Directed evolution of colE1 plasmid replication compatibility: a fast tractable tunable model for investigating biological orthogonality.

Plasmids of the ColE1 family are among the most frequently used in molecular biology. They were adopted early for many biotechnology applications, and as models to study plasmid biology. Their mechanism of replication is well understood, involving specific interactions between a plasmid encoded sense-antisense gene pair (RNAI and RNAII). Due to such mechanism, two plasmids with the same origin cannot be stably maintained in cells-a process known as incompatibility. While mutations in RNAI and RNAII can make colE1 more compatible, there has been no systematic effort to engineer new compatible colE1 origins, which could bypass technical design constraints for multi-plasmid applications. Here, we show that by diversifying loop regions in RNAI (and RNAII), it is possible to select new viable colE1 origins compatible with the wild-type one. We demonstrate that sequence divergence is not sufficient to enable compatibility and pairwise interactions are not an accurate guide for higher order interactions. We identify potential principles to engineer plasmid copy number independently from other regulatory strategies and we propose plasmid compatibility as a tractable model to study biological orthogonality.

Development of an open hardware 3-D printed conveyor device for continuous cryopreservation of non-batched samples.

A great challenge among communities participating in germplasm repository development is to obtain suitable cryopreservation equipment and devices. Commercial programmable freezers are costly and thus unaffordable to many users. Self-made devices have substantial variability among users, resulting in few opportunities for standardization across communities. The development of open hardware with the increasing accessibility of three-dimensional (3-D) printing offers rapid prototyping and easy fabrication of devices by users around the world at low cost. The present study explored the feasibility of developing operational prototypes of 3-D printed motorized cryopreservation devices for continuous freezing of non-batched samples. A controlled cooling conveyor device (CCCD) was designed and fabricated to cryopreserve sperm samples in straws that were loaded onto chain links suspended over liquid nitrogen held in a Styrofoam box. Cooling rates of 5 to 34 °C/min for 0.5-ml French straws were produced by adjusting the height of conveyor chains, slopes, and liquid nitrogen mass. The plunge temperature (-47 °C to -61 °C) was controlled by adjustment of conveyor speed. The cooling curves from the CCCD were comparable to a commercial programmable freezer. There were no significant differences in post-thaw motility of sperm from ornamental (Koi) common carp (Cyprinus carpio) among samples frozen with the CCCD and those frozen with a commercial programmable freezer. The post-thaw sperm motility was consistent among samples frozen in the CCCD across a 15-min time span. The CCCD prototypes in the present study proved to be feasible and functional as low-cost, customizable, portable, and yet standardizable options for freezing of individual (non-batched) samples. Additional design alternatives are proposed to facilitate further adaptation and development by diverse user communities.

Sperm quality biomarkers complement reproductive and endocrine parameters in investigating environmental contaminants in common carp (Cyprinus carpio) from the Lake Mead National Recreation Area.

Lake Mead National Recreational Area (LMNRA) serves as critical habitat for several federally listed species and supplies water for municipal, domestic, and agricultural use in the Southwestern U.S. Contaminant sources and concentrations vary among the sub-basins within LMNRA. To investigate whether exposure to environmental contaminants is associated with alterations in male common carp (Cyprinus carpio) gamete quality and endocrine- and reproductive parameters, data were collected among sub-basins over 7 years (1999-2006). Endpoints included sperm quality parameters of motility, viability, mitochondrial membrane potential, count, morphology, and DNA fragmentation; plasma components were vitellogenin (VTG), 17ß-estradiol, 11-keto-testosterone, triiodothyronine, and thyroxine. Fish condition factor, gonadosomatic index, and gonadal histology parameters were also measured. Diminished biomarker effects were noted in 2006, and sub-basin differences were indicated by the irregular occurrences of contaminants and by several associations between chemicals (e.g., polychlorinated biphenyls, hexachlorobenzene, galaxolide, and methyl triclosan) and biomarkers (e.g., plasma thyroxine, sperm motility and DNA fragmentation). By 2006, sex steroid hormone and VTG levels decreased with subsequent reduced endocrine disrupting effects. The sperm quality bioassays developed and applied with carp complemented endocrine and reproductive data, and can be adapted for use with other species.

