Catechol biosensing using a nanostructured layer-by-layer film containing Cl-catechol 1,2-dioxygenase

The detection of aromatic compounds from pesticides and industrial wastewater has become of great interest, since these compounds withstand chemical oxidation and biological degradation, accumulating in the environment. In this work, a highly sensitive biosensor for detecting catechol was obtained with the immobilization of Cl-catechol 1,2-dioxygenase (CCD) in nanostructured films. CCD layers were alternated with poly(amidoamine) generation 4 (PAMAM G4) dendrimer using the electrostatic layer-by-layer (LbL) technique. Circular dichroism (CD) measurements indicated that the immobilized CCD preserved the same conformation as in solution. The thickness of the very first CCD layers in the LbL films was estimated at ca. 3.6 nm, as revealed by surface plasmon resonance (SPR). PAMAM/CCD 10-bilayer films were employed in detecting diluted catechol solutions using either an optical or electrical approach. Due to the mild immobilization conditions employed, especially regarding the pH and ionic strength of the dipping solutions, CCD remained active in the films for periods longer than 3 weeks. The optical detection comprised absorption experiments in which the formation of cis-cis muconic acid, resulting from the reaction between CCD and catechol, was monitored by measuring the absorbance at 260 nm after film immersion in catechol solutions. The electrical detection was carried out using LbL films deposited onto gold-interdigitated electrodes immersed in aqueous solutions at different catechol concentrations. Using impedance spectroscopy in a broad frequency range (1Hz-1kHz), we could detect catechol in solutions at concentrations as low as 10(-10) M.     

Replication of hexitol oligonucleotides as a prelude to the propagation of a third type of nucleic acid in vivo

No backbone motif other than phospho-ribose and phospho-deoxyribose has been found in natural nucleic acids, currently restricting the molecular types of replicable biopolymers to DNA and RNA. With the aim of propagating and expressing a third type of nucleic acid in vivo, we assessed the replicability of polynucleotides with a phospho-hexitol backbone (HNA) in vivo and in vitro. Faithful polymerisation of up to four deoxynucleotides templated by hexitol oligonucleotides was established in vitro using DNA polymerase from Escherichia coli (PolA Klenow exo-fragment) and Thermus aquaticus (Taq polymerase). Condensation of up to three successive hTTPs (hexitol thymidine triphosphate) in responses to a pentameric hexitol template (hA)5 could also be demonstrated in vitro. Such a marginal HNA-dependent HNA polymerase activity of natural polymerases may be evolved in the future to catalyse in vitro amplification of HNA. The transmission of a two-codon-long genetic message carried on a hexameric hexitol template was also established using a selection screen for restoring thymidylate synthase activity in E. coli. These results exemplify the potential that can be explored by converting artificial substrates with natural enzymes in the field of informational polymer synthesis.     

Acinetobacter sp. ADP1: an ideal model organism for genetic analysis and genome engineering

Acinetobacter sp. strain ADP1 is a naturally transformable gram-negative bacterium with simple culture requirements, a prototrophic metabolism and a compact genome of 3.7 Mb which has recently been sequenced. Wild-type ADP1 can be genetically manipulated by the direct addition of linear DNA constructs to log-phase cultures. This makes it an ideal organism for the automation of complex strain construction. Here, we demonstrate the flexibility and versatility of ADP1 as a genetic model through the construction of a broad variety of mutants. These include marked and unmarked insertions and deletions, complementary replacements, chromosomal expression tags and complex combinations thereof. In the process of these constructions, we demonstrate that ADP1 can effectively express a wide variety of foreign genes including antibiotic resistance cassettes, essential metabolic genes, negatively selectable catabolic genes and even intact operons from highly divergent bacteria. All of the described mutations were achieved by the same process of splicing PCR, direct transformation of growing cultures and plating on selective media. The simplicity of these tools make genetic analysis and engineering with Acinetobacter ADP1 accessible to laboratories with minimal microbial genetics expertise and very little equipment. They are also compatible with complete automation of genetic analysis and engineering protocols.     

Sulfated glycosaminoglycans in two hematophagous arthropod vectors of Chagas disease, Triatoma brasiliensis and Rhodnius prolixus (Hemiptera: Reduviidae)

The characterization of sulfated glycosaminoglycans (GAGs) in hematophagous arthropod vectors in general has been limited, with the exception of the studies in the triatomine Rhodnius prolixus. Heparan sulfate (HS) and chondroitin sulfate (CS) were previously identified and structurally characterized in extracts of whole bodies of fourth instar larvae of R. prolixus. Recently, we showed the expression of these two sulfated GAGs in specific body tissues of adult males and females and in embryos of R. prolixus. In the present work, we identified and compared the sulfated GAG composition in specific tissues of adult insects and in embryos of another triatomine species, Triatoma brasiliensis. Sulfated GAGs were isolated from the fat body, intestinal tract, and the reproductive tracts of adult insects and from embryos. Only HS and CS were found in the tissues analyzed. The present results extend the initial observations on the sulfated GAG composition in R. prolixus by showing that these molecules are widely distributed among internal organs of triatomines. These observations may be useful for future investigations aiming to evaluate the possible implication of these compounds in physiological events that take place in a specific organ(s) in these insects.     

