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.

The Need for a Framework Addressing the Temporal Aspects of Fish Sperm Motility Leading to Community-Level Standardization.

Motility is a widely available parameter that can be used to assess sperm quality of aquatic species. Sperm from fishes with external fertilization usually undergo a dynamic and short-lived period of motility after activation. The common practice of assigning a single value at an arbitrary peak of motility presents challenges for reproducibility, community-level standardization, and comparisons across studies. This study aimed to explore statistical approaches to standardize motility reporting, and to develop an initial framework for community-level standards. Sperm samples from 14 zebrafish (Danio rerio) with a total of 21,705 cells were analyzed by use of computer-assisted sperm analysis with data collection starting at 10 s after activation at 5-s intervals for 50 s. Four common motility variables were selected for analyses: curvilinear velocity, straight-line velocity, beat cross frequency, and amplitude of lateral head displacement. Cluster analysis was used to evaluate sperm subpopulations within and among males over time, least-square means was used to explore temporal aspects, and the first derivative of the regression equations was used to calculate the rate of change for the motility parameters. Cluster analysis proved informative, but overlapping ephemeral clusters were not valuable for providing standardization options. Analysis of temporal aspects and rate of change indicated opportunities for standardization by reporting the overall motility-time functions or reporting during stable time windows instead of peak motility or at random times. These approaches could minimize the inconsistencies caused by male-to-male variation and dynamic changes of subpopulations while providing comparable information. An overall temporal framework was identified for motility reporting along the collection-processing-cryopreservation-thawing sequence to provide a basis to support efforts of community-level standardization.

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.

Antibody response to a new member of the DBL family (EBP2) after a brief Plasmodium vivax exposure.

Plasmodium vivax blood-stage invasion into reticulocyte is critical for parasite development. Thus, validation of novel parasite invasion ligands is essential for malaria vaccine development. Recently, we demonstrated that EBP2, a Duffy binding protein (DBP) paralog, is antigenically distinct from DBP and could not be functionally inhibited by anti-DBP antibodies. Here, we took advantage of a small outbreak of P.vivax malaria, located in a non-malarious area of Brazil, to investigate for the first time IgM/IgG antibodies against EBP2 and DEKnull-2 (an engineering DBPII vaccine) among individuals who had their first and brief exposure to P.vivax (16 cases and 22 non-cases). Our experimental approach included 4 cross sectional surveys at 3-month interval (12-month follow-up). The results demonstrated that while a brief initial P.vivax infection was not efficient to induce IgM/ IgG antibodies to either EBP2 or DEKnull-2, IgG antibodies against DEKnull-2 (but not EBP2) were boosted by recurrent blood-stage infections following treatment. Of interest, in most recurrent P. vivax infections (4 out of 6 patients) DEKnull-2 IgG antibodies were sustained for 6 to 12 months. Polymorphisms in the ebp2 gene does not seem to explain EBP2 low immunogenicity as the ebp2 allele associated with the P.vivax outbreak presented high identity to the original EBP2 isolate used as recombinant protein. Although EBP2 antibodies were barely detectable after a primary episode of P.vivax infection, EBP2 was highly recognized by serum IgG from long-term malaria-exposed Amazonians (range from 35 to 92% according to previous malaria episodes). Taken together, the results showed that individuals with a single and brief exposure to P.vivax infection develop very low anti-EBP2 antibodies, which tend to increase after long-term malaria exposure. Finally, the findings highlighted the potential of DEKnull-2 as a vaccine candidate, as in non-immune individuals anti-DEKnull-2 IgG antibodies were boosted even after a brief exposure to P.vivax blood stages.

