LuTHy: a double‐readout bioluminescence‐based two‐hybrid technology for quantitative mapping of protein–protein interactions in mammalian cells

Information on protein–protein interactions (PPIs) is of critical importance for studying complex biological systems and developing therapeutic strategies. Here, we present a double‐readout bioluminescence‐based two‐hybrid technology, termed LuTHy, which provides two quantitative scores in one experimental procedure when testing binary interactions. PPIs are first monitored in cells by quantification of bioluminescence resonance energy transfer (BRET) and, following cell lysis, are again quantitatively assessed by luminescence‐based co‐precipitation (LuC). The double‐readout procedure detects interactions with higher sensitivity than traditional single‐readout methods and is broadly applicable, for example, for detecting the effects of small molecules or disease‐causing mutations on PPIs. Applying LuTHy in a focused screen, we identified 42 interactions for the presynaptic chaperone CSPα, causative to adult‐onset neuronal ceroid lipofuscinosis (ANCL), a progressive neurodegenerative disease. Nearly 50% of PPIs were found to be affected when studying the effect of the disease‐causing missense mutations L115R and ?L116 in CSPα with LuTHy. Our study presents a robust, sensitive research tool with high utility for investigating the molecular mechanisms by which disease‐associated mutations impair protein activity in biological systems.     

Fast protocol for the production of Histoplasma capsulatum antigens for antibody detection in the immunodiagnosis of histoplasmosis

Background

Current methods for the production of Histoplasma capsulatum antigens are problematic in terms of standardization, specificity, stability, repeatability and reproducibility.

ArfGAP1 restricts Mycobacterium tuberculosis entry by controlling the actin cytoskeleton

The interaction of Mycobacterium tuberculosis (Mtb) with pulmonary epithelial cells is critical for early stages of bacillus colonization and during the progression of tuberculosis. Entry of Mtb into epithelial cells has been shown to depend on F‐actin polymerization, though the molecular mechanisms are still unclear. Here, we demonstrate that mycobacterial uptake into epithelial cells requires rearrangements of the actin cytoskeleton, which are regulated by ADP‐ribosylation factor 1 (Arf1) and phospholipase D1 (PLD1), and is dependent on the M3 muscarinic receptor (M₃R). We show that this pathway is controlled by Arf GTPase‐activating protein 1 (ArfGAP1), as its silencing has an impact on actin cytoskeleton reorganization leading to uncontrolled uptake and replication of Mtb. Furthermore, we provide evidence that this pathway is critical for mycobacterial entry, while the cellular infection with other pathogens, such as Shigella flexneri and Yersinia pseudotuberculosis, is not affected. Altogether, these results reveal how cortical actin plays the role of a barrier to prevent mycobacterial entry into epithelial cells and indicate a novel role for ArfGAP1 as a restriction factor of host–pathogen interactions.     

Contribution of crop model structure,parameters and climate projections to uncertainty in climate change impact assessments

Climate change impact assessments are plagued with uncertainties from many sources, such as climate projections or the inadequacies in structure and parameters of the impact model. Previous studies tried to account for the uncertainty from one or two of these. Here, we developed a triple‐ensemble probabilistic assessment using seven crop models, multiple sets of model parameters and eight contrasting climate projections together to comprehensively account for uncertainties from these three important sources. We demonstrated the approach in assessing climate change impact on barley growth and yield at Jokioinen, Finland in the Boreal climatic zone and Lleida, Spain in the Mediterranean climatic zone, for the 2050s. We further quantified and compared the contribution of crop model structure, crop model parameters and climate projections to the total variance of ensemble output using Analysis of Variance (ANOVA). Based on the triple‐ensemble probabilistic assessment, the median of simulated yield change was ?4% and +16%, and the probability of decreasing yield was 63% and 31% in the 2050s, at Jokioinen and Lleida, respectively, relative to 1981–2010. The contribution of crop model structure to the total variance of ensemble output was larger than that from downscaled climate projections and model parameters. The relative contribution of crop model parameters and downscaled climate projections to the total variance of ensemble output varied greatly among the seven crop models and between the two sites. The contribution of downscaled climate projections was on average larger than that of crop model parameters. This information on the uncertainty from different sources can be quite useful for model users to decide where to put the most effort when preparing or choosing models or parameters for impact analyses. We concluded that the triple‐ensemble probabilistic approach that accounts for the uncertainties from multiple important sources provide more comprehensive information for quantifying uncertainties in climate change impact assessments as compared to the conventional approaches that are deterministic or only account for the uncertainties from one or two of the uncertainty sources.     