Cryopreservation of sperm bundles (spermatozeugmata) from endangered livebearing goodeids.

More than half of fishes in the family Goodeidae are considered to be endangered, threatened, or vulnerable. Sperm cryopreservation is an effective tool for conserving genetic resources of imperiled populations, but development of protocols with livebearing fishes faces numerous challenges including the natural packaging of sperm into bundles. In this study the cryopreservation of sperm bundles (spermatozeugmata) of three goodeids species was evaluated. Sperm quality was evaluated by activation with NaCl-NaOH solution (at 300 mOsmol/kg and pH 11.8), and analysis of dissociable bundles and dissociation duration. Using Redtail Splitfin (Xenotoca eiseni) as a model, the effects of cryoprotectants (dimethyl sulfoxide, methanol, and glycerol) with different concentrations (5-15% v/v %), equilibration exposure times (1-60 min), cooling rates (5-40 °C/min), concentrations (4 × 104-4 × 106 bundles/ml), buffers (HBSS, PBS and NaCl), and buffer osmolalities (200-400 mOsmol/kg) were investigated. After cooling and thawing, sperm bundles maintained their packed form. A specific protocol was developed (10% dimethyl sulfoxide, 20-min equilibration, 10 °C/min cooling rate, 4 × 106 bundles/ml, and 300 mOsmol/kg HBSS). This protocol yielded 89 ±â€¯5% of post-thaw dissociable bundles with 209 ±â€¯10 s of dissociation duration for X. eiseni, 96 ±â€¯9% with 814 ±â€¯14 s for Blackfin Goodea (Goodea atripinni), and 66 ±â€¯2% with 726 ±â€¯25 s for Striped Goodeid (Ataeniobius toweri). This is the first study of cryopreservation of sperm within bundles for livebearing fishes and provides a basis for establishment of germplasm repositories for goodeids and other livebearers.

Activation of free sperm and dissociation of sperm bundles (spermatozeugmata) of an endangered viviparous fish, Xenotoca eiseni.

Knowledge of sperm motility activation for viviparous fishes has been limited to study of several species in Poeciliidae, and the dissociation of sperm bundles is even less understood. The goal of this study was to use the endangered Redtail Splitfin (Xenotoca eiseni) as a model to investigate the activation of sperm from viviparous fishes by study of free sperm and spermatozeugmata (unencapsulated sperm bundles). The specific objectives were to evaluate the effects of: (1) osmotic pressure and refrigerated storage (4 °C) on activation of free sperm, (2) osmotic pressure, ions, and pH on dissociation of spermatozeugmata, and (3) CaCl2 concentration and pH on sperm membrane integrity. Free sperm were activated in Ca2+-free Hanks' balanced salt solution at 81-516 mOsmol/kg. The highest motility (19 ±â€¯6%) was at 305 mOsmol/kg and swim remained for 84 h. Glucose (300-700 mOsmol/kg), NaCl (50-600 mOsmol/kg), and KCl, MgCl2, and MnCl2 at 5-160 mM activated sperm within spermatozeugmata, but did not dissociate spermatozeugmata. CaCl2 at 5-160 mM dissociated spermatozeugmata within 10 min. Solutions of NaCl-NaOH at pH 11.6 to 12.4 dissociated spermatozeugmata within 1 min. The percentage of viable cells had no significant differences (P = 0.2033) among different concentrations of CaCl2, but it was lower (P < 0.0001) at pH 12.5 than at pH between 7.0 and 12.0. Overall, this study provided a foundation for quality evaluation of sperm and spermatozeugmata from livebearing fishes, and for development of germplasm repositories for imperiled goodeids.

Identification of Two Auxin-Regulated Potassium Transporters Involved in Seed Maturation.