Safety with engineering control and treatment to standard precaution in peripheral venous catheterization: observational study / Seguridad con control de ingeniería y tratamiento según precaución estándar en cateterismo venoso periférico: estudio observacional / Segurança com controle de engenharia e adesão à precaução padrão no cateterismo venoso periférico: estudo observacional

Objective: to analyze the handling of the safety device with engineering control in peripheral venous catheter and the adherence to Standard Precautions by nursing professionals during peripheral venous puncture in adults. Method: observational, descriptive study, with quantitative approach, performed in a surgical clinical hospitalization unit. Data collected through a semi-structured instrument, containing variables related to the catheter and the observation of the procedure. The data were analyzed using descriptive statistics. Results: the study sample consisted of 28 professionals, and 235 procedures were observed. Hand hygiene with antiseptic was performed in 23% (54); alcohol solution in 10.6% (25). Procedure gloves and goggles were used in 56.6% (133) and 2.1% (5), respectively. The catheter with retractable device was incorrectly activated in 45.1% (106). Conclusion: most professionals used the peripheral venous catheter with inadequate engineering control and the standard precautions were low.

Objetivo: analizar el manejo del dispositivo de seguridad con control de ingeniería en catéter venoso periférico y el seguimiento de las precauciones estándar por parte de los profesionales de enfermería durante la punción venosa periférica en adultos. Método: abordaje observacional, descriptivo, cuantitativo, realizado en una unidad de hospitalización clínica quirúrgica. Datos recogidos a través de un instrumento semiestructurado, que contiene variables relacionadas con el catéter y la observación del procedimiento. Los datos fueron analizados mediante estadística descriptiva. Resultados: la muestra del estudio estuvo constituida por 28 profesionales, y se observaron 235 procedimientos. La higiene de manos con antiséptico se realizó en el 23% (54); solución de alcohol en 10,6% (25). Se utilizaron guantes y gafas de procedimiento en el 56,6% (133) y el 2,1% (5), respectivamente. El catéter con dispositivo retráctil se activó incorrectamente en el 45,1% (106). Conclusión: la mayoría de los profesionales utilizaron el catéter venoso periférico con un control de ingeniería inadecuado y las precauciones estándar fueron bajas.

Objetivo: analisar o manuseio do dispositivo de segurança com controle de engenharia em cateter venoso periférico e adesão às Precauções Padrão por profissionais de enfermagem durante a punção venosa periférica em adultos. Método: observacional, descritivo, de abordagem quantitativa, realizado em unidade de internação clínica cirúrgica. Dados coletados por meio de instrumento semiestruturado, contendo variáveis referentes ao cateter e a observação do procedimento. Os dados foram analisados mediante estatística descritiva. Resultados: a amostra do estudo foi composta por 28 profissionais, sendo observados 235 procedimentos. A higienização das mãos com antisséptico foi realizada em 23% (54); solução alcoólica em 10,6% (25). Luvas de procedimento e óculos de proteção foram utilizados em 56,6% (133) e 2,1% (5), respectivamente. O cateter com dispositivo retrátil foi acionado incorretamente em 45,1% (106). Conclusão: a maioria dos profissionais utilizou o cateter venoso periférico com controle de engenharia de maneira inadequada e a adesão às precauções padrão foi baixa.

Multiplexed Microsphere-Based Flow Cytometric Assay to Assess Strain Transcending Antibodies to Plasmodium vivax Duffy Binding Protein II Reveals an Efficient Tool to Identify Binding-Inhibitory Antibody Responders.

Malaria remains a major public health problem worldwide, and Plasmodium vivax is the most widely distributed malaria parasite. Naturally acquired binding inhibitory antibodies (BIAbs) to region II of the Duffy binding protein (DBPII), a P. vivax ligand that is critical for reticulocyte invasion, are associated with a reduced risk of clinical malaria. Owing to methodological issues in evaluating antibodies that inhibit the DBPII-DARC interaction, a limited number of studies have investigated DBPII BIAbs in P. vivax-exposed populations. Based on the assumption that individuals with a consistent BIAb response are characterized by strain-transcending immune responses, we hypothesized that detecting broadly reactive DBPII antibodies would indicate the presence of BIAb response. By taking advantage of an engineered DBPII immunogen targeting conserved DBPII neutralizing epitopes (DEKnull-2), we standardized a multiplex flow cytometry-based serological assay to detect broadly neutralizing IgG antibodies. For this study, a standard in vitro cytoadherence assay with COS-7 cells expressing DBPII was used to test for DBPII BIAb response in long-term P. vivax-exposed Amazonian individuals. Taken together, the results demonstrate that this DBPII-based multiplex assay facilitates identifying DBPII BIAb carriers. Of relevance, the ability of the multiplex assay to identify BIAb responders was highly accurate when the positivity for all antigens was considered. In conclusion, the standardized DBPII-based flow cytometric assay confirmed that DBPII-BIAb activity was associated with the breadth rather than the magnitude of anti-DBPII antibodies. Altogether, our results suggest that multiplex detection of broadly DBPII-reactive antibodies facilitates preliminary screening of BIAb responders.

