Sm29, but Not Sm22.6 Retains its Ability to Induce a Protective Immune Response in Mice Previously Exposed to a Schistosoma mansoni Infection

Background

A vaccine against schistosomiasis would have a great impact in disease elimination. Sm29 and Sm22.6 are two parasite tegument proteins which represent promising antigens to compose a vaccine. These antigens have been associated with resistance to infection and reinfection in individuals living in endemic area for the disease and induced partial protection when evaluated in immunization trials using naïve mice.

Methodology/principals findings

In this study we evaluated rSm29 and rSm22.6 ability to induce protection in Balb/c mice that had been previously infected with S. mansoni and further treated with Praziquantel. Our results demonstrate that three doses of the vaccine containing rSm29 were necessary to elicit significant protection (26%–48%). Immunization of mice with rSm29 induced a significant production of IL-2, IFN-γ, IL-17, IL-4; significant production of specific antibodies; increased percentage of CD4+ central memory cells in comparison with infected and treated saline group and increased percentage of CD4+ effector memory cells in comparison with naïve Balb/c mice immunized with rSm29. On the other hand, although immunization with Sm22.6 induced a robust immune response, it failed to induce protection.

Conclusion/significance

Our results demonstrate that rSm29 retains its ability to induce protection in previously infected animals, reinforcing its potential as a vaccine candidate.     

Changes in satellite‐derived spring vegetation green‐up date and its linkage to climate in China from 1982 to 2010: a multimethod analysis

The change in spring phenology is recognized to exert a major influence on carbon balance dynamics in temperate ecosystems. Over the past several decades, several studies focused on shifts in spring phenology; however, large uncertainties still exist, and one understudied source could be the method implemented in retrieving satellite‐derived spring phenology. To account for this potential uncertainty, we conducted a multimethod investigation to quantify changes in vegetation green‐up date from 1982 to 2010 over temperate China, and to characterize climatic controls on spring phenology. Over temperate China, the five methods estimated that the vegetation green‐up onset date advanced, on average, at a rate of 1.3 ± 0.6 days per decade (ranging from 0.4 to 1.9 days per decade) over the last 29 years. Moreover, the sign of the trends in vegetation green‐up date derived from the five methods were broadly consistent spatially and for different vegetation types, but with large differences in the magnitude of the trend. The large intermethod variance was notably observed in arid and semiarid vegetation types. Our results also showed that change in vegetation green‐up date is more closely correlated with temperature than with precipitation. However, the temperature sensitivity of spring vegetation green‐up date became higher as precipitation increased, implying that precipitation is an important regulator of the response of vegetation spring phenology to change in temperature. This intricate linkage between spring phenology and precipitation must be taken into account in current phenological models which are mostly driven by temperature.     

Drug repositioning for orphan diseases

The need and opportunity to discover therapeutics for rare or orphan diseases are enormous. Due to limited prevalence and/or commercial potential, of the approximately 6000 orphan diseases (defined by the FDA Orphan Drug Act as <200 000 US prevalence), only a small fraction (5%) is of interest to the biopharmaceutical industry. The fact that drug development is complicated, time-consuming and expensive with extremely low success rates only adds to the low rate of therapeutics available for orphan diseases. An alternative and efficient strategy to boost the discovery of orphan disease therapeutics is to find connections between an existing drug product and orphan disease. Drug Repositioning or Drug Repurposing--finding a new indication for a drug--is one way to maximize the potential of a drug. The advantages of this approach are manifold, but rational drug repositioning for orphan diseases is not trivial and poses several formidable challenges--pharmacologically and computationally. Most of the repositioned drugs currently in the market are the result of serendipity. One reason the connection between drug candidates and their potential new applications are not identified in an earlier or more systematic fashion is that the underlying mechanism 'connecting' them is either very intricate and unknown or indirect or dispersed and buried in an ever-increasing sea of information, much of which is emerging only recently and therefore is not well organized. In this study, we will review some of these issues and the current methodologies adopted or proposed to overcome them and translate chemical and biological discoveries into safe and effective orphan disease therapeutics.     