The seed is the most important plant reproductive unit responsible for the evolutionary success of flowering plants. Aside from its essential function in the sexual reproduction of plants, the seed also represents the most economically important agricultural product worldwide, providing energy, nutrients, and raw materials for human nutrition, livestock feed, and countless manufactured goods. Hence, improvements in seed quality or size are highly valuable, due to their economic potential in agriculture. Recently, the importance of indolic compounds in regulating these traits has been reported for Arabidopsis thaliana. The transcriptional and physiological mechanisms involved, however, remain largely undisclosed. Potassium transporters have been suggested as possible mediators of embryo cell size, controlling turgor pressure during seed maturation. In addition, it has been demonstrated that the expression of K⁺ transporters is effectively regulated by auxin. Here, we provide evidence for the identification of two Arabidopsis K⁺ transporters, HAK/KT12 (At1g60160) and KUP4 (At4g23640), that are likely to be implicated in determining seed size during seed maturation and, at the same time, show a differential regulation by indole-3-acetic acid and indole-3-acetamide.

Addressing Reproducibility in Cryopreservation, and Considerations Necessary for Commercialization and Community Development in Support of Genetic Resources of Aquatic Species.

For the past six decades a repeated cycle of developing new cryopreservation protocols or simply reinventing them to counteract a lack of reproducibility has led to hundreds of published studies that have offered little to the establishment of a genetic resources community for aquatic species. This has hampered repository development and inhibited industrial application. Most protocols were developed without standardized approaches, leading to irreproducible studies and questionable or meaningless comparisons. Thus cryopreservation of germplasm in aquatic species would greatly benefit from strategies to facilitate reproducibility. Our objectives were to: (1) identify major sources of irreproducibility across research, small-scale, repository, and commercial-scale development levels, (2) provide recommendations to address reproducibility challenges, and (3) offer suggestions on how researchers can directly influence commercial development and application of cryopreservation research. Sources of irreproducibility include lack of standardized procedural approaches, lack of standardized terminology, and lack of reporting guidelines. To address these challenges, we propose implementation of standard operating procedures (SOP), support of stock centers and internet content for development of training programs, and strengthening of the role of scientific journals and reviewers in reducing the frequency of irreproducible outcomes. Reproducibility is the foundation for quality management programs and product reliability, and therefore standardization is necessary to assure efficient transition to commercial-scale application and repository development. Progress can only be possible through community-based approaches focused on coalescence and consensus of disparate groups involved in aquatic species cryopreservation and management of genetic resources.

Hexagons to Ribbons: Flipping Cyanide on Au{111}.

Cyanide monolayers on Au{111} restructure from a hexagonal close-packed lattice to a mixed-orientation "ribbon" structure through thermal annealing. The new surface structure loses most of the observed surface features characterizing the initial as-adsorbed system with "ribbon" domain boundaries isolating rotationally offset surface regions where the orientation is guided by the underlying gold lattice. A blue shift to higher frequencies of the CN vibration to 2235 cm-1 with respect to the as-adsorbed CN/Au{111} vibration at 2146 cm-1 is observed. In addition, a new low-frequency mode is observed at 145 cm-1, suggesting a chemical environment change similar to gold-cyanide crystallization. We discuss this new structure with respect to a mixed cyanide/isocyanide monolayer and propose a bonding scheme consisting of Au-CN and Au-NC bound molecules that are oriented normal to the Au{111} surface.

Selection platforms for directed evolution in synthetic biology.

Life on Earth is incredibly diverse. Yet, underneath that diversity, there are a number of constants and highly conserved processes: all life is based on DNA and RNA; the genetic code is universal; biology is limited to a small subset of potential chemistries. A vast amount of knowledge has been accrued through describing and characterizing enzymes, biological processes and organisms. Nevertheless, much remains to be understood about the natural world. One of the goals in Synthetic Biology is to recapitulate biological complexity from simple systems made from biological molecules-gaining a deeper understanding of life in the process. Directed evolution is a powerful tool in Synthetic Biology, able to bypass gaps in knowledge and capable of engineering even the most highly conserved biological processes. It encompasses a range of methodologies to create variation in a population and to select individual variants with the desired function-be it a ligand, enzyme, pathway or even whole organisms. Here, we present some of the basic frameworks that underpin all evolution platforms and review some of the recent contributions from directed evolution to synthetic biology, in particular methods that have been used to engineer the Central Dogma and the genetic code.

Cryopreservation in fish: current status and pathways to quality assurance and quality control in repository development.

