Discovery and Contribution of Nontypeable Haemophilus influenzae NTHI1441 to Human Respiratory Epithelial Cell Invasion.

Nontypeable Haemophilus influenzae (NTHi) is the primary cause of bacterially induced acute exacerbations of chronic obstructive pulmonary disease (COPD). NTHi adheres to and invades host respiratory epithelial cells as a means to persist in the lower airways of adults with COPD. Therefore, we mined the genomes of NTHi strains isolated from the airways of adults with COPD to identify novel proteins to investigate their role in adherence and invasion of human respiratory epithelial cells. An isogenic knockout mutant of the open reading frame NTHI1441 showed a 76.6% ± 5.5% reduction in invasion of human bronchial and alveolar epithelial cells at 1, 3, and 6 h postinfection. Decreased invasion of the NTHI1441 mutant was independent of either intracellular survival or adherence to cells. NTHI1441 is conserved among NTHi genomes. Results of whole-bacterial-cell enzyme-linked immunosorbent assay (ELISA) and flow cytometry experiments identified that NTHI1441 has epitopes expressed on the bacterial cell surface. Adults with COPD develop increased serum IgG against NTHI1441 after experiencing an exacerbation with NTHi. This study reveals NTHI1441 as a novel NTHi virulence factor expressed during infection of the COPD lower airways that contributes to invasion of host respiratory epithelial cells. The role in host cell invasion, conservation among strains, and expression of surface-exposed epitopes suggest that NTHI1441 is a potential target for preventative and therapeutic interventions for disease caused by NTHi.

Large protein as a potential target for use in rabies diagnostics.

Rabies is a zoonotic viral disease that remains a serious threat to public health worldwide. The rabies lyssavirus (RABV) genome encodes five structural proteins, multifunctional and significant for pathogenicity. The large protein (L) presents well-conserved genomic regions, which may be a good alternative to generate informative datasets for development of new methods for rabies diagnosis. This paper describes the development of a technique for the identification of L protein in several RABV strains from different hosts, demonstrating that MS-based proteomics is a potential method for antigen identification and a good alternative for rabies diagnosis.

Photo-identification as a simple tool for studying invasive lionfish Pterois volitans populations.

Photo-tagging, i.e. using a specific software to match colour patterns on photographs, was tested as a means to identify individual Indo-Pacific Pterois volitans to assist with population and movement studies of this invasive species. The stripe pattern on the flank of adult P. volitans (n = 48) was the most individually distinctive of three body regions tested, leading to correct individual identification on 68 and 82% of tests with a single and two images of the reference individual, respectively. Photo-tagging is inexpensive, logistically simple and can involve citizen scientists, making it a viable alternative to traditional tagging to provide information on P. volitans distribution, movement patterns and recolonization rates after removals.

Diagnosis of the pinworm Syphacia muris in the Wistar rat Rattus norvegicus.

This study aimed to compare three qualitative parasitological methods for the diagnosis of Syphacia muris infection in 30 Wistar rats (Rattus norvegicus) infected naturally. Methods of spontaneous sedimentation (Hoffman, Pons and Janer, or HPJ) and spontaneous flotation (Willis) for faecal samples and a method of taping (Graham) were performed and compared. The Graham and Willis methods were more sensitive than the HPJ method (P< 0.05). The Graham method was able to detect S. muris eggs in 100% of the samples. Eggs were detected in 83% and 60% of the samples using the Willis and HPJ methods, respectively. Method choice is important for screening for parasites of rats kept under laboratory conditions, as accurate diagnosis helps prevent future environmental contamination and infection. We concluded that the Graham method was the most efficient of those tested in this study for detection of S. muris infection in rats. This method is also rapid, inexpensive and practical, and should be implemented as a necessary measure for infection control.

Population dynamics of Armigeres subalbatus (Diptera: Culicidae) across a temperate altitudinal gradient.

Understanding the impacts of weather fluctuations, and environmental gradients, on the abundance of vectors is fundamental to grasp the dynamic nature of the entomological risk for disease transmission. The mosquito Armigeres subalbatus (Coquillet) is a common vector of filariasis. Nevertheless, its population dynamics have been relatively poorly studied. Here, we present results from a season long study where we studied spatio-temporal abundance patterns of Ar. subalbatus across the altitudinal gradient of Mt. Konpira in Nagasaki, Japan. Spatially, we found that abundance of adult Ar. subalbatus decreased with altitude and increased in areas where the ground was rich in leaf litter. Similarly, adult activity was observed only when relative humidity was over 65%. Temporally, we found that peaks in abundance followed large rainfall events. Nevertheless, this mosquito was under significant density dependence regulation. Our results suggest that Ar. subalbatus population peaks following large rainfall events could reflect the recruitment of individuals that were dormant as dry eggs. We did not find a clear signal of temperature on abundance changes of this mosquito, but only on its phenology. Since ground cover seemed more critical than temperature to its spatial distribution, we propose that this mosquito might have some degree of autonomy to changes in temperature.