Profiling Humoral Immune Response Against Pre-Erythrocytic and Erythrocytic Antigens of Malaria Parasites Among Neotropical Primates in the Brazilian Atlantic Forest.

Human malaria due to zoonotic transmission has been recorded in the Atlantic Forest, an extra-Amazonian area in Brazil, which are a challenge for malaria control. Naturally acquired humoral immune response against pre-erythrocytic and erythrocytic antigens of Neotropical primates (NP) was evaluated here to improve the knowledge about the exposure of those animals to the malaria transmission and support the identification of the potential reservoirs of the disease in the Atlantic Forest. Blood samples of 154 monkeys from three areas of the Atlantic Forest were used to identify IgG antibodies against peptides of the repeat region of the major pre-erythrocytic antigen, the circumsporozoite protein (CSP), of Plasmodium vivax (PvCSP), Plasmodium brasilianum/Plasmodium malariae (Pb/PmCSP), and Plasmodium falciparum (PfCSP) by ELISA. Antibodies against erythrocytic recombinant antigens of P. vivax, Apical membrane antigen 1 (PvAMA-1), Erythrocyte binding protein 2 (PvEBP-2) and domain II of Duffy binding protein (PvDBPII) were also evaluated. Parameters, such as age, sex, PCR positivity, and captivity, potentially associated with humoral immune response were analyzed. Eighty-five percent of NP had antibodies against at least one CSP peptide, and 76% against at least one P. vivax erythrocytic antigen. A high percentage of adults compared to non-adults were seropositive and showed increased antibody levels. Neotropical primates with PCR positive for P. simium had a significantly higher frequency of positivity rate for immune response against PvEBP-2, PvDBPII and also higher antibody levels against PvDBPII, compared to PCR negative NPs for this species. Monkeys with PCR positive for P. brasilianum/P. malariae showed higher frequency of seropositivity and antibody levels against Pb/PmCSP. Levels of antibodies against Pb/PmCSP, PvEBP-2 and PvDBPII were higher in free-living than in captive monkeys from the same area. All Platyrrhine families showed antibodies against CSP peptides, however not all showed IgG against erythrocytic antigens. These findings showed a high prevalence of naturally acquired antibodies against CSP repeats in all studied areas, suggesting an intense exposure to infected-mosquitoes bites of NP from all families. However, mainly monkeys of Atelidae family showed antibodies against P. vivax erythrocytic antigens, suggesting blood infection, which might serve as potential reservoirs of malaria in the Atlantic Forest.

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.

Cumprimento às precauções-padrão por profissionais de enfermagem e fatores associados / Observancia de precauciones estándar por parte de profesionales de enfermería y factores asociados / Compliance with standard precautions by nursing professionals and related factors

Resumo Objetivo Verificar o cumprimento às Precauções-Padrão por profissionais de enfermagem e fatores associados. Método Estudo descritivo, transversal, com 522 profissionais de enfermagem, realizado em dois hospitais brasileiros, entre janeiro de 2017 a março de 2018. Os dados foram coletados por meio de um formulário contendo variáveis demográficas e profissionais e a Compliance with Standard Precautions Scale (versão Português-Brasil); posteriormente, analisados por estatísticas descritivas e exploratórias e um modelo de regressão linear múltiplo. Resultados O escore médio global 12,9 (DP=2,5). Técnicos de enfermagem tiveram escores médios estatisticamente significativos maiores (p <0,01) do que enfermeiros. Houve diferença significativa quanto à categoria profissional (p <0,01) e escolaridade (p <0,01), e, marginalmente significativa em relação à idade (p = 0,06). Não houve diferenças quanto à experiência profissional (p = 077), participação em treinamentos (p = 0,79), tipo de hospital (p = 0,13), respectivamente. A escolaridade não contribuiu para um maior cumprimento às medidas (p <0,01), assim como o ensino superior (p ≤ 0,01). Conclusão e Implicações para a prática O aumento na escolaridade e experiência profissional não contribuiu para maior cumprimento às Precações. Por contemplar aspectos da prática de enfermagem, estratégias de prevenção de exposição ocupacional podem ser revistas e aperfeiçoadas.