Cryopreservation in aquatic species in general has been constrained to research activities for more than 60 years. Although the need for application and commercialisation pathways has become clear, the lack of comprehensive quality assurance and quality control programs has impeded the progress of the field, delaying the establishment of germplasm repositories and commercial-scale applications. In this review we focus on the opportunities for standardisation in the practices involved in the four main stages of the cryopreservation process: (1) source, housing and conditioning of fish; (2) sample collection and preparation; (3) freezing and cryogenic storage of samples; and (4) egg collection and use of thawed sperm samples. In addition, we introduce some key factors that would assist the transition to commercial-scale, high-throughput application.

Amine reactive dyes: an alternative to estimate membrane integrity in fish sperm cells.

Fluorescent dyes that binds irreversibly to cellular amines, come in several available emission spectra, and do not poses health concerns were used to evaluate membrane integrity in fish sperm cells. The objectives of the present study were to determine: (1) a working dye concentration for fish sperm samples, and (2) if the traditional propidium iodide/SYBR-14 staining combination was comparable with the amine reactive dye (ARD) methods at identifying cell populations with intact and compromised membranes after sperm activation, refrigerated storage, and exposure to cryoprotectant and surfactant. Zebrafish (Danio rerio) sperm were obtained by stripping, and pooled samples (in triplicate) were used in all tests. Six dilutions of the amine dye (ranging from 0.625 to 0.02 µL/mL) were evaluated, and compared with the traditional staining protocol. A concentration of 0.5 µL/mL ARD was selected to be used in subsequent assays. Sperm suspensions were activated with deionized water to simulate urine contamination. After 10 sec, osmolality was increased to stop activation, and the procedure was repeated in 10-sec intervals until the sperm remained activated for 120 consecutive sec; membrane integrity was analyzed at each time interval. For the storage assay, sperm suspensions were prepared in Hanks' balanced salt solution at 302 mOsm/kg osmolality (HBSS302), HBSS354 and HBSS402, and evaluated every 2 hr for 8 hr, and every 24 hr for 72 hr. Cryoprotectant toxicity was tested by diluting sperm suspensions in HBSS340 with methanol at 5, 10 and 15% final concentrations. Surfactant toxicity was tested by diluting sperm suspensions in HBSS354 with Triton X-100 at 0.2, 0.15 and 0.1 mM final concentrations. In each toxicity assay, membrane integrity was tested every 20 min for 80 min. The number of membrane-intact cells significantly decreased across time in all treatments (p < 0.05). Significant differences between staining protocols were observed after activation and after exposure to methanol at 10 and 15%, and to Triton X-100 (p < 0.05). The average difference, however, was minor (between 1 and 6% in average) in relation to the typical values used for decision making based on this assay. Results showed that this method has the potential to contribute greatly to the standardization of cryopreservation in aquatic species.

Are endocrine and reproductive biomarkers altered in contaminant-exposed wild male Largemouth Bass (Micropterus salmoides) of Lake Mead, Nevada/Arizona, USA?

Male Largemouth Bass were sampled from two locations in Lake Mead (USA), a site influenced by treated municipal wastewater effluent and urban runoff (Las Vegas Bay), and a reference site (Overton Arm). Samples were collected in summer (July '07) and spring (March '08) to assess general health, endocrine and reproductive biomarkers, and compare contaminant body burdens by analyzing 252 organic chemicals. Sperm count and motility were measured in spring. Contaminants were detected at much higher frequencies and concentrations in fish from Las Vegas Bay than Overton Arm. Those with the highest concentrations included PCBs, DDTs, PBDEs, galaxolide, and methyl triclosan. Fish from Las Vegas Bay also had higher Fulton condition factor, hepatosomatic index, and hematocrit, and lower plasma 11-ketotestosterone concentration (KT). Gonadosomatic index (GSI) and sperm motility did not differ between sites, but sperm count was lower by nearly 50% in fish from Las Vegas Bay. A positive association between KT and GSI was identified, but this association was nonlinear. On average, maximal GSI was reached at sub-maximal KT concentrations. In conclusion, the higher concentration of contaminant body burdens coupled with reduced levels of KT and sperm count in fish from Las Vegas Bay suggest that male reproductive condition was influenced by contaminant exposures. Also, the nonlinear KT-GSI association provided a framework to understand why GSI was similar between male bass from both sites despite their large difference in KT, and also suggested the existence of post-gonadal growth functions of KT at high concentrations.