Development of microsatellite markers for Hancornia speciosa Gomes (Apocynaceae).

Herein, we describe 34 microsatellite loci developed using an enrichment genomic library for the tree species Hancornia speciosa Gomes (Apocynaceae). Thirty-five individuals were genotyped using 34 primers to analyze the polymorphisms at each locus. The number of alleles per locus ranged from 4 to 20. The average number of alleles was 8.11, and the expected heterozygosity ranged from 0.62 to 0.94. These microsatellite primers will be useful in population genetics studies for this species.

New and Common Haplotypes Shape Genetic Diversity in Asian Tiger Mosquito Populations from Costa Rica and Panamá.

The Asian tiger mosquito, Aedes albopictus (Skuse) (Diptera: Culicidae), is a vector of several human pathogens. Ae. albopictus is also an invasive species that, over recent years, has expanded its range out of its native Asia. Ae. albopictus was suspected to be present in Central America since the 1990s, and its presence was confirmed by most Central American nations by 2010. Recently, this species has been regularly found, yet in low numbers, in limited areas of Panamá and Costa Rica (CR). Here, we report that short sequences (∼558 bp) of the mitochondrial cytochrome oxidase subunit 1 (COI) and NADH dehydrogenase subunit 5 genes of Ae. albopictus, had no haplotype diversity. Instead, there was a common haplotype for each gene in both CR and Panamá. In contrast, a long COI sequence (∼1,390 bp) revealed that haplotype diversity (±SD) was relatively high in CR (0.72±0.04) when compared with Panamá (0.33±0.13), below the global estimate for reported samples (0.89±0.01). The long COI sequence allowed us to identify seven (five new) haplotypes in CR and two (one new) in Panamá. A haplotype network for the long COI gene sequence showed that samples from CR and Panamá belong to a single large group. The long COI gene sequences suggest that haplotypes in Panamá and CR, although similar to each other, had a significant geographic differentiation (Kst=1.33; P<0.001). Thus, most of our results suggest a recent range expansion in CR and Panamá.

Effectiveness of mosquito magnet in preserved area on the coastal Atlantic rainforest: implication for entomological surveillance.

A variety of traps are used for sampling, surveillance, and monitoring of mosquito vector species associated with parasite and pathogen transmission. Here, we assessed the performance of the Mosquito Magnet Independence trap with Lurex3 (MMI), by comparing its effectiveness with those of a Centers forDisease Control and Prevention light trap (CDC-LT) and CDC with CO2 and Lurex3 (CDC-A) in a dense tropical rainforest. Multivariate generalized linear models revealed significant differences among the traps regarding mosquito composition and abundance (deviance = 768; P = 0.016). Variance analyses indicated that the MMI captured significantly more mosquitoes compared with CDC-LT (P < 0.01) and CDC-A (P < 0.03). The abundance values did not significantly differ between the CDC-LT and CDC-A traps (P = 0.7). Mosquito species richness was higher from the MMI than from the CDC-LT and CDC-A traps. Furthermore, medically important mosquito species captured by the three traps showed high association with MMI. These results suggest the potential to use the MMI in studies aiming to obtain entomological surveillance information about medically important mosquitoes that occur in tropical rainforest areas. The MMI could also be used in faunal studies focusing on increasing knowledge about mosquito diversity. Considering the present positive results, the effectiveness of the MMI should additionally be evaluated in other Brazilian natural ecosystems. Further studies are also needed to address demographic data from the mosquito population sampled by the MMI.

Demographic history and the low genetic diversity in Dipteryx alata (Fabaceae) from Brazilian Neotropical savannas.