Dominance–diversity relationships in ant communities differ with invasion

The relationship between levels of dominance and species richness is highly contentious, especially in ant communities. The dominance‐impoverishment rule states that high levels of dominance only occur in species‐poor communities, but there appear to be many cases of high levels of dominance in highly diverse communities. The extent to which dominant species limit local richness through competitive exclusion remains unclear, but such exclusion appears more apparent for non‐native rather than native dominant species. Here we perform the first global analysis of the relationship between behavioral dominance and species richness. We used data from 1,293 local assemblages of ground‐dwelling ants distributed across five continents to document the generality of the dominance‐impoverishment rule, and to identify the biotic and abiotic conditions under which it does and does not apply. We found that the behavioral dominance–diversity relationship varies greatly, and depends on whether dominant species are native or non‐native, whether dominance is considered as occurrence or relative abundance, and on variation in mean annual temperature. There were declines in diversity with increasing dominance in invaded communities, but diversity increased with increasing dominance in native communities. These patterns occur along the global temperature gradient. However, positive and negative relationships are strongest in the hottest sites. We also found that climate regulates the degree of behavioral dominance, but differently from how it shapes species richness. Our findings imply that, despite strong competitive interactions among ants, competitive exclusion is not a major driver of local richness in native ant communities. Although the dominance‐impoverishment rule applies to invaded communities, we propose an alternative dominance‐diversification rule for native communities.     

Temperature sensitivities of extracellular enzyme Vmax and Km across thermal environments

The magnitude and direction of carbon cycle feedbacks under climate warming remain uncertain due to insufficient knowledge about the temperature sensitivities of soil microbial processes. Enzymatic rates could increase at higher temperatures, but this response could change over time if soil microbes adapt to warming. We used the Arrhenius relationship, biochemical transition state theory, and thermal physiology theory to predict the responses of extracellular enzyme V_max and K_m to temperature. Based on these concepts, we hypothesized that V_max and K_m would correlate positively with each other and show positive temperature sensitivities. For enzymes from warmer environments, we expected to find lower V_max, K_m, and K_m temperature sensitivity but higher V_max temperature sensitivity. We tested these hypotheses with isolates of the filamentous fungus Neurospora discreta collected from around the globe and with decomposing leaf litter from a warming experiment in Alaskan boreal forest. For Neurospora extracellular enzymes, V_max Q₁₀ ranged from 1.48 to 2.25, and K_m Q₁₀ ranged from 0.71 to 2.80. In agreement with theory, V_max and K_m were positively correlated for some enzymes, and V_max declined under experimental warming in Alaskan litter. However, the temperature sensitivities of V_max and K_m did not vary as expected with warming. We also found no relationship between temperature sensitivity of V_max or K_m and mean annual temperature of the isolation site for Neurospora strains. Declining V_max in the Alaskan warming treatment implies a short‐term negative feedback to climate change, but the Neurospora results suggest that climate‐driven changes in plant inputs and soil properties are important controls on enzyme kinetics in the long term. Our empirical data on enzyme V_max, K_m, and temperature sensitivities should be useful for parameterizing existing biogeochemical models, but they reveal a need to develop new theory on thermal adaptation mechanisms.     