Novel associations between contaminant body burdens and biomarkers of reproductive condition in male Common Carp along multiple gradients of contaminant exposure in Lake Mead National Recreation Area, USA.

Adult male Common Carp were sampled in 2007/08 over a full reproductive cycle at Lake Mead National Recreation Area. Sites sampled included a stream dominated by treated wastewater effluent, a lake basin receiving the streamflow, an upstream lake basin (reference), and a site below Hoover Dam. Individual body burdens for 252 contaminants were measured, and biological variables assessed included physiological [plasma vitellogenin (VTG), estradiol-17ß (E2), 11-ketotestosterone (11KT)] and organ [gonadosomatic index (GSI)] endpoints. Patterns in contaminant composition and biological condition were determined by Principal Component Analysis, and their associations modeled by Principal Component Regression. Three spatially distinct but temporally stable gradients of contaminant distribution were recognized: a contaminant mixture typical of wastewaters (PBDEs, methyl triclosan, galaxolide), PCBs, and DDTs. Two spatiotemporally variable patterns of biological condition were recognized: a primary pattern consisting of reproductive condition variables (11KT, E2, GSI), and a secondary pattern including general condition traits (condition factor, hematocrit, fork length). VTG was low in all fish, indicating low estrogenic activity of water at all sites. Wastewater contaminants associated negatively with GSI, 11KT and E2; PCBs associated negatively with GSI and 11KT; and DDTs associated positively with GSI and 11KT. Regression of GSI on sex steroids revealed a novel, nonlinear association between these variables. Inclusion of sex steroids in the GSI regression on contaminants rendered wastewater contaminants nonsignificant in the model and reduced the influence of PCBs and DDTs. Thus, the influence of contaminants on GSI may have been partially driven by organismal modes-of-action that include changes in sex steroid production. The positive association of DDTs with 11KT and GSI suggests that lifetime, sub-lethal exposures to DDTs have effects on male carp opposite of those reported by studies where exposure concentrations were relatively high. Lastly, this study highlighted advantages of multivariate/multiple regression approaches for exploring associations between complex contaminant mixtures and gradients and reproductive condition in wild fishes.

Specimen self-collection and HPV DNA screening in a pilot study of 100,242 women.

Since cervical cancer remains common in Mexico despite an established cytology screening program, the Ministry of Health recently introduced pilot front-line HPV testing into the Mexican cervical cancer screening program (CCSP). Here, we present the key field performance metrics of this population-based study. High-risk HPV DNA (hrHPV) testing was conducted on self-collected vaginal specimens from 100,242 women aged 25-75 years residing in Morelos State. All hrHPV positive women and a random sample of 3.2% (n = 2,864) of hrHPV negative participants were referred for colposcopic examination. The main disease endpoint of interest was cervical intraepithelial neoplasia grade 2 or higher (CIN2+). We calculated relative risk, positive predictive value and negative predictive value adjusted for screening test verification bias. The overall prevalence of hrHPV was 10.8% (95% CI 10.6-11.0). Women positive for hrHPV had a relative risk of 15.7 for histologically detectable CIN2+. The adjusted positive predictive value of the hrHPV test was 2.4% (95% CI 2.1-2.7); whereas the adjusted negative predictive value was 99.8% (95% CI 99.8-99.9). These findings suggest that large-scale vaginal hrHPV testing in a middle-income country can identify women at greater risk of advanced cervical abnormalities in a programmatically meaningful way but care is warranted to ensure that disease not detectable at colposcopy is kept to a minimum. PASS shows areas that need improvement and sets the stage for wider use of hrHPV screening of self-collected vaginal specimens in Mexico.

Cost analysis of different cervical cancer screening strategies in Mexico.