The effect of protein a cycle number on the performance and lifetime of an anion exchange polishing step

Most mAb platform purification processes consist of an affinity capture step followed by one or two polishing steps. An understanding of the performance linkages between the unit operations can lead to robust manufacturing processes. In this study, a weak‐partitioning anion‐exchange chromatography polishing step used in a mAb purification process was characterized through high‐throughput screening (HTS) experiments, small‐scale experiments including a cycling study performed on qualified scale‐down models, and large‐scale manufacturing runs. When material from a Protein A column that had been cycled <10× was loaded on the AEX resin, early breakthrough of impurities and premature loss of capacity was observed. As the cycle number on the Protein A resin increased, the capacity of the subsequent AEX step increased. Different control strategies were considered for preventing impurity breakthrough and improving AEX resin lifetimes. Depth filtration of the Protein A peak pool significantly improved the AEX resin capacity, robustness, and lifetime. Further, the turbidity of the Protein A pool has the potential for use as an in‐process control parameter for monitoring the performance of the AEX step. Biotechnol. Bioeng. 2013; 110: 1142–1152. © 2012 Wiley Periodicals, Inc.     

Immunoanalysis of dehydrins in Araucaria angustifolia embryos

The aim of this study was to describe the dehydrin content of mature Araucaria angustifolia embryos, a species of endangered and economically important conifers, native to southern Brazil, northeastern Argentina, and eastern Paraguay. The A. angustifolia seeds have been categorized as recalcitrant. Dehydrins were studied by western blot analysis and in situ immunolocalization microscopy using antibodies raised against the K segment, a highly conserved lysine-rich 15-amino acid sequence extensively used to recognize proteins immunologically related to the dehydrin family. Western blot analysis of the heat-stable protein fraction, as estimated by 15 % SDS-PAGE, revealed three main bands of approximately 20-, 26-, and 29-kDa; when 17.5 % SDS-PAGE was used, each band resolved into two other bands. Two thermosensitive dehydrin bands of around 16 and 35 kDa were common to the axis and cotyledons, and another thermosensitive band, with molecular mass of approximately 10 kDa, was present in the cotyledons only. Following alkaline phosphatase (AP) treatment, a gel mobility shift was detected for each one of the four main bands that can be due to phosphorylation. Dehydrins were detected in all axis and cotyledon tissues using in situ immunolocalization microscopy. At the subcellular level, dehydrins were immunolocalized in the nuclei, protein bodies, and microbodies. In the nucleus, dehydrins were found to be associated with chromatin. We concluded that the gel mobility shift for the four main bands (probably due to phosphorylation), the presence of thermosensitive bands, and the specific localizations in nuclei and protein bodies provide key starting points to understand the function of dehydrins in the embryo cells of this species.     

Aluminum inhibits root growth and induces hydrogen peroxide accumulation in Plantago algarbiensis and P. almogravensis seedlings

We have evaluated the impact of aluminum (Al) on germination, relative root growth, Al accumulation in roots tips, H₂O₂ levels, plasma membrane integrity, pigment levels, protein content, and the activities of superoxide dismutase (SOD) and catalase (CAT) in seedlings of the endangered Portuguese species Plantago algarbiensis and Plantago almogravensis. We found that up to 400 μM Al had no impact on the germination percentage in either species but inhibited root growth in a concentration-dependent manner (more severely in P. algarbiensis). Al accumulation in the root tips of both species was concentration dependent up to 200 μM but declined thereafter despite the absence of membrane damage. We observed a concentration-dependent induction of SOD activity but no change in CAT activity resulting in the accumulation of H₂O₂ (a known growth inhibitor), although its impact in P. almogravensis may be partially ameliorated by the accumulation of carotenoid pigments. Our data suggest an association between Al uptake, H₂O₂ production, and the inhibition of root growth during early seedling development in P. algarbiensis and P. almogravensis, although the latter is more tolerant towards higher concentrations of the metal.     

Structural and component characterization of meristem cells in Araucaria angustifolia (Bert.) O. Kuntze zygotic embryo

Araucaria angustifolia, the Brazilian pine, is an endangered native conifer with economic and ecological importance. The female cone develops seeds containing the zygotic embryo, which, at cotyledonary stage, shows well-developed meristems. Little is known about the structure of gymnosperm meristems. In the present work, the composition and morphological organization of Araucaria angustifolia shoot and root apical meristems were studied during embryo development, using histochemical and microscope analyses. Histochemical evaluation revealed the presence of cellulose within the cell wall, cells with the presence of total proteins that react with Coomassie Brilliant Blue, starch grains, and large nuclei with evident nucleoli in the cytoplasm. Scanning electron microscopy showed apical meristem surface morphology, and both scanning and transmission microscopy revealed a thin and irregular cell wall with plasmodesmata and within the cells, mitochondria, many vacuoles, lipid bodies, Golgi bodies, and many amyloplasts with endoplasmic reticulum surrounding them and large nuclei. Similar to angiosperm cells, A. angustifolia meristem cells exhibit pluripotent characteristics, such as apparatus for intercellular communication and differentiation.     

