In Situ μGISAXS: I. Experimental Setup for Submicron Study of Protein Nucleation and Growth

In this study, we used microbeam grazing-incidence small-angle x-ray scattering (μGISAXS) to investigate in situ protein nucleation and crystal growth assisted by a protein nanotemplate, and introduced certain innovations to improve the method. Our aim was to understand the protein nanotemplate method in detail, as this method has been shown to be capable of accelerating and increasing crystal size and quality, as well as inducing crystallization of proteins that are not crystallizable by classical methods. The nanotemplate experimental setup was used for drops containing growing protein crystals at different stages of nucleation and growth. Two model proteins, lysozyme and thaumatin, were used under unique flow conditions to differentially probe protein crystal nucleation and growth.     

Oak seedling survival and growth along resource gradients in Mediterranean forests: implications for regeneration in current and future environmental scenarios

Understanding seedling performance across resource gradients is crucial for defining the regeneration niche of plant species under current environmental conditions and for predicting potential changes under a global change scenario. A 2‐year field experiment was conducted to determine how seedling survival and growth of two evergreen and two deciduous Quercus species vary along gradients of light and soil properties in two Mediterranean forests with contrasting soils and climatic conditions. Half the seedlings were subjected to an irrigation treatment during the first year to quantify the effects on performance of an alteration in the summer drought intensity. Linear and non‐linear models were parameterized and compared to identify major resources controlling seedling performance. We found both site‐specific and general patterns of regeneration. Strong site‐specificity was found in the identity of the best predictors of seedling survival: survival decreased linearly with increasing light (i.e. increasing desiccation risk) in the drier site, whereas it decreased logistically with increasing spring soil water content (i.e. increasing waterlogging risk) in the wetter site. We found strong empirical support for multiple resource limitation at the drier site, the response to light being modulated by the availability of soil resources (water and P). Evidence for regeneration niche partitioning among Quercus species was only found at the wetter site. However, at both sites Quercus species shared the same response to summer drought alleviation through water addition: increased first‐year survival but not final survival (i.e. after two years). This suggests that extremely dry summers (i.e. the second summer in the experiment) can cancel out the positive effects of previous wetter summers. Therefore, an increase in the intensity and frequency of summer drought with climate change might cause a double negative impact on Quercus regeneration, due to a general reduction in survival probability and the annulment of the positive effects of (infrequent) ‘wet’ years. Overall, results presented in this study are a major step towards the development of a mechanistic model of Mediterranean forest dynamics that incorporates the idiosyncrasies and generalities of tree regeneration in these systems, and that allow simulation and prediction of the ecological consequences of resource level alterations due to global change.     

Phosphatidylethanolamine synthesis is required for optimal virulence of Brucella abortus

The Brucella cell envelope contains the zwitterionic phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Synthesis of PC occurs exclusively via the PC synthase pathway, implying that the pathogen depends on the choline synthesized by the host cell to form PC. Notably, PC is necessary to sustain a chronic infection process, which suggests that the membrane lipid content is relevant for Brucella virulence. In this study we investigated the first step of PE biosynthesis in B. abortus, which is catalyzed by phosphatidylserine synthase (PssA). Disruption of pssA abrogated the synthesis of PE without affecting the growth in rich complex medium. In minimal medium, however, the mutant required choline supplementation for growth, suggesting that at least PE or PC is necessary for Brucella viability. The absence of PE altered cell surface properties, but most importantly, it impaired several virulence traits of B. abortus, such as intracellular survival in both macrophages and HeLa cells, the maturation of the replicative Brucella-containing vacuole, and mouse colonization. These results suggest that membrane phospholipid composition is critical for the interaction of B. abortus with the host cell.     

Size-Related Differential Seed Predation in a Heavily Defaunated Neotropical Rain Forest

Hunting and habitat loss represent an increasingly common anthropogenic impact. Available evidence suggests that defaunation is typically differential: medium/large mammals are most affected while small rodents are less affected, or even favored. In heavily impacted areas, such as Los Tuxtlas, the residual mammalian fauna is dominated by small rodents. We tested the expectation that if small rodents will preferentially attack small-seeded species, large-seeded species may escape predation in the absence of medium/large seed predators and that this may affect germination and, possibly, recruitment. Experiments with caged rodents ( Heteromys desmarestianus ) under laboratory conditions showed a preference for smaller seeds. A field experiment involving seeds of contrasting size showed that small, unprotected seeds experienced a predation risk 30-times larger than protected seeds, while the effect of protection was not significant for large seeds. These patterns of predation led to significant differences in germination: protected small-seeded species had a fourfold greater germination than unprotected small-seeded species, while germination was not significantly different between exposed and protected large seeds. The observed contrasts in germination suggest that under heavy defaunation, small-seeded species are likely to be penalized by the overabundance of small rodents, while large-seeded species escape predation. Our results are consistent with the observation of dense seedling carpets dominated by large-seeded species in the understory of Los Tuxtlas. We speculate that the patterns we uncovered may explain, at least partly, the impoverished diversity of the understory vegetation that characterizes heavily defaunated understories and that this has the potential to affect forest regeneration.     

