Recombinant renewable polyclonal antibodies

Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.     

Comparison of different delivery systems of DNA vaccination for the induction of protection against tuberculosis in mice and guinea pigs

The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly (DL-lactide-co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in mice as well as in guinea pigs by comparison with the efficacy and toxicity induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in both mice and guinea pigs.     

Socioeconomic disparities associated with symptomatic Zika virus infections in pregnancy and congenital microcephaly: A spatiotemporal analysis from Goiânia,Brazil (2016 to 2020)

The Zika virus (ZIKV) epidemic, which was followed by an unprecedented outbreak of congenital microcephaly, emerged in Brazil unevenly, with apparent pockets of susceptibility. The present study aimed to detect high-risk areas for ZIKV infection and microcephaly in Goiania, a large city of 1.5 million inhabitants in Central-West Brazil. Using geocoded surveillance data from the Brazilian Information System for Notifiable Diseases (SINAN) and from the Public Health Event Registry (RESP-microcefalia), we analyzed the spatiotemporal distribution and socioeconomic indicators of laboratory confirmed (RT-PCR and/or anti-ZIKV IgM ELISA) symptomatic ZIKV infections among pregnant women and clinically confirmed microcephaly in neonates, from 2016 to 2020. We investigated temporal patterns by estimating the risk of symptomatic maternal ZIKV infections and microcephaly per 1000 live births per month. We examined the spatial distribution of maternal ZIKV infections and microcephaly cases across the 63 subdistricts of Goiania by manually plotting the geographical coordinates. We used spatial scan statistics estimated by discrete Poisson models to detect high clusters of maternal ZIKV infection and microcephaly and compared the distributions by socioeconomic indicators measured at the subdistrict level. In total, 382 lab-confirmed cases of maternal ZIKV infections, and 31 cases of microcephaly were registered in the city of Goiania. More than 90% of maternal cases were reported between 2016 and 2017. The highest incidence of ZIKV cases among pregnant women occurred between February and April 2016. A similar pattern was observed in the following year, although with a lower number of cases, indicating seasonality for ZIKV infection, during the local rainy season. Most congenital microcephaly cases occurred with a time-lag of 6 to 7 months after the peak of maternal ZIKV infection. The highest estimated incidence of maternal ZIKV infections and microcephaly were 39.3 and 2.5 cases per 1000 livebirths, respectively. Districts with better socioeconomic indicators and with higher proportions of self-identified white inhabitants were associated with lower risks of maternal ZIKV infection. Overall, the findings indicate heterogeneity in the spatiotemporal patterns of maternal ZIKV infections and microcephaly, which were correlated with seasonality and included a high-risk geographic cluster. Our findings identified geographically and socio-economically underprivileged groups that would benefit from targeted interventions to reduce exposure to vector-borne infections.     

The antibody mining toolbox: An open source tool for the rapid analysis of antibody repertoires

In vitro selection has been an essential tool in the development of recombinant antibodies against various antigen targets. Deep sequencing has recently been gaining ground as an alternative and valuable method to analyze such antibody selections. The analysis provides a novel and extremely detailed view of selected antibody populations, and allows the identification of specific antibodies using only sequencing data, potentially eliminating the need for expensive and laborious low-throughput screening methods such as enzyme-linked immunosorbant assay. The high cost and the need for bioinformatics experts and powerful computer clusters, however, have limited the general use of deep sequencing in antibody selections. Here, we describe the AbMining ToolBox, an open source software package for the straightforward analysis of antibody libraries sequenced by the three main next generation sequencing platforms (454, Ion Torrent, MiSeq). The ToolBox is able to identify heavy chain CDR3s as effectively as more computationally intense software, and can be easily adapted to analyze other portions of antibody variable genes, as well as the selection outputs of libraries based on different scaffolds. The software runs on all common operating systems (Microsoft Windows, Mac OS X, Linux), on standard personal computers, and sequence analysis of 1–2 million reads can be accomplished in 10–15 min, a fraction of the time of competing software. Use of the ToolBox will allow the average researcher to incorporate deep sequence analysis into routine selections from antibody display libraries.     

Biotic and Abiotic Factors Affecting Brevicoryne brassicae (L.) (Hemiptera: Aphididae) and the Associated Hyperparasitoid Alloxysta fuscicornis Hartig (Hymenoptera: Figitidae) Morphologies

This study investigates the influence of biotic and abiotic factors associated with the morphological development of Brevicoryne brassicae (Linnaeus) and the associated hyperparasitoid Alloxysta fuscicornis (Hartig). The experiment involved the examination of whether aphid size was influenced by their vertical distribution and density on the host plant, as well as whether variations in hyperparasitoid size and symmetry were correlated with those of their aphid hosts. An aphid multivariate size index was obtained using principal component analysis, while symmetry was evaluated in terms of fluctuating asymmetry (FA). Samples were collected in 2007 on cabbage plants cultivated at an experimental farm located in Uberlandia, Minas Gerais, Brazil (18°56??54???S; 48°12??46???W). The results demonstrated that the size of B. brassicae was negatively associated with temperature, but not with its vertical distribution on the host plant. Temperature was also negatively correlated with hyperparasitoid size. During warmer periods, females produced large quantities of small-sized offspring, whereas an opposite pattern, i.e. the production of fewer offspring of larger size took place during colder periods. This type of adjustment involving trade-offs between physiological and morphological mechanisms, as well as individual interaction with abiotic environmental factors, such as temperature, can be considered an adaptive plastic response in order to increase the chances of survival at a given locality. The encountered relationship between aphid and hyperparasitoid sizes may be an after effect of their indirectly biotic interaction. Hyperparasitoid FA was dependent on the width of the mummified aphids. However, the hypothesis that temperature and vertical distribution on the host plant might influence FA was not confirmed.     

