Genetic diversity and structure of an endangered medicinal plant species (Pilocarpus microphyllus) in eastern Amazon: implications for conservation

Few studies have evaluated the genetic status of medicinal plants exposed to commercial harvesting. Here, we examine the genetic variability of Pilocarpus microphyllus, an endemic and threatened medicinal plant species from the eastern Amazon, across its largest remaining wild population. Popularly known as jaborandi, species of Pilocarpus genus are the unique known natural source of pilocarpine, an alkaloid used to treat glaucoma and xerostomia. However, Populations of P. microphyllus has experienced a severe decline in the last decades. Using RAD sequencing, we identified a total of 5,266 neutral and independent SNPs in 277 individuals collected from the Carajás National Forest (CNF). We quantified genetic diversity and gene flow patterns and estimated the minimum number of individuals necessary to establish a germplasm bank. Our results revealed high genetic diversity and four spatially distinct clusters of P. microphyllus with substantial admixture among them. Geographic distance and temperature dissimilarity were the factors that best explained the relatedness patterns among individuals. Additionally, our findings indicate that at least 40 matrices sampled randomly from each population would be required to conserve genetic diversity in the long term. In short, P. microphyllus showed high levels of genetic diversity and an effective population size (N_E) sufficient to reduce the likelihood of extinction due to inbreeding depression. Our results indicate that diversity has been maintained despite the continuous harvesting of raw leaf material in the area over recent decades. Finally, the results provide information essential for the design of a germplasm bank to protect the endangered medicinal plant species.

     

Analysis of factors affecting the periplasmic production of recombinant proteins in Escherichia coli

Five fusion proteins between Z domains derived from Staphylococcal Protein A and Green Fluorescent Protein or Human Proinsulin were produced on the periplasm of Escherichia coli. The effects of the molecular weight and amino acid composition of the translocated peptide, culture medium composition, and growth phase of the bacterial culture were analyzed regarding the expression and periplasmic secretion of the recombinant proteins. It was found that secretion was not affected by the size of the translocated peptide (17-42 kDa) and that the highest periplasmic production values were obtained on the exponential phase of growth. Moreover, the highest periplasmic values were obtained in minimal medium, showing the relevance of the culture medium composition on secretion. In silico prediction analysis suggested that with respect to the five proteins used in this study, those that are prone to form alpha-helix structures are more translocated to the periplasm.     

On the stability of plasmid DNA vectors during cell culture and purification

Gene therapy and DNA vaccination applications have increased the demand for highly purified plasmid DNA (pDNA) in the last years. One of the main problems related to the scale-up of pDNA purification is the degradation of the supercoiled (sc) isoforms during cell culture and multi-stage purification. In this work, a systematic study of the stability of two model plasmids (3,697 and 6,050 bp) during a mid-scale production process, which includes fermentation, alkaline lysis, isopropanol and ammonium sulphate precipitation and hydrophobic interaction chromatography, was performed. Results indicate that by extending cell culture (up to 26 h) and cell lysis (up to 2 h) it is possible to significantly reduce the amounts of RNA, without significantly compromising the yields of the sc pDNA isoform, a feature that could be conveniently exploited for downstream processing purposes. The stability of pDNA upon storage of E. coli pellets at different temperatures indicates that, differently from RNA, pDNA is remarkably stable when stored in cell pellets (>3 weeks at 4°C, >12 weeks at −20°C) prior to processing. With alkaline lysates, however, storage at −20°C is mandatory to avoid sc pDNA degradation within the first 8 weeks. Furthermore, the subsequent purification steps could be carried out at room temperature without significant pDNA degradation. Since the unit operations and process conditions studied in this work are similar to those generally used for plasmid DNA production, the results presented here may contribute to improve the current knowledge on plasmid stability and process optimization. Authors Freitas and Azzoni contributed equally to this work.     

Reactivation of tuberculosis by tumor necrosis factor neutralization

Tumor necrosis factor (TNF) is required in the control of infection with Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. TNF is essential and non-redundant for forming microbiocidal granulomas, and cannot be replaced by other members of the TNF family. We established a model of latent Mtb infection in mice, allowing investigation of the reactivation of latent Mtb as observed in patients receiving TNF-neutralizing therapy used in rheumatoid arthritis and Crohn's disease. Antibody neutralization of TNF is able to reactivate clinically silent Mtb infection. Using mutant mice expressing solely membrane, but not soluble TNF, we demonstrated that membrane TNF is sufficient to control acute Mtb infection. Therefore, we hypothesize that TNF-neutralizing therapy, sparing membrane TNF, may have an advantage as compared to complete neutralization. In conclusion, endogenous TNF is critical for the control of tuberculosis infection. Genetic absence or pharmacological neutralization of TNF results in uncontrolled infection, while selective neutralization might retain the desired anti-inflammatory effect but reduce the infectious risk.     