Anti-VSG antibodies induce an increase in Trypanosoma evansi intracellular Ca2+ concentration

Trypanosoma evansi and Trypanosoma vivax have shown a very high immunological cross-reactivity. Anti-T. vivax antibodies were used to monitor changes in the T. evansi intracellular Ca2+ concentration ([Ca2+]i) by fluorometric ratio imaging from single parasites. A short-time exposure of T. evansi parasites to sera from T. vivax-infected bovines induced an increase in [Ca2+]i, which generated their complete lysis. The parasite [Ca2+]i boost was reduced but not eliminated in the absence of extracellular Ca2+ or following serum decomplementation. Decomplemented anti-T. evansi VSG antibodies also produced an increase in the parasite [Ca2+]i, in the presence of extracellular Ca2+. Furthermore, this Ca2+ signal was reduced following blockage with Ni2+ or in the absence of extracellular Ca2+, suggesting that this response was a combination of an influx of Ca2+ throughout membrane channels and a release of this ion from intracellular stores. The observed Ca2+ signal was specific since (i) it was completely eliminated following pre-incubation of the anti-VSG antibodies with the purified soluble VSG, and (ii) affinity-purified anti-VSG antibodies also generated an increase in [Ca2+]i by measurements on single cells or parasite populations. We also showed that an increase of the T. evansi [Ca2+]i by the calcium A-23187 ionophore led to VSG release from the parasite surface. In addition, in vivo immunofluorescence labelling revealed that anti-VSG antibodies induced the formation of raft patches of VSG on the parasite surface. This is the first study to identify a ligand that is coupled to calcium flux in salivarian trypanosomes.     

N2 fixation and nitrogen allocation to above and below ground plant parts in red clover-grasslands

Leys, used for grazing or production of forage to be conserved as silage or hay, are very important crops in northern areas. In order to measure the N₂ fixation in leys of varying ages and during different parts of the season, detailed measurements were taken of yield, N₂ fixation and the amounts of N remaining in the field after harvesting red clover (Trifolium pratense L.)-grass leys at a site in northern Sweden, where they are generally harvested twice per growing season. Entire plants, including stubble and roots, were sampled at the time of first and second harvest and, in addition, at the end of the growing season in three neighbouring fields, carrying a first, a second and a third year ley, respectively. N₂ fixation was measured by both ¹⁵N isotope dilution (ID) and ¹⁵N natural abundance (NA) methods. The proportion of clover dry matter (DM) in the stands increased from the first to the second harvest, but the grasses dominated throughout the entire season, especially below ground. The N concentrations, in both herbage and whole plants, were about twice as high in the clover as in the grasses. Seasonal variations in N concentrations were minor, and total N contents followed the same trends as DM. The clover acquired nearly all of its N from N₂ fixation: the proportion of N in clover herbage derived from N₂ fixation was often >0.8 throughout the season. The variations in the amounts of N₂ fixed during the course of the season corresponded well to the seasonal changes in clover biomass. Amounts of fixed N₂ allocated to clover herbage during the whole season were in the range 4 to 6 g N m⁻² in this unusually rainy year. Calculations of daily N allocation rates to herbage showed that N uptake rates were similar, and high, in grasses during May–June and July–August, while N₂ fixation rates in clover were about 10-fold as high in July–August as in May–June, reflecting the need for N in clover growth. The proportion of N remaining in clover stubble and roots after the first and second harvests was about 60 and 25%, respectively, while about 60% of the N in grasses remained in stubble and roots after both harvests. The considerable amounts of biomass and N that were left in field after harvesting red clover-grass leys are important for re-growth of the plants and provide substantial N fertilization for the next crop in the crop rotation.     