Genetic effects of habitat fragmentation may be undetectable because they are generally a recent event in evolutionary time or because of confounding effects such as historical bottlenecks and historical changes in species' distribution. To assess the effects of demographic history on the genetic diversity and population structure in the Neotropical tree Dipteryx alata (Fabaceae), we used coalescence analyses coupled with ecological niche modeling to hindcast its distribution over the last 21 000 years. Twenty-five populations (644 individuals) were sampled and all individuals were genotyped using eight microsatellite loci. All populations presented low allelic richness and genetic diversity. The estimated effective population size was small in all populations and gene flow was negligible among most. We also found a significant signal of demographic reduction in most cases. Genetic differentiation among populations was significantly correlated with geographical distance. Allelic richness showed a spatial cline pattern in relation to the species' paleodistribution 21 kyr BP (thousand years before present), as expected under a range expansion model. Our results show strong evidences that genetic diversity in D. alata is the outcome of the historical changes in species distribution during the late Pleistocene. Because of this historically low effective population size and the low genetic diversity, recent fragmentation of the Cerrado biome may increase population differentiation, causing population decline and compromising long-term persistence.

Biotechnology advances: a perspective on the diagnosis and research of Rabies Virus.

Rabies is a widespread zoonotic disease responsible for approximately 55,000 human deaths/year. The direct fluorescent antibody test (DFAT) and the mouse inoculation test (MIT) used for rabies diagnosis, have high sensitivity and specificity, but are expensive and time-consuming. These disadvantages and the identification of new strains of the virus encourage the use of new techniques that are rapid, sensitive, specific and economical for the detection and research of the Rabies Virus (RABV). Real-time RT-PCR, phylogeographic analysis, proteomic assays and DNA recombinant technology have been used in research laboratories. Together, these techniques are effective on samples with low virus titers in the study of molecular epidemiology or in the identification of new disease markers, thus improving the performance of biological assays. In this context, modern advances in molecular technology are now beginning to complement more traditional approaches and promise to revolutionize the diagnosis of rabies. This brief review presents some of the recent molecular tools used for RABV analysis, with emphasis on rabies diagnosis and research.

Development and characterization of new microsatellites for Eugenia dysenterica DC (Myrtaceae).

Microsatellite markers were developed for population genetic analyses of the Neotropical tree Eugenia dysenterica DC (Myrtaceae), after construction of a shotgun genomic library for microsatellite discovery. Nine primers were designed, of which 5 yielded amplified product. These primers were polymorphic for 97 individuals collected in 3 distinct localities. The number of alleles per locus (primer) ranged from 3 to 11 and expected heterozygosities varied from 0.309 to 0.884. The probability of locus identity was ~1.88 x 10(-4) and the probability of paternity exclusion was ~0.9367. The 5 microsatellite primer pairs may be suitable for population genetic studies such as parentage and fine-scale genetic analyses of this species.

Pan-cancer genomic analysis shows hemizygous PTEN loss tumors are associated with immune evasion and poor outcome.

In tumors, somatic mutations of the PTEN suppressor gene are associated with advanced disease, chemotherapy resistance, and poor survival. PTEN loss of function may occur by inactivating mutation, by deletion, either affecting one copy (hemizygous loss) leading to reduced gene expression or loss of both copies (homozygous) with expression absent. Various murine models have shown that minor reductions in PTEN protein levels strongly influence tumorigenesis. Most PTEN biomarker assays dichotomize PTEN (i.e. presence vs. absence) ignoring the role of one copy loss. We performed a PTEN copy number analysis of 9793 TCGA cases from 30 different tumor types. There were 419 (4.28%) homozygous and 2484 (25.37%) hemizygous PTEN losses. Hemizygous deletions led to reduced PTEN gene expression, accompanied by increased levels of instability and aneuploidy across tumor genomes. Outcome analysis of the pan-cancer cohort showed that losing one copy of PTEN reduced survival to comparable levels as complete loss, and was associated with transcriptomic changes controlling immune response and the tumor microenvironment. Immune cell abundances were significantly altered for PTEN loss, with changes in head and neck, cervix, stomach, prostate, brain, and colon more evident in hemizygous loss tumors. These data suggest that reduced expression of PTEN in tumors with hemizygous loss leads to tumor progression and influences anticancer immune response pathways.

Morphophysiology and production of bell pepper grown under salt stress and salicylic acid foliar application.

Considering the relevance of bell pepper and the limitations imposed by the deleterious effects of salt stress, especially in semi-arid regions, it is extremely important to establish strategies that can facilitate the use of saline water in vegetable production. In this scenario, this study aimed to evaluate the effect of the frequency of foliar application of salicylic acid on the morphophysiology and production of the bell pepper cv. "All Big" irrigated with saline water. The study was conducted at a greenhouse in Campina Grande - PB. The treatments were distributed in a completely randomized design and set up in a 4 × 4 factorial arrangement with three replications, corresponding to four application frequencies of salicylic acid (F1- No application of salicylic acid, F2 - Weekly application, F3- fortnightly application, and F4- monthly application) and four levels of electrical conductivity of irrigation water - ECw (0.8, 1.6, 2.4 and 3.2 dS m-1). The fortnightly application of salicylic acid at a concentration of 1.0 mM mitigated the effects of salt stress on the morphophysiology and production components of bell pepper cv. All Big cultivated with ECw of up to 2.4 dS m-1, which reinforces the hypothesis that salicylic acid can act as a signaling molecule and reduce the effects of saline stress in bell pepper, enabling the use of brackish water in agricultural activity, mainly in semi-arid regions of northeastern Brazil, which have a shortage of fresh water.