OBJECTIVE: To compare the costs and number of undetected cases of four cervical cancer screening strategies (CCSS) in Mexico. MATERIALS AND METHODS: We estimated the costs and outcomes of the following CCSS: a) conventional Papanicolaou smear (Pap) alone; b) high-risk human papilloma virus testing (HR-HPV) as primary screening with Pap as reflex triage; c) HR-HPV as primary screening with HPV-16/18 typing, liquid-based cytology (LBC) and immunostaining for p16/Ki67 testing as reflex triage, and d) co-testing with HR-HPV and LBC with HPV-16/18 typing and immunostaining for p16/Ki67 as reflex triage. The outcome of interest was high-grade cervical lesions or cervical cancer. RESULTS: HR-HPV testing, HPV typing, LBC testing and immunostaining is the best alternative because it is the least expensive option with an acceptable number of missed cases. CONCLUSIONS: The opportunity costs of a poor quality CCSS is many false negatives. Combining multiple tests may be a more cost-effective way to screen for cervical cancer in Mexico.

Natural genetic diversity of the DBL domain of a novel member of the Plasmodium vivax erythrocyte binding-like proteins (EBP2) in the Amazon rainforest.

In malaria parasites, the erythrocyte binding-like proteins (EBL) are a family of invasion proteins that are attractive vaccine targets. In the case of Plasmodium vivax, the widespread malaria parasite, blood-stage vaccines have been largely focused on a single EBL candidate, the Duffy binding-like domain (DBL) of the Duffy binding protein (DBPII), due to its well-characterized role in the reticulocyte invasion. A novel P. vivax EBL family member, the Erythrocyte binding protein (EBP2, also named EBP or DBP2), binds preferentially to reticulocytes and may mediate an alternative P. vivax invasion pathway. To gain insight into the natural genetic diversity of the DBL domain of EBP2 (region II; EBP2-II), we analyzed ebp2-II gene sequences of 71 P. vivax isolates collected in different endemic settings of the Brazilian Amazon rainforest, where P. vivax is the predominant malaria-associated species. Although most of the substitutions in the ebp2-II gene were non-synonymous and suggested positive selection, the results showed that the DBL domain of the EBP2 was much less polymorphic than that of DBPII. The predominant EBP2 haplotype in the Amazon region corresponded to the C127 reference sequence first described in Cambodia (25% C127-like haplotype). An overview of ebp2-II gene sequences available at GenBank (n = 352) from seven countries (Cambodia, Madagascar, Myanmar, PNG, South Korea, Thailand, Vietnam) confirmed the C127-like haplotype as highly prevalent worldwide. Two out of 43 haplotypes (5 to 20 inferred per country) showed a global frequency of 60%. The results presented here open new avenues of research pursuit while suggesting that a vaccine based on the DBL domain of EBP2 should target a few haplotypes for broad coverage.

Engineering the substrate specificity of a thermophilic penicillin acylase from thermus thermophilus.

A homologue of the Escherichia coli penicillin acylase is encoded in the genomes of several thermophiles, including in different Thermus thermophilus strains. Although the natural substrate of this enzyme is not known, this acylase shows a marked preference for penicillin K over penicillin G. Three-dimensional models were created in which the catalytic residues and the substrate binding pocket were identified. Through rational redesign, residues were replaced to mimic the aromatic binding site of the E. coli penicillin G acylase. A set of enzyme variants containing between one and four amino acid replacements was generated, with altered catalytic properties in the hydrolyses of penicillins K and G. The introduction of a single phenylalanine residue in position α188, α189, or ß24 improved the K(m) for penicillin G between 9- and 12-fold, and the catalytic efficiency of these variants for penicillin G was improved up to 6.6-fold. Structural models, as well as docking analyses, can predict the positioning of penicillins G and K for catalysis and can demonstrate how binding in a productive pose is compromised when more than one bulky phenylalanine residue is introduced into the active site.

Effects of dietary exposure to brominated flame retardant BDE-47 on thyroid condition, gonadal development and growth of zebrafish.