The diversity of pathogenesis-related proteins decreases during grape maturation

It was recently shown that wines contain typically a huge diversity of structurally similar polypeptides that exhibit a high degree of homology to pathogenesis-related (PR) proteins. This observation suggested the existence of one or a few precursors in mature grapes, common to most or all the wine PR proteins. Limited proteolysis and chemical modification of the precursor(s) during fruit ripening and winemaking could then generate the large number of distinct wine polypeptides. However, the patterns of PR proteins extracted from grape berries regularly harvested from the onset of development until maturity did not confirm the previous hypothesis. Two different methodologies, involving 2-D immunoblotting and a combination of FPLC cation/anion exchange chromatographies with 1-D immunoblotting, indicate that the total concentration of PR proteins is increased but its diversity is reduced from the early stages of berry development until maturity. These results indicate that PR proteins are synthesized in a wide variety of forms from the early stages of grape development, eliminating the hypothesis previously formulated on the existence of one or few precursors common to the wine proteins.     

Parasitism capacity of Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae) reared under different temperatures on Bonagota salubricola (Meyrick) (Lepidoptera: Tortricidae) eggs

The parasitism capacity of Trichogramma pretiosum Riley strain bonagota on Bonagota salubricola (Meyrick) eggs was studied under the temperatures of 18, 20, 22, 25, 28, 30 and 32 degrees C. The number of days with parasitism, accumulated parasitism, total number of eggs parasitized per female and parasitoid longevity was evaluated. In the first 24h, parasitism ranged from 1.6 (32 degrees C) to 8.8 (22 degrees C) eggs of B. salubricola. Accumulated egg parasitism of B. salubricola reached 80% in 1st to 4th day at 20 degrees C to 32 degrees C, respectively, and in the 7th day at 18 degrees C. Temperatures from 18 degrees C to 22 degrees C were the best suited for the total eggs parasitized for female, resulting in 35.4 and 24.6 eggs/male respectively. T. pretiosum female longevity ranged from 7.8 to 2.5 days, at 18 degrees C and 32 degrees C, respectively. The results showed that T. pretiosum strain bonagota is better adapted to temperatures from 18 degrees C to 22 degrees C.     

Eclosion rate, development and survivorship of Aedes albopictus (Skuse)(Diptera: Culicidae) under different water temperatures

In tropical areas, where vector insects populations are particularly numerous, temperature usually range between 25 degrees C and 35 degrees C. Considering the importance of such temperature variation in determining mosquitoes population dynamics, in this work the developmental, eclosion and survival rates of the immature stages of Aedes albopictus (Skuse) were compared under constant 25, 30 and 35 degrees C (using acclimatized chambers) and environmental (25 degrees C to 29 degrees C) temperatures. The hatching rate was considered as total number of larvae recovered after 24h. The development period as well as larval and pupal survival rate were evaluated daily. Eclosion rate was significantly higher under environmental temperature than under the studied constant temperatures, suggesting that temperature variation may be an eclosion-stimulating factor. The mean eclosion time increased with the temperature, ranging from 2.8h (25 degrees C) to 5.2h (35 degrees C). The larval period was greatly variable inside each group, although it did not differ significantly amongst groups (11.0 +/- 4.19 days), with individuals showing longer larval stages in water at 35 degrees C (12.0 +/- 4.95 days) and environmental temperature (13.6 +/- 5.98 days). Oppositely, survival was strongly affected by the higher temperature, where only one individual lived through to adult phase. The results suggest that population of Ae. albopictus from Recife may be adapting to increasing of environmental temperatures and that the limiting temperature to larval development is around 35 degrees C.     