Relationships between four measures of genetic distance and breeding behavior in spring wheat

We estimated the genetic distances among 10 spring wheat genotypes based on pedigree data, morphological traits and AFLP markers, used individually and combined with morphological traits, to find the best predictors of general- and specific-combining abilities among parental genotypes. Ten wheat parents were crossed in a diallel form, disregarding reciprocal hybrids, totaling 45 combinations. The F(1) hybrids, F(2) populations and parents were evaluated in the field in 2007. The experimental plots consisted of 20 plants for F(1) hybrids and 40 plants for parental and F(2) populations. All methods (pedigree data, AFLP markers and morphological traits, used individually and combined) were found to be useful for the assessment of genetic diversity. The significant coefficient correlations ranged from low (0.45) to moderate (0.67) between the distance measures and hybrid performance. There was significant agreement between the distance measures based on AFLP markers vs morphological traits + AFLP markers (r = 0.47) and between pedigree data vs morphological traits + AFLP markers (r = 0.43). The pedigree distance was positively associated with traits 100-kernel weight and grain yield per plant in F(1) (correlations of 0.67 and 0.62, respectively) and F(2) (correlations of 0.62 and 0.59, respectively) generations. These correlation values indicate that the genetic distance, based on pedigree data, could replace diallel crosses for the selection of parents with higher combining ability and with moderate reliability.     

Non‐timber Forest Product Harvest does not Affect the Genetic Diversity of a Tropical Tree Despite Negative Effects on Population Fitness

The level of genetic diversity in a population can affect ecological processes and plant responses to disturbance. In turn, disturbance can alter population genetic diversity and structure. Populations in fragmented and logged habitats often show reduced genetic diversity and increased inbreeding and differentiation. Long‐term harvesting of wild plants (for foliage, bark, and roots), can affect population genetic diversity by altering individual fitness and genetic contribution. Our understanding of these changes in genetic diversity due to the harvesting of plant organs is still limited. We used nine microsatellite markers to study the effect of long‐term bark and foliage harvest by Fulani people on the genetic diversity and structure of 12 populations of African mahogany (Khaya senegalensis) in Benin. We sampled 20 individuals in each population to test the effect of harvesting. For each population, we divided the samples equally between seedling and adults to test if the effects are stronger in seedlings. We found moderate genetic diversity (H_e = 0.53 ± 0.04) and weak but significant differentiation among local populations (F_ST = 0.043, P < 0.001). There was no significant effect of harvest on genetic diversity or structure, although previous work found significant negative effects of harvest on the reproduction of adults, offspring density, and population fitness. Our results suggest that demographic responses to disturbance precede a detectable genetic response. Future studies should focus on using parentage analysis to test if genotypes of harvested parents are directly represented in the offspring populations.     

Do the antiherbivore traits of expanding leaves in the Neotropical tree Inga paraensis (Fabaceae) vary with light availability?

Treefall gaps in tropical forests have a profound effect on plants growing in the understory, primarily due to increased light availability. In higher light, mature leaves typically have increased anti-herbivore defenses. However, since the majority of herbivory occurs while leaves are expanding, it is important to determine whether defense expression during the short period of leaf expansion is canalized (invariant) or plastic in response to variation in light. Therefore, we examined young leaves of Inga paraensis (Fabaceae) saplings growing along a light gradient in a terra-firme forest in Central Amazonia. We quantified leaf production and expansion time, dry mass of phenolics, saponins, and nitrogen, ants attracted to extrafloral nectaries, and leaf consumption. Over the entire light gradient, the number of leaves produced per flush increased by 50?% and the mass of phenolic compounds by 20?%, but no other traits changed. On average, 39?% of leaf area was consumed with no difference across the light gradient. Alone, none of the leaf traits was a significant predictor of leaf consumption, except for phenolics, which showed a positive relationship. Multiple regressions showed that leaf consumption was positively related to more leaves per flush and a higher concentration of phenolics in leaves. Unlike studies of mature leaves, young leaves of I. paraensis show low plasticity in defense traits across a light gradient, suggesting that leaf development is canalized.     

Clade‐specific consequences of climate change to amphibians in Atlantic Forest protected areas

The rapid global decline of amphibian population is alarming because many occur for apparently unknown or enigmatic reasons, even inside protected areas (PAs). Some studies have predicted the effects of climate change on amphibians’ distribution and extinction, but the relationship and consequences of climate change to the phylogenetic structure of amphibian assemblages remain obscure. By applying robust techniques for ecological niche modeling and a cutting‐edge approach on community phylogenetics, here, we evaluate how climate change affects the geographical pattern of amphibian species richness and phylogenetic diversity in the Atlantic Forest Biodiversity Hotspot, Brazil, as well as how the phylogenetic composition of amphibian assemblages respond to climate change. We found that most species contracted their ranges and that such responses are clade specific. Basal amphibian clades (e.g. Gymnophiona and Pipidae) were positively affected by climate change, whereas late‐divergent clades (e.g. Cycloramphidae, Centrolenidae, Eleutherodactylidae, Microhylidae) were severely impacted. Identifying major changes in the phylogenetic pool represents a first step towards a better understanding of how assembly processes related to climate change will affect ecological communities. A deep analysis of the impacts of climate change not only on species, but also on the evolutionary relationships among species might foster the discussion on clade‐level conservation priorities for this imperiled fauna.