Little is known about the effects of brominated flame retardants in teleosts and some of the information currently available is inconsistent. This study examined effects of dietary exposure to 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) on thyroid condition, body mass and size, and gonadal development of zebrafish. Pubertal, 49-day-old (posthatch) fish were fed diets without BDE-47 (control) or with 1, 5 or 25 µg/g BDE-47/diet. Treatments were conducted in triplicate 30-L tanks each containing 50 zebrafish, and 15 fish per treatment (5 per tank) were sampled at days 40, 80 and 120 of exposure. Measurements were taken of body mass, standard length, head depth and head length. Sex (at 40-120 days of exposure), germ cell stage (at 40 days) and thyroid condition (at 120 days; follicular cell height, colloid depletion, angiogenesis) were histologically determined. Whole-body BDE-47 levels at study completion were within the high end of levels reported in environmentally exposed (wild) fishes. Analysis of variance was used to determine differences among treatments at each sampling time. No effects were observed on thyroid condition or germ cell stage in either sex. Reduced head length was observed in females exposed to BDE-47 at 80 days but not at 40 or 120 days. In males, no apparent effects of BDE-47 were observed at 40 and 80 days, but fish exposed to 25 µg/g had lower body mass at 120 days compared to control fish. These observations suggest that BDE-47 at environmentally relevant whole-body concentrations does not affect thyroid condition or pubertal development of zebrafish but does affect growth during the juvenile-to-adult transition, especially in males.

IgM antibody responses against Plasmodium antigens in neotropical primates in the Brazilian Atlantic Forest.

Introduction: Zoonotic transmission is a challenge for the control and elimination of malaria. It has been recorded in the Atlantic Forest, outside the Amazon which is the endemic region in Brazil. However, only very few studies have assessed the antibody response, especially of IgM antibodies, in Neotropical primates (NP). Therefore, in order to contribute to a better understanding of the immune response in different hosts and facilitate the identification of potential reservoirs, in this study, naturally acquired IgM antibody responses against Plasmodium antigens were evaluated, for the first time, in NP from the Atlantic Forest. Methods: The study was carried out using 154 NP samples from three different areas of the Atlantic Forest. IgM antibodies against peptides of the circumsporozoite protein (CSP) from different Plasmodium species and different erythrocytic stage antigens were detected by ELISA. Results: Fifty-nine percent of NP had IgM antibodies against at least one CSP peptide and 87% against at least one Plasmodium vivax erythrocytic stage antigen. Levels of antibodies against PvAMA-1 were the highest compared to the other antigens. All families of NP showed IgM antibodies against CSP peptides, and, most strikingly, against erythrocytic stage antigens. Generalized linear models demonstrated that IgM positivity against PvCSP and PvAMA-1 was associated with PCR-detectable blood-stage malaria infection and the host being free-living. Interestingly, animals with IgM against both PvCSP and PvAMA-1 were 4.7 times more likely to be PCR positive than animals that did not have IgM for these two antigens simultaneously. Discussion: IgM antibodies against different Plasmodium spp. antigens are present in NP from the Atlantic Forest. High seroprevalence and antibody levels against blood-stage antigens were observed, which had a significant association with molecular evidence of infection. IgM antibodies against CSP and AMA-1 may be used as a potential marker for the identification of NP infected with Plasmodium, which are reservoirs of malaria in the Brazilian Atlantic Forest.

Sucrose synthase in unicellular cyanobacteria and its relationship with salt and hypoxic stress.

Higher plants and cyanobacteria metabolize sucrose (Suc) by a similar set of enzymes. Suc synthase (SuS, A/UDP-glucose: D: -fructose 2-α-D: -glucosyl transferase) catalyzes a reversible reaction. However, it is in the cleavage of Suc that this enzyme plays an important role in vivo, providing sugar nucleotides for polysaccharide biosynthesis. In cyanobacteria, SuS occurrence has been reported in heterocyst-forming strains, where it was shown to be involved also in nitrogen fixation. We investigated the presence of sequences homologous to SuS-encoding genes (sus) in recently sequenced cyanobacterial genomes. In this work, we show for the first time the presence of SuS in unicellular cyanobacterium strains (Microcystis aeruginosa PCC 7806, Gloebacter violaceus PCC 7421, and Thermosynechococcus elongatus BP-1). After functional characterization of SuS encoding genes, we demonstrated an increase in their transcript levels after a salt treatment or hypoxic stress in M. aeruginosa and G. violaceus cells. Based on phylogenetic analysis and on the presence of sus homologs in the most recently radiated cyanobacterium strains, we propose that sus genes in unicellular cyanobacteria may have been acquired through horizontal gene transfer. Taken together, our data indicate that SuS acquisition by cyanobacteria might be related to open up new ecological niches.