Resumen Objetivo Verificar la observancia de las Precauciones Estándar por parte de profesionales de enfermería, y sus factores asociados. Método Estudio descriptivo, transversal, con 522 profesionales de enfermería, realizado en dos hospitales brasileños entre enero de 2017 y marzo de 2018. Datos recolectados mediante formulario incluyendo variables sociodemográficas y profesionales, y Compliance with Standard Precautions Scale (versión Portugués-Brasil); analizados por estadística descriptiva y exploratoria y un modelo de regresión lineal múltiple. Resultados Puntaje medio global de 12,9 (DS=2,5). Los auxiliares de enfermería obtuvieron puntajes promedio mayores, estadísticamente significantes (p<0,01) respecto de los enfermeros. Existió diferencia significante respecto de la categoría profesional (p<0,01) y la escolarización (p<0,01); y marginalmente significante en relación a la edad (p=0,06). No hubo diferencias relativas a la experiencia profesional (p=0,77), participación en capacitaciones (p=0,79) y tipos de hospital (p=0,13). La escolarización no contribuyó a una mayor observancia de las medidas (p<0,01), al igual que los estudios superiores (p≤0,01). Conclusión e Implicaciones para la práctica Mayores grados de escolarización y experiencia profesional no contribuyeron a la observancia de las Precauciones. En razón de contemplar aspectos de la práctica de enfermería, las estrategias de prevención y exposición profesional merecen ser revisadas y perfeccionadas.

Abstract Objective To verify compliance with the Standard Precautions by nursing professionals and associated factors. Method A descriptive, cross-sectional study was carried out with 522 nursing professionals, in two Brazilian hospitals, between January 2017 and March 2018. Data were collected using a form containing demographic and professional variables and the Compliance with Standard Precautions Scale (Portuguese-Brazilian version); later, analyzed by descriptive and exploratory statistics and a multiple linear regression model. Results The global mean score was 12.9 (SD = 2.5). Nursing professionals had statistically significant higher scores (p <0.01) than nurses. There was a significant difference in terms of professional category (p < 0.01)) and education (p <0.01), and marginally significant in relation to age (p = 0.06). There were no differences regarding professional experience (p = 077), participation in training (p = 0.79), and type of hospital (p = 0.13), respectively. Education did not contribute to greater compliance with the measures (p <0.01), nor did higher education (p ≤ 0.01). Conclusion and implications for practice Increased education and professional experience did not contribute to greater compliance with the Standards Precautions. By considering aspects of nursing practice, occupational exposure prevention strategies can be reviewed and improved.

Dynamics of IgM and IgG responses to the next generation of engineered Duffy binding protein II immunogen: Strain-specific and strain-transcending immune responses over a nine-year period.

