revealed that this source shows a spectral index a= 3.6 between 2 and m, which is typical of a YSO immersed in a protostellar envelope. Lower limits to the bolometric luminosity and the mass of the embedded protostar have been estimated as L= 7.7 x 10(3) L? and M= 10 M?, respectively, which correspond to a B0B1 V zero-age main sequence star.

Germline mutation analysis of Tpit in Poodle dogs with ACTH-dependent hyperadrenocorticism

There is a high incidence of pituitary-dependent hyperadrenocorticism (PDH) in Poodle dogs, with family members being affected by the disease, suggesting a genetic involvement. Tpit is an obligate transcription factor for the expression of pro-opiomelanocortingene and for corticotroph terminal differentiation. The aim of the present study was to screen the Tpit gene for germline mutations in Poodles with PDH. Fifty Poodle dogs (33 female, 8.71 +/- 2.8 years) with PDH and 50 healthy Poodle dogs (32 females, 9.4241 2.8 years) were studied. Genomic DNA was isolated from peripheral blood, amplified by PCR and submitted to automatic sequence. No mutation in the coding region of Tpit was found, whereas the new single nucleotide polymorphism p.S343G, in heterozygous state, was found in the same frequency in both PDH and control groups. We concluded that Tpit gain-of-function mutations are not involved in the etiology of PDH in Poodle dogs.

Sequence and Copy Number Analyses of HEXB Gene in Patients Affected by Sandhoff Disease: Functional Characterization of 9 Novel Sequence Variants

Sandhoff disease (SD) is a lysosomal disorder caused by mutations in the HEXB gene. To date, 43 mutations of HEXB have been described, including 3 large deletions. Here, we have characterized 14 unrelated SD patients and developed a Multiplex Ligation-dependent Probe Amplification (MLPA) assay to investigate the presence of large HEXB deletions. Overall, we identified 16 alleles, 9 of which were novel, including 4 sequence variation leading to aminoacid changes [c.626C>T (p.T209I), c.634C>A (p.H212N), c.926G>T (p.C309F), c.1451G>A (p.G484E)] 3 intronic mutations (c.1082+5G>A, c.1242+1G>A, c.1169+5G>A), 1 nonsense mutation c.146C>A (p.S49X) and 1 small in-frame deletion c.1260_1265delAGTTGA (p.V421_E422del). Using the new MLPA assay, 2 previously described deletions were identified. In vitro expression studies showed that proteins bearing aminoacid changes p.T209I and p.G484E presented a very low or absent activity, while proteins bearing the p.H212N and p.C309F changes retained a significant residual activity. The detrimental effect of the 3 novel intronic mutations on the HEXB mRNA processing was demonstrated using a minigene assay. Unprecedentedly, minigene studies revealed the presence of a novel alternative spliced HEXB mRNA variant also present in normal cells. In conclusion, we provided new insights into the molecular basis of SD and validated an MLPA assay for detecting large HEXB deletions.

Lithium in M 67: From the main sequence to the red giant branch

Context. Lithium abundances in open clusters are a very effective probe of mixing processes, and their study can help us to understand the large depletion of lithium that occurs in the Sun. Owing to its age and metallicity, the open cluster M 67 is especially interesting on this respect. Many studies of lithium abundances in M 67 have been performed, but a homogeneous global analysis of lithium in stars from subsolar masses and extending to the most massive members, has yet to be accomplished for a large sample based on high-quality spectra. Aims. We test our non-standard models, which were calibrated using the Sun with observational data. Methods. We collect literature data to analyze, for the first time in a homogeneous way, the non-local thermal equilibrium lithium abundances of all observed single stars in M 67 more massive than similar to 0.9 M-circle dot. Our grid of evolutionary models is computed assuming a non-standard mixing at metallicity [Fe/H] = 0.01, using the Toulouse-Geneva evolution code. Our analysis starts from the entrance into the zero-age main-sequence. Results. Lithium in M 67 is a tight function of mass for stars more massive than the Sun, apart from a few outliers. A plateau in lithium abundances is observed for turn-off stars. Both less massive (M >= 1.10 M-circle dot) and more massive (M >= 1.28 M-circle dot) stars are more depleted than those in the plateau. There is a significant scatter in lithium abundances for any given mass M <= 1.1 M-circle dot. Conclusions. Our models qualitatively reproduce most of the features described above, although the predicted depletion of lithium is 0.45 dex smaller than observed for masses in the plateau region, i.e. between 1.1 and 1.28 solar masses. More work is clearly needed to accurately reproduce the observations. Despite hints that chromospheric activity and rotation play a role in lithium depletion, no firm conclusion can be drawn with the presently available data.