Bradykinin B2 Receptors of dendritic cells, acting as sensors of kinins proteolytically released by Trypanosoma cruzi, are critical for the development of protective type-1 responses

Although the concept that dendritic cells (DCs) recognize pathogens through the engagement of Toll-like receptors is widely accepted, we recently suggested that immature DCs might sense kinin-releasing strains of Trypanosoma cruzi through the triggering of G-protein-coupled bradykinin B2 receptors (B2R). Here we report that C57BL/6.B2R-/- mice infected intraperitoneally with T. cruzi display higher parasitemia and mortality rates as compared to B2R+/+ mice. qRT-PCR revealed a 5-fold increase in T. cruzi DNA (14 d post-infection [p.i.]) in B2R-/- heart, while spleen parasitism was negligible in both mice strains. Analysis of recall responses (14 d p.i.) showed high and comparable frequencies of IFN-gamma-producing CD4+ and CD8+ T cells in the spleen of B2R-/- and wild-type mice. However, production of IFN-gamma by effector T cells isolated from B2R-/- heart was significantly reduced as compared with wild-type mice. As the infection continued, wild-type mice presented IFN-gamma-producing (CD4+CD44+ and CD8+CD44+) T cells both in the spleen and heart while B2R-/- mice showed negligible frequencies of such activated T cells. Furthermore, the collapse of type-1 immune responses in B2R-/- mice was linked to upregulated secretion of IL-17 and TNF-alpha by antigen-responsive CD4+ T cells. In vitro analysis of tissue culture trypomastigote interaction with splenic CD11c+ DCs indicated that DC maturation (IL-12, CD40, and CD86) is controlled by the kinin/B2R pathway. Further, systemic injection of trypomastigotes induced IL-12 production by CD11c+ DCs isolated from B2R+/+ spleen, but not by DCs from B2R-/- mice. Notably, adoptive transfer of B2R+/+ CD11c+ DCs (intravenously) into B2R-/- mice rendered them resistant to acute challenge, rescued development of type-1 immunity, and repressed TH17 responses. Collectively, our results demonstrate that activation of B2R, a DC sensor of endogenous maturation signals, is critically required for development of acquired resistance to T. cruzi infection.     

Development of native forest species of the Atlantic forest in soil contaminated with hormonal herbicides

Abstract

Hormonal herbicides, used in pastures, can suffer drift and reach forests. The sensitivity and potential phytoremediation of native species to herbicide residues should be evaluated. The objective of this study is to evaluate the initial development of native Atlantic Forest tree species in soil contaminated with hormonal herbicides. The experiment was conducted in a greenhouse in a 4 x 8 factorial scheme. The first factor had the control and the herbicide Tordon^® in three doses (0.166, 0.333 and 0.666?L ha^?1) and the second consisted of the forest species Anadenanthera colubrina (Vell.), Cassia ferruginea (Schrad.) Schrad. ex DC., Dalbergia villosa (Benth.) Benth., Machaerium nyctitans (Vell.) Benth., Machaerium opacum Vogel, Piptadenia gonoacantha (Mart.) JF Macbr., Senegalia polyphylla (DC.) Britton and Rose, Senna macranthera (DC Collad.) HS Irwin and Barnaby. The emergence, height, survival, emergence speed index, intoxication, root volume, stem diameter, root and shoot dry mass, leaf area and leaf numbers of the forest species were evaluated. The A. colubrina, D. villosa and M. opacum initial development was reduced by the herbicides 2.4-D plus picloram residues. S. macranthera and P. gonoacantha are tolerant to this mixture and, therefore, show potential for phytoremediation of degraded areas containing residues of these compounds.     

Effects of the herbicide trifluralin in the initial development of Piptadenia gonoacantha (Fabales: Fabaceae)

Abstract

Trifluralin, a pre-emergent herbicide, is widely used in Brazil in the weed grass management in restoration areas. The objective was to evaluate the tolerance of Piptadenia gonoacantha to trifluralin. The treatments had three trifluralin doses (445, 890, and 1335?g a.i. ha^?1), applied in pre-sowing, as well as the control, without herbicide. Visual intoxication, seed germination, survival rate, emergence speed index (EMI), mean germination period, seedling height, and diameter, micromorphometric parameters of plant roots collected at 60 d after sowing, root length (RL) and volume, leaf area (LA), leaf numbers, root and shoot dry matter, and fluorescence of chlorophyll a at 30, 45, and 60 d after sowing were analyzed. Visual intoxication values above 50% were observed only with 1335?g a.i. ha^?1. The herbicide did not affect seed germination, EMI, average germination period, seedling height, and diameter, root micromorphometric parameters, length, dry matter or root volume, and chlorophyll a fluorescence. The dose 1335?g a.i. ha^?1 caused a reduction of 41.5% in survival, 50.3% in the LA, 36.7% in the number of leaves (LN), and 59.8% in the aerial dry mass of seedlings. The trifluralin presents potential for restoration programs of degraded areas with this forest species.