IL-10 Inhibits the NF-κB and ERK/MAPK-Mediated Production of Pro-Inflammatory Mediators by Up-Regulation of SOCS-3 in Trypanosoma cruzi-Infected Cardiomyocytes

Trypanosoma cruzi (T. cruzi) infection produces an intense inflammatory response which is critical for the control of the evolution of Chagas’ disease. Interleukin (IL)-10 is one of the most important anti-inflammatory cytokines identified as modulator of the inflammatory reaction. This work shows that exogenous addition of IL-10 inhibited ERK1/2 and NF-κB activation and reduced inducible nitric oxide synthase (NOS2), metalloprotease (MMP) -9 and MMP-2 expression and activities, as well as tumour necrosis factor (TNF)-α and interleukin (IL)-6 expression, in T. cruzi-infected cardiomyocytes. We found that T. cruzi and IL-10 promote STAT3 phosphorylation and up-regulate the expression of suppressor of cytokine signalling (SOCS)-3 thereby preventing NF-κB nuclear translocation and ERK1/2 phosphorylation. Specific knockdown of SOCS-3 by small interfering RNA (siRNA) impeded the IL-10-mediated inhibition of NF-κB and ERK1/2 activation. As a result, the levels of studied pro-inflammatory mediators were restored in infected cardiomyocytes. Our study reports the first evidence that T. cruzi up- regulates SOCS-3 expression and highlights the relevance of IL-10 in the modulation of pro-inflammatory response of cardiomyocytes in Chagas’ disease.     

Arrestin-3 Binds c-Jun N-terminal Kinase 1 (JNK1) and JNK2 and Facilitates the Activation of These Ubiquitous JNK Isoforms in Cells via Scaffolding

Non-visual arrestins scaffold mitogen-activated protein kinase (MAPK) cascades. The c-Jun N-terminal kinases (JNKs) are members of MAPK family. Arrestin-3 has been shown to enhance the activation of JNK3, which is expressed mainly in neurons, heart, and testes, in contrast to ubiquitous JNK1 and JNK2. Although all JNKs are activated by MKK4 and MKK7, both of which bind arrestin-3, the ability of arrestin-3 to facilitate the activation of JNK1 and JNK2 has never been reported. Using purified proteins we found that arrestin-3 directly binds JNK1α1 and JNK2α2, interacting with the latter comparably to JNK3α2. Phosphorylation of purified JNK1α1 and JNK2α2 by MKK4 or MKK7 is increased by arrestin-3. Endogenous arrestin-3 interacted with endogenous JNK1/2 in different cell types. Arrestin-3 also enhanced phosphorylation of endogenous JNK1/2 in intact cells upon expression of upstream kinases ASK1, MKK4, or MKK7. We observed a biphasic effect of arrestin-3 concentrations on phosphorylation of JNK1α1 and JNK2α2 both in vitro and in vivo. Thus, arrestin-3 acts as a scaffold, facilitating JNK1α1 and JNK2α2 phosphorylation by MKK4 and MKK7 via bringing JNKs and their activators together. The data suggest that arrestin-3 modulates the activity of ubiquitous JNK1 and JNK2 in non-neuronal cells, impacting the signaling pathway that regulates their proliferation and survival.     

Serogroups, K1 antigen, and antimicrobial resistance patterns of Aeromonas spp. strains isolated from different sources in Mexico

A total of 221 strains of Aeromonas species isolated in Mexico from clinical (161), environmental (40), and food (20) samples were identified using the automated system bioMérieux-Vitek. Antisera for serogroups O1 to 044 were tested using the Shimada and Sakazaki scheme. The K1 antigen was examined using as antiserum the O7:K1C of Escherichia coli. Besides, we studied the antimicrobial patterns according to Vitek AutoMicrobic system. Among the 161 clinical strains 60% were identified as A. hydrophila, 20.4% as A. caviae, and 19.25% as A. veronii biovar sobria. Only A. hydrophila and A. veronii biovar sobria were found in food (55 and 90% respectively) and environmental sources (45 and 10% respectively). Using "O" antisera, only 42.5% (94/221) of the strains were serologically identified, 55% (121/221) were non-typable, and 2.5% (6/221) were rough strains. Twenty-two different serogroups were found, O14, O16, O19, O22, and O34 represented 60% of the serotyped strains. More than 50% of Aeromonas strain examined (112/221) expressed K1 encapsulating antigen; this characteristic was predominant among Aeromonas strains of clinical origin. Resistance to ampicillin/sulbactam and cephazolin was detected in 100 and 67% of Aeromonas strain tested for their susceptibility to antibiotics. In conclusion, antibiotic-resistant Aeromonas species that possess the K1 encapsulating antigen and represent serogroups associated with clinical syndrome in man are not uncommon among Aeromonas strains isolated from clinical, food and environmental sources in Mexico.