Phylogenetic and Similarity Analysis of HTLV-1 Isolates from HIV-Coinfected Patients from the South and Southeast Regions of Brazil

HTLV-1 is endemic in Brazil and HIV/ HTLV-1 coinfection has been detected, mostly in the northeast region. Cosmopolitan HTLV-1a is the main subtype that circulates in Brazil. This study characterized 17 HTLV-1 isolates from HIV coinfected patients of southern (n = 7) and southeastern (n = 10) Brazil. HTLV-1 provirus DNA was amplified by nested PCR (env and LTR) and sequenced. Env sequences (705 bp) from 15 isolates and LTR sequences (731 bp) from 17 isolates showed 99.5% and 98.8% similarity among sequences, respectively. Comparing these sequences with ATK (HTLV-1a) and Mel5 (HTLV-1c) prototypes, similarities of 99% and 97.4%, respectively, for env and LTR with ATK, and 91.6% and 90.3% with Mel5, were detected. Phylogenetic analysis showed that all sequences belonged to the transcontinental subgroup A of the Cosmopolitan subtype, clustering in two Latin American clusters.

Multiple independent origins of mitochondrial control region duplications in the order Psittaciformes

mitochondrial genomes are generally thought to be under selection for compactness, due to their small size, consistent gene content, and a lack of introns or intergenic spacers. As more animal mitochondrial genomes are fully sequenced, rearrangements and partial duplications are being identified with increasing frequency, particularly in birds (Class Ayes). In this study, we investigate the evolutionary history of mitochondrial control region states within the avian order Psittaciformes (parrots and cockatoos). To this aim, we reconstructed a comprehensive multi-locus phylogeny of parrots, used PCR of three diagnostic fragments to classify the mitochondrial control region state as single or duplicated, and mapped these states onto the phylogeny. We further sequenced 44 selected species to validate these inferences of control region state. Ancestral state reconstruction using a range of weighting schemes identified six independent origins of mitochondrial control region duplications within Psittaciformes. Analysis of sequence data showed that varying levels of mitochondrial gene and tRNA homology and degradation were present within a given clade exhibiting duplications. Levels of divergence between control regions within an individual varied from 0-10.9% with the differences occurring mainly between 51 and 225 nucleotides 3' of the goose hairpin in domain I. Further investigations into the fates of duplicated mitochondrial genes, the potential costs and benefits of having a second control region, and the complex relationship between evolutionary rates, selection, and time since duplication are needed to fully explain these patterns in the mitochondrial genome. (C) 2012 Elsevier Inc. All rights reserved.

SmTRC1, a novel Schistosoma mansoni DNA transposon, discloses new families of animal and fungi transposons belonging to the CACTA superfamily

Abstract Background The CACTA (also called En/Spm) superfamily of DNA-only transposons contain the core sequence CACTA in their Terminal Inverted Repeats (TIRs) and so far have only been described in plants. Large transcriptome and genome sequence data have recently become publicly available for Schistosoma mansoni, a digenetic blood fluke that is a major causative agent of schistosomiasis in humans, and have provided a comprehensive repository for the discovery of novel genes and repetitive elements. Despite the extensive description of retroelements in S. mansoni, just a single DNA-only transposon belonging to the Merlin family has so far been reported in this organism. Results We describe a novel S. mansoni transposon named SmTRC1, for S. mansoni Transposon Related to CACTA 1, an element that shares several characteristics with plant CACTA transposons. Southern blotting indicates approximately 30–300 copies of SmTRC1 in the S. mansoni genome. Using genomic PCR followed by cloning and sequencing, we amplified and characterized a full-length and a truncated copy of this element. RT-PCR using S. mansoni mRNA followed by cloning and sequencing revealed several alternatively spliced transcripts of this transposon, resulting in distinct ORFs coding for different proteins. Interestingly, a survey of complete genomes from animals and fungi revealed several other novel TRC elements, indicating new families of DNA transposons belonging to the CACTA superfamily that have not previously been reported in these kingdoms. The first three bases in the S. mansoni TIR are CCC and they are identical to those in the TIRs of the insects Aedes aegypti and Tribolium castaneum, suggesting that animal TRCs may display a CCC core sequence. Conclusion The DNA-only transposable element SmTRC1 from S. mansoni exhibits various characteristics, such as generation of multiple alternatively-spliced transcripts, the presence of terminal inverted repeats at the extremities of the elements flanked by direct repeats and the presence of a Transposase_21 domain, that suggest a distant relationship to CACTA transposons from Magnoliophyta. Several sequences from other Metazoa and Fungi code for proteins similar to those encoded by SmTRC1, suggesting that such elements have a common ancestry, and indicating inheritance through vertical transmission before separation of the Eumetazoa, Fungi and Plants.