Physiological responses of Plantago algarbiensis and P. almogravensis shoots and plantlets to low pH and aluminum stress

We investigated the impact of low pH and aluminum (Al) stress on the growth, nutrients concentration, chlorophyll a fluorescence, photosynthetic pigment contents, proline and carbohydrate accumulation in shoots and plantlets (leaves and roots) of Plantago almogravensis and P. algarbiensis. Both species accumulated considerable and similar amounts of Al in their tissues, mainly in the roots. The presence of Al caused a significant reduction on root elongation in P. algarbiensis. Low pH and Al induced significant changes on nutrient accumulation, but no significant alterations on the maximum efficiency of PSII (F _v/F _m), quantum yield of PSII photochemistry (?_PSII), quantum yield of regulated energy dissipation (?_NPQ) and quantum yield of non-regulated energy dissipation (?_NO) were detected in both species in response to these stresses. However, Al increased significantly the non-photochemical quenching and the chlorophyll b content and decreased the PSII excitation pressure (1 ? q _p) in P. almogravensis leaves. Both stress treatments induced carbohydrate accumulation in the shoots and roots of this species, but not in leaves. In P. algarbiensis, low pH and Al decreased the photosynthetic pigment contents in the shoots, whereas Al stimulated the carbohydrate accumulation in the leaves. Although our data showed that both species are tolerant to Al³⁺ and H⁺, P. almogravensis appeared to be more adapted to maintain cellular physiology and growth under those conditions.     

Antischistosomal activity of a calcium channel antagonist on schistosomula and adult Schistosoma mansoni worms

Current schistosomiasis control strategies are largely based on chemotherapeutic agents and a limited number of drugs are available today. Praziquantel (PZQ) is the only drug currently used in schistosomiasis control programs. Unfortunately, this drug shows poor efficacy in patients during the earliest infection phases. The effects of PZQ appear to operate on the voltage-operated Ca2+channels, which are located on the external Schistosoma mansoni membrane. Because some Ca2+channels have dihydropyridine drug class (a class that includes nifedipine) sensitivity, an in vitro analysis using a calcium channel antagonist (clinically used for cardiovascular hypertension) was performed to determine the antischistosomal effects of nifedipine on schistosomula and adult worm cultures. Nifedipine demonstrated antischistosomal activity against schistosomula and significantly reduced viability at all of the concentrations used alone or in combination with PZQ. In contrast, PZQ did not show significant efficacy when used alone. Adult worms were also affected by nifedipine after a 24 h incubation and exhibited impaired motility, several lesions on the tegument and intense contractility. These data support the idea of Ca2+channels subunits as drug targets and favour alternative therapeutic schemes when drug resistance has been reported. In this paper, strong arguments encouraging drug research are presented, with a focus on exploring schistosomal Ca2+channels.     

Synthesis and antifungal activities of miltefosine analogs

Miltefosine is an alkylphosphocholine that shows broad-spectrum in vitro antifungal activities and limited in vivo efficacy in mouse models of cryptococcosis. To further explore the potential of this class of compounds for the treatment of systemic mycoses, nine analogs (3a?3i) were synthesized by modifying the choline structural moiety and the alkyl chain length of miltefosine. In vitro testing of these compounds against the opportunistic fungal pathogens Candida albicans, Candida glabrata, Candida krusei, Aspergillus fumigatus, and Cryptococcus neoformans revealed that N-benzyl-N,N-dimethyl-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3a), N,N-dimethyl-N-(4-nitrobenzyl)-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3d), and N-(4-methoxybenzyl)-N,N-dimethyl-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3e) exhibited minimum inhibitory concentrations (MIC) of 2.5–5.0 μg/mL against all tested pathogens, when compared to miltefosine with MICs of 2.5–3.3 μg/mL. Compound 3a showed low in vitro cytotoxicity against three mammalian cell lines similar to miltefosine. In vivo testing of 3a and miltefosine against C. albicans in a mouse model of systemic infection did not demonstrate efficacy. The results of this study indicate that further investigation will be required to determine the potential usefulness of the alkylphosphocholines in the treatment of invasive fungal infections.