BACKGROUND: A low proportion of P. vivax-exposed individuals acquire protective strain-transcending neutralizing IgG antibodies that are able to block the interaction between the Duffy binding protein II (DBPII) and its erythrocyte-specific invasion receptor. In a recent study, a novel surface-engineered DBPII-based vaccine termed DEKnull-2, whose antibody response target conserved DBPII epitopes, was able to induce broadly binding-inhibitory IgG antibodies (BIAbs) that inhibit P. vivax reticulocyte invasion. Toward the development of DEKnull-2 as an effective P. vivax blood-stage vaccine, we investigate the relationship between naturally acquired DBPII-specific IgM response and the profile of IgG antibodies/BIAbs activity over time. METHODOLOGY/PRINCIPAL FINDINGS: A nine-year follow-up study was carried-out among long-term P. vivax-exposed Amazonian individuals and included six cross-sectional surveys at periods of high and low malaria transmission. DBPII immune responses associated with either strain-specific (Sal1, natural DBPII variant circulating in the study area) or conserved epitopes (DEKnull-2) were monitored by conventional serology (ELISA-detected IgM and IgG antibodies), with IgG BIAbs activity evaluated by functional assays (in vitro inhibition of DBPII-erythrocyte binding). The results showed a tendency of IgM antibodies toward Sal1-specific response; the profile of Sal1 over DEKnull-2 was not associated with acute malaria and sustained throughout the observation period. The low malaria incidence in two consecutive years allowed us to demonstrate that variant-specific IgG (but not IgM) antibodies waned over time, which resulted in IgG skewed to the DEKnull-2 response. A persistent DBPII-specific IgM response was not associated with the presence (or absence) of broadly neutralizing IgG antibody response. CONCLUSIONS/SIGNIFICANCE: The current study demonstrates that long-term exposure to low and unstable levels of P. vivax transmission led to a sustained DBPII-specific IgM response against variant-specific epitopes, while sustained IgG responses are skewed to conserved epitopes. Further studies should investigate on the role of a stable and persistent IgM antibody response in the immune response mediated by DBPII.

Blood-stage Plasmodium vivax antibody dynamics in a low transmission setting: A nine year follow-up study in the Amazon region.

Plasmodium vivax remains a global health problem and its ability to cause relapses and subpatent infections challenge control and elimination strategies. Even in low malaria transmission settings, such as the Amazon basin, where progress in malaria control has caused a remarkable reduction in case incidence, a recent increase in P. vivax transmission demonstrates the continued vulnerability of P.vivax-exposed populations. As part of a search for complementary approaches to P.vivax surveillance in areas in which adults are the majority of the exposed-population, here we evaluated the potential of serological markers covering a wide range of immunogenicity to estimate malaria transmission trends. For this, antibodies against leading P. vivax blood-stage vaccine candidates were assessed during a 9 year follow-up study among adults exposed to unstable malaria transmission in the Amazon rainforest. Circulating antibody levels against immunogenic P. vivax proteins, such as the Apical Membrane Antigen-1, were a sensitive measure of recent P. vivax exposure, while antibodies against less immunogenic proteins were indicative of naturally-acquired immunity, including the novel engineered Duffy binding protein II immunogen (DEKnull-2). Our results suggest that the robustness of serology to estimate trends in P.vivax malaria transmission will depend on the immunological background of the study population, and that for adult populations exposed to unstable P.vivax malaria transmission, the local heterogeneity of antibody responses should be considered when considering use of serological surveillance.

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.

Susceptibility to Plasmodium vivax malaria associated with DARC (Duffy antigen) polymorphisms is influenced by the time of exposure to malaria.

Malaria has provided a major selective pressure and has modulated the genetic diversity of the human genome. The variants of the Duffy Antigen/Receptor for Chemokines (DARC) gene have probably been selected by malaria parasites, particularly the FY*O allele, which is fixed in sub-Saharan Africa and confers resistance to Plasmodium vivax infection. Here, we showed the influence of genomic ancestry on the distribution of DARC genotypes in a highly admixed Brazilian population and confirmed the decreased susceptibility of the FY*A/FY*O genotype to clinical P. vivax malaria. FY*B/FY*O individuals were associated with a greater risk of developing clinical malaria. A remarkable difference among DARC variants concerning the susceptibility to clinical malaria was more evident for individuals who were less exposed to malaria, as measured by the time of residence in the endemic area. Additionally, we found that DARC-negative and FY*A/FY*O individuals had a greater chance of acquiring high levels of antibodies against the 19-kDa C-terminal region of the P. vivax merozoite surface protein-1. Altogether, our results provide evidence that DARC polymorphisms modulate the susceptibility to clinical P. vivax malaria and influence the naturally-acquired humoral immune response to malaria blood antigens, which may interfere with the efficacy of a future vaccine against malaria.

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.

Xenobiotic Nucleic Acid (XNA) Synthesis by Phi29 DNA Polymerase.