Radiation of the Tnt1 retrotransposon superfamily in three Solanaceae genera

Abstract Background Tnt1 was the first active plant retrotransposon identified in tobacco after nitrate reductase gene disruption. The Tnt1 superfamily comprises elements from Nicotiana (Tnt1 and Tto1) and Lycopersicon (Retrolyc1 and Tlc1) species. The study presented here was conducted to characterise Tnt1-related sequences in 20 wild species of Solanum and five cultivars of Solanum tuberosum. Results Tnt1-related sequences were amplified from total genomic DNA using a PCR-based approach. Purified fragments were cloned and sequenced, and clustering analysis revealed three groups that differ in their U3 region. Using a network approach with a total of 453 non-redundant sequences isolated from Solanum (197), Nicotiana (140) and Lycopersicon (116) species, it is demonstrated that the Tnt1 superfamily can be treated as a population to resolve previous phylogenetic multifurcations. The resulting RNAseH network revealed that sequences group according to the Solanaceae genus, supporting a strong association with the host genome, whereas tracing the U3 region sequence association characterises the modular evolutionary pattern within the Tnt1 superfamily. Within each genus, and irrespective of species, nearly 20% of Tnt1 sequences analysed are identical, indicative of being part of an active copy. The network approach enabled the identification of putative "master" sequences and provided evidence that within a genus these master sequences are associated with distinct U3 regions. Conclusion The results presented here support the hypothesis that the Tnt1 superfamily was present early in the evolution of Solanaceae. The evidence also suggests that the RNAseH region of Tnt1 became fixed at the host genus level whereas, within each genus, propagation was ensured by the diversification of the U3 region. Different selection pressures seemed to have acted on the U3 and RNAseH modules of ancestral Tnt1 elements, probably due to the distinct functions of these regions in the retrotransposon life cycle, resulting in both co evolution and adaptation of the element population with its host.

Trypanosoma cruzi mitochondrial maxicircles display species- and strain-specific variation and a conserved element in the non-coding region

Abstract Background The mitochondrial DNA of kinetoplastid flagellates is distinctive in the eukaryotic world due to its massive size, complex form and large sequence content. Comprised of catenated maxicircles that contain rRNA and protein-coding genes and thousands of heterogeneous minicircles encoding small guide RNAs, the kinetoplast network has evolved along with an extreme form of mRNA processing in the form of uridine insertion and deletion RNA editing. Many maxicircle-encoded mRNAs cannot be translated without this post-transcriptional sequence modification. Results We present the complete sequence and annotation of the Trypanosoma cruzi maxicircles for the CL Brener and Esmeraldo strains. Gene order is syntenic with Trypanosoma brucei and Leishmania tarentolae maxicircles. The non-coding components have strain-specific repetitive regions and a variable region that is unique for each strain with the exception of a conserved sequence element that may serve as an origin of replication, but shows no sequence identity with L. tarentolae or T. brucei. Alternative assemblies of the variable region demonstrate intra-strain heterogeneity of the maxicircle population. The extent of mRNA editing required for particular genes approximates that seen in T. brucei. Extensively edited genes were more divergent among the genera than non-edited and rRNA genes. Esmeraldo contains a unique 236-bp deletion that removes the 5'-ends of ND4 and CR4 and the intergenic region. Esmeraldo shows additional insertions and deletions outside of areas edited in other species in ND5, MURF1, and MURF2, while CL Brener has a distinct insertion in MURF2. Conclusion The CL Brener and Esmeraldo maxicircles represent two of three previously defined maxicircle clades and promise utility as taxonomic markers. Restoration of the disrupted reading frames might be accomplished by strain-specific RNA editing. Elements in the non-coding region may be important for replication, transcription, and anchoring of the maxicircle within the kinetoplast network.