Directed Evolution of a Model Primordial Enzyme Provides Insights into the Development of the Genetic Code

The contemporary proteinogenic repertoire contains 20 amino acids with diverse functional groups and side chain geometries. Primordial proteins, in contrast, were presumably constructed from a subset of these building blocks. Subsequent expansion of the proteinogenic alphabet would have enhanced their capabilities, fostering the metabolic prowess and organismal fitness of early living systems. While the addition of amino acids bearing innovative functional groups directly enhances the chemical repertoire of proteomes, the inclusion of chemically redundant monomers is difficult to rationalize. Here, we studied how a simplified chorismate mutase evolves upon expanding its amino acid alphabet from nine to potentially 20 letters. Continuous evolution provided an enhanced enzyme variant that has only two point mutations, both of which extend the alphabet and jointly improve protein stability by >4 kcal/mol and catalytic activity tenfold. The same, seemingly innocuous substitutions (Ile→Thr, Leu→Val) occurred in several independent evolutionary trajectories. The increase in fitness they confer indicates that building blocks with very similar side chain structures are highly beneficial for fine-tuning protein structure and function.     

Modeling Disease Vector Occurrence When Detection Is Imperfect II: Drivers of Site-Occupancy by Synanthropic Triatoma brasiliensis in the Brazilian Northeast

Background

Understanding the drivers of habitat selection by insect disease vectors is instrumental to the design and operation of rational control-surveillance systems. One pervasive yet often overlooked drawback of vector studies is that detection failures result in some sites being misclassified as uninfested; naïve infestation indices are therefore biased, and this can confound our view of vector habitat preferences. Here, we present an initial attempt at applying methods that explicitly account for imperfect detection to investigate the ecology of Chagas disease vectors in man-made environments.

Methodology

We combined triplicate-sampling of individual ecotopes (n = 203) and site-occupancy models (SOMs) to test a suite of pre-specified hypotheses about habitat selection by Triatoma brasiliensis. SOM results were compared with those of standard generalized linear models (GLMs) that assume perfect detection even with single bug-searches.

Principal Findings

Triatoma brasiliensis was strongly associated with key hosts (native rodents, goats/sheep and, to a lesser extent, fowl) in peridomestic environments; ecotope structure had, in comparison, small to negligible effects, although wooden ecotopes were slightly preferred. We found evidence of dwelling-level aggregation of infestation foci; when there was one such focus, same-dwelling ecotopes, whether houses or peridomestic structures, were more likely to become infested too. GLMs yielded negatively-biased covariate effect estimates and standard errors; both were, on average, about four times smaller than those derived from SOMs.

Conclusions/Significance

Our results confirm substantial population-level ecological heterogeneity in T. brasiliensis. They also suggest that, at least in some sites, control of this species may benefit from peridomestic rodent control and changes in goat/sheep husbandry practices. Finally, our comparative analyses highlight the importance of accounting for the various sources of uncertainty inherent to vector studies, including imperfect detection. We anticipate that future research on infectious disease ecology will increasingly rely on approaches akin to those described here.     

Germinable soil seed bank of a gallery forest in Brazilian Cerrado

The soil seed bank is a dynamic biotic component of plant communities that represents the population’s memory in relation to selective events. Few studies have investigated the natural stock of germinable seeds in the gallery forests to evaluate their regeneration potential, although they are target of anthropogenic action. Thus, seasonal, horizontal, and vertical qualitative and quantitative variations of the seed bank of a gallery forest in the Brazilian Cerrado were studied to test the influence of the climatic seasonality, the influence of the physical structure, and depth of the soil in different microhabitats of the forest in this natural seed store. It was also compared the richness and abundance of species in the germinable seed bank with the above-ground vegetation. Three hundred and seventy-five soil samples were collected at the beginning (April 1998 and 1999) and at the end of the dry season (September 1998). These samples were collected in three microhabitats distributed transversally in relation to drainage line of a large stream, at five depths. The density of germinable seeds decreases with depth, and is similar among microhabitats and seasons. In 24,690 cm³ of soil and 4.05 m² of litter, 1390 seedlings emerged, being 743 dicotyledons and 647 monocotyledons. From 761 survivors, 263 were Cyperaceae, 206 Melastomataceae, 153 Poaceae, and 82 Onagraceae, the most abundant families. This study suggests that the diversity of the germinable soil seed bank is lower than that of the above-ground vegetation of the forest, and that the soil seed bank is not the principal regeneration form of this environment.     

Recently differentiated epimastigotes from Trypanosoma cruzi are infective to the mammalian host

Trypanosoma cruzi, the etiologic agent of Chagas disease, has a complex life cycle in which four distinct developmental forms alternate between the insect vector and the mammalian host. It is assumed that replicating epimastigotes present in the insect gut are not infective to mammalian host, a paradigm corroborated by the widely acknowledged fact that only this stage is susceptible to the complement system. In the present work, we establish a T. cruzi in vitro and in vivo epimastigogenesis model to analyze the biological aspects of recently differentiated epimastigotes (rdEpi). We show that both trypomastigote stages of T. cruzi (cell‐derived and metacyclic) are able to transform into epimastigotes (processes termed primary and secondary epimastigogenesis, respectively) and that rdEpi have striking properties in comparison to long‐term cultured epimastigotes: resistance to complement‐mediated lysis and both in vitro (cell culture) and in vivo (mouse) infectivity. Proteomics analysis of all T. cruzi stages reveled a cluster of proteins that were up‐regulated only in rdEpi (including ABC transporters and ERO1), suggesting a role for them in rdEpi virulence. The present work introduces a new experimental model for the study of host‐parasite interactions, showing that rdEpi can be infective to the mammalian host.