Genetic structure in fragmented populations of Solanum lycocarpum A. St.-Hil. with distinct anthropogenic histories in a Cerrado region of Brazil.

Solanum lycocarpum is a woody tree widely distributed in the Cerrado that reaches high population densities in disturbed environments. We examined the genetic diversity and population differentiation of six S. lycocarpum populations with different degrees of human disturbance in order to determine if they are negatively affected by anthropogenic activity. Three populations located in southern and three located in southeastern regions of Goiás State, Central Brazil, were genotyped with five microsatellite markers. The population located in a protected area had higher number of alleles (26) than the remaining populations (19 to 21 alleles). It indicates that extensive and continuous areas of preserved native vegetation contribute positively to the conservation of genetic diversity, even with S. lycocarpum that easily adapts to disturbed environments. The three southeastern populations, although fragmented, had preserved native vegetation and were not significantly different from each other (θp = 0.002). All other population pairs compared were significantly divergent (θp varied from 0.03 to 0.11 between pairs, P < 0.05). We found three distinct sets of allele frequencies. The three southeastern populations shared similar gene pools, as well as the two disturbed southern populations, which are secondary vegetation. The southern population located in protected area had the most dissimilar gene pool. In conclusion, populations showing a higher degree of human disturbance tends to show a larger population differentiation than expected from the isolation by distance model, which in the current scenario of the Cerrado destruction points out to a threat to the long-term conservation of the species.

Geographic shifts in climatically suitable areas and loss of genetic variability in Dipteryx alata ("Baru" Tree; Fabaceae).

Many species are expected to suffer strong shifts in their geographic ranges due to climate changes in the next 50 years, with severe consequences for biodiversity patterns and population structure. We used here an ensemble forecast approach for obtaining species' range in which multiple species distribution models and climatic models were combined to model loss of genetic variability in Baru, Dipteryx alata (Fabaceae), an economically important Neotropical tree native to the Cerrado of Brazil. We estimated a series of genetic parameters (number of alleles per locus, expected heterozygosity under Hardy-Weinberg equilibrium and mutation-drift equilibrium) for this species based on eight microsatellite loci. We then recalculated these parameters assuming that local populations in areas of low future environmental suitability will go extinct. All genetic parameters remained approximately constant up to a 50% threshold of climatic suitability in the future; after this critical threshold there is an abrupt reduction in all parameters, although the magnitude of shift is only about 10% of current values, on average. Thus, despite the shifts in geographic range and climatically suitable areas towards southeastern Brazil, our analyses do not predict a strong loss of genetic diversity in D. alata because of the broad tolerance of this species, which ensures large future ranges, contrasting with other Cerrado species that have been analyzed in a similar manner.

Sexual dimorphism in Kinosternon scorpioides (Linnaeus, 1766) from the Brazilian Amazon.

The sexual dimorphism of the Kinosternon scorpioides was evaluated using two different techniques (linear and geometric morphometry) from images and linear measurements of the carapace and plastron of adults (male and female). Linear morphometry indicated that the height and width of carapace and plastron are statistically different between sexes, with females being wider and taller. In the evaluation of geometric morphometry, ANOVA demonstrated variation in the size of the plastron and the shape of the carapace and plastron, expressing a tendency in shape for each sex. Sexual dimorphism, therefore, is verified for this species, notably by the plastron. This study indicates an additional tool for the phenotypic knowledge of animals, contributing to the study of threatened populations.

Sequence of a turkey BAC clone identifies MHC Class III orthologs and supports ancient origins of immunological gene clusters.