Phi29 DNA polymerase (DNAP) is the replicative enzyme of the Bacillus subtilis bacteriophage Phi29. Its extraordinary processivity and its ability to perform isothermal amplification of DNA are central to many molecular biology applications, including high-sensitivity detection and large-scale production of DNA. We present here Phi29 DNAP as an efficient catalyst for the production of various artificial nucleic acids (XNAs) carrying backbone modifications such as 1,5-anhydrohexitol nucleic acid (HNA), 2'-deoxy-2'-fluoro-arabinonucleic acid (FANA), and 2'-fluoro-2'-deoxyribonucleic acid (2'-fluoro-DNA). A full protocol for the synthesis of HNA polymers by an exonuclease-deficient variant (D12A) of Phi29 DNAP plus a detailed guide for the design and test of novel XNA synthetase reactions performed by Phi29 DNAP are provided. © 2018 by John Wiley & Sons, Inc.

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.

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.

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.

Quality evaluation of sperm from livebearing fishes: Standardized assessment of sperm bundles (spermatozeugmata) from Xenotoca eiseni (Goodeidae).

Standardized evaluation of sperm quality is essential for research, commercial-scale cryopreservation, and induced spawning. However, standardized methods for evaluation of sperm bundles (spermatozeugmata or spermatophores) have not been established. The purpose of the present study was to use Redtail Splitfin (Xenotoca eiseni) as a model for freshwater livebearing fishes to establish initial standardized methods to collect sperm bundles, and quantitatively and qualitatively evaluate quality-related attributes. No sperm or sperm bundles were able to be collected by stripping. Testes were removed, rinsed, weighed, placed in 50 µL of buffer solution on a glass slide, and crushed gently 3-5 times with angled spade-tip forceps. Sperm bundles were released into the buffer solution and collected with a pipette into 1.5-mL centrifuge tubes. To quantify size and shape, images of bundles were captured with a CCD camera connected to a microscope, and measured with computer software. There was no significant correlation between body wet weight and major bundle axis length (P = 0.6759), minor axis length (P = 0.5658), average axis length (P = 0.5869), aspect ratio (P = 0.7839), and observed area (P = 0.5727). The concentrations of sperm bundles, estimated with the three methods (Makler® counting chamber, a hemocytometer, and direct counting) were significantly different (P < 0.0001). Hemocytometers were suitable for estimation of bundles from X. eiseni. To evaluate activation of sperm, bundles were viewed with a microscope, and classified into one of five phases by evaluating morphology of the bundles and motion of sperm within the bundles as Phase 0 through Phase 4 that represented early through late activation stages. The frequencies and duration of each activation phase were used to evaluate dissociation of sperm bundles and motility capability of sperm within the bundles. Within 180 min of activation, all five phases were observed. Overall, this study for the first time established standardized methods to collect and evaluate quality-related attributes of sperm bundles. These standardized evaluations provide a basis for further modification, standardization, and generalization, which are useful in research on livebearing fishes involving male gametes, such as studies on cryopreservation, artificial insemination, and in development of germplasm repositories for imperiled species including goodeids.

Challenges in Development of Sperm Repositories for Biomedical Fishes: Quality Control in Small-Bodied Species.

Quality control (QC) is essential for reproducible and efficient functioning of germplasm repositories. However, many biomedical fish models present significant QC challenges due to small body sizes (<5 cm) and miniscule sperm volumes (<5 µL). Using minimal volumes of sperm, we used Zebrafish to evaluate common QC endpoints as surrogates for fertilization success along sequential steps of cryopreservation. First, concentrations of calibration bead suspensions were evaluated with a Makler® counting chamber by using different sample volumes and mixing methods. For sperm analysis, samples were initially diluted at a 1:30 ratio with Hanks' balanced salt solution (HBSS). Motility was evaluated by using different ratios of sperm and activation medium, and membrane integrity was analyzed with flow cytometry at different concentrations. Concentration and sperm motility could be confidently estimated by using volumes as small as 1 µL, whereas membrane integrity required a minimum of 2 µL (at 1 × 106 cells/mL). Thus, <5 µL of sperm suspension (after dilution to 30-150 µL with HBSS) was required to evaluate sperm quality by using three endpoints. Sperm quality assessment using a combination of complementary endpoints enhances QC efforts during cryopreservation, increasing reliability and reproducibility, and reducing waste of time and resources.