Two genetically unlinked gene clusters currently define the turkey major histocompatibility complex (MHC). Previous studies identified turkey bacterial artificial chromosome (BAC) clones hypothesized as orthologs of the MHC-B and MHC-Y regions of the chicken. Physical mapping assigned these clones to the same microchromosome (MGA18) and sequencing of the MHC-B BAC found near synteny with a portion of the chicken B-locus. This study examines the sequence of the second MHC BAC clone that was hypothesized, based on subclone sequences, to be orthologous to the MHC-Y. Sequencing of this clone identified a class I locus and orthologs of additional genes found in the mammalian class III region. Approximately 50% of the BAC insert is comprised of sequence corresponding to the centromeric repeat, MGASat2. This turkey MHC BAC sequence is unique from sequences assigned to the MHC-Y in the chicken. Based on sequence comparisons, the class I gene appears to be a nonfunctional pseudogene. The class III genes (BAT1, BAT3, STK19, and a G4-like locus) represent the second class III gene cluster identified in the galliform genome. This cluster appears to be of ancient origin and provides insight into the evolution of the avian MHC.

A locus-wide approach to assessing variation in the avian MHC: the B-locus of the wild turkey.

Studies of major histocompatibility complex (MHC) diversity in non-model vertebrates typically focus on structure and sequence variation in the antigen-presenting loci: the highly variable and polymorphic class I and class IIB genes. Although these studies provide estimates of the number of genes and alleles/locus, they often overlook variation in functionally related and co-inherited genes important in the immune response. This study utilizes the sequence of the MHC B-locus derived from a commercial turkey to investigate MHC variation in wild birds. Sequences were obtained for nine interspersed MHC amplicons (non-class I/II) from each of 40 birds representing 3 subspecies of wild turkey (Meleagris gallopavo). Analysis of aligned sequences identified 238 single-nucleotide variants approximately one-third of which had minor allele frequencies >0.2 in the sampled birds. PHASE analysis identified 70 prospective MHC haplotypes in the wild turkeys, whereas a combined analysis with commercial birds identified almost 100 haplotypes in the species. Denaturing gradient gel electrophoresis (DGGE) of the class IIB loci was used to test the efficacy of single-nucleotide polymorphism (SNP) haplotyping to capture locus-wide variation. Diversity in SNP haplotypes and haplotype sharing among individuals was directly reflected in the DGGE patterns. Utilization of a reference haplotype to sequence interspersed regions of the MHC has significant advantages over other methods of surveying diversity while identifying high-frequency SNPs for genotyping. SNP haplotyping provides a means to identify both divergent haplotypes and homozygous individuals for assessment of immunological variation in wild and domestic populations.

Weather variability impacts on oviposition dynamics of the southern house mosquito at intermediate time scales.

Oviposition is a major event in the life history of mosquitoes, shaping both individual fitness and vectorial capacity. Several exogenous factors have been shown as important for the dynamic forcing of oviposition at finely (hourly) and coarsely (monthly or season to season) grained temporal scales. However, field studies addressing the interplay of weather factors on oviposition dynamics at the intermediate (days to weeks) time scale are missing. Here, we present the results from a field study that showed the oviposition dynamics of the southern house mosquito, Culex quinquefasciatus Say (Diptera: Culicidae), to be: (i) primarily dictated by relative humidity; and (ii) disrupted by rainfall events that resulted in a modified sensitivity to relative humidity. Rainfall changed the concentration of ammonia, a major limiting resource of microbes used as food by mosquito larvae. Following major rainfall events, the importance of relative humidity in forcing the oviposition dynamics also changed. Finally, our results indicate that qualitative changes in oviposition habitats modify the importance of weather variables as predictors of mosquito oviposition dynamics.

Genetic structure based on nuclear and chloroplast microsatellite loci of Solanum lycocarpum A. St. Hil. (Solanaceae) in Central Brazil.

Solanum lycocarpum (Solanaceae) is a woody species found in the Brazilian Cerrado. The flowers are pollinated by Xylocopa spp bees, and seeds are dispersed by mammals with distinct home range sizes. As a consequence, relative contributions of pollen and seeds to overall gene flow can vary according to different spatial scales. We studied the genetic structure of four natural populations of S. lycocarpum separated by 19 to 128 km, including individuals located along dirt roads that interlink three of the populations. A total of 294 individuals were genotyped with five nuclear and six chloroplast microsatellite markers. Significant spatial genetic structure was found in the total set of individuals; the Sp statistic was 0.0086. Population differentiation based on the six chloroplast microsatellite markers (θ(pC) = 0.042) was small and similar to that based on the five nuclear microsatellite markers (θ(p) = 0.054). For this set of populations, pollen and seed flow did not differ significantly from one another (pollen-to-seed flow ratio = 1.22). Capability for long distance seed dispersion and colonization of anthropogenic sites contributes to the ability of S. lycocarpum to maintain genetic diversity. Seed dispersion along dirt roads may be critical in preserving S. lycocarpum genetic diversity in fragmented landscapes.