Comparative proteomic analysis of Xanthomonas citri ssp. citri periplasmic proteins reveals changes in cellular envelope metabolism during in vitro pathogenicity induction

Citrus canker is a plant disease caused by Gram‐negative bacteria from the genus Xanthomonas. The most virulent species is Xanthomonas citri ssp. citri (XAC), which attacks a wide range of citrus hosts. Differential proteomic analysis of the periplasm‐enriched fraction was performed for XAC cells grown in pathogenicity‐inducing (XAM‐M) and pathogenicity‐non‐inducing (nutrient broth) media using two‐dimensional electrophoresis combined with liquid chromatography‐tandem mass spectrometry. Amongst the 40 proteins identified, transglycosylase was detected in a highly abundant spot in XAC cells grown under inducing condition. Additional up‐regulated proteins related to cellular envelope metabolism included glucose‐1‐phosphate thymidylyltransferase, dTDP‐4‐dehydrorhamnose‐3,5‐epimerase and peptidyl‐prolyl cis–trans‐isomerase. Phosphoglucomutase and superoxide dismutase proteins, known to be involved in pathogenicity in other Xanthomonas species or organisms, were also detected. Western blot and quantitative real‐time polymerase chain reaction analyses for transglycosylase and superoxide dismutase confirmed that these proteins were up‐regulated under inducing condition, consistent with the proteomic results. Multiple spots for the 60‐kDa chaperonin and glyceraldehyde‐3‐phosphate dehydrogenase were identified, suggesting the presence of post‐translational modifications. We propose that substantial alterations in cellular envelope metabolism occur during the XAC infectious process, which are related to several aspects, from defence against reactive oxygen species to exopolysaccharide synthesis. Our results provide new candidates for virulence‐related proteins, whose abundance correlates with the induction of pathogenicity and virulence genes, such as hrpD6, hrpG, hrpB7, hpa1 and hrpX. The results present new potential targets against XAC to be investigated in further functional studies.     

An approach based on the total‐species accumulation curve and higher taxon richness to estimate realistic upper limits in regional species richness

Most of accumulation curves tend to underestimate species richness, as they do not consider spatial heterogeneity in species distribution, or are structured to provide lower bound estimates and limited extrapolations. The total‐species (T–S) curve allows extrapolations over large areas while taking into account spatial heterogeneity, making this estimator more prone to attempt upper bound estimates of regional species richness. However, the T–S curve may overestimate species richness due to (1) the mismatch among the spatial units used in the accumulation model and the actual units of variation in β‐diversity across the region, (2) small‐scale patchiness, and/or (3) patterns of rarity of species. We propose a new framework allowing the T–S curve to limit overestimation and give an application to a large dataset of marine mollusks spanning over 11 km² of subtidal bottom (W Mediterranean). As accumulation patterns are closely related across the taxonomic hierarchy up to family level, improvements of the T–S curve leading to more realistic estimates of family richness, that is, not exceeding the maximum number of known families potentially present in the area, can be considered as conducive to more realistic estimates of species richness. Results on real data showed that improvements of the T–S curve to accounts for true variations in β‐diversity within the sampled areas, small‐scale patchiness, and rarity of families led to the most plausible richness when all aspects were considered in the model. Data on simulated communities indicated that in the presence of high heterogeneity, and when the proportion of rare species was not excessive (>2/3), the procedure led to almost unbiased estimates. Our findings highlighted the central role of variations in β‐diversity within the region when attempting to estimate species richness, providing a general framework exploiting the properties of the T–S curve and known family richness to estimate plausible upper bounds in γ‐diversity.     

A novel <Emphasis Type="Italic">PAX5</Emphasis> rearrangement in <Emphasis Type="Italic">TCF3-PBX1</Emphasis> acute lymphoblastic leukemia: a case report

Background

Chromosome translocations are a hallmark of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Additional genomic aberrations are also crucial in both BCP-ALL leukemogenesis and treatment management. Herein, we report the phenotypic and molecular cytogenetic characterization of an extremely rare case of BCP-ALL harboring two concomitant leukemia-associated chromosome translocations: t(1;19)(q23;q13.3) and t(9;17)(p13;q11.2). Of note, we described a new rearrangement between exon 6 of PAX5 and a 17q11.2 region, where intron 3 of SPECC1 is located. This rearrangement seems to disrupt PAX5 similarly to a PAX5 deletion. Furthermore, a distinct karyotype between diagnosis and relapse samples was observed, disclosing a complex clonal evolution during leukemia progression.

Case presentation

A 16-year-old boy was admitted febrile with abdominal and joint pain. At clinical investigation, he presented with anemia, splenomegaly, low white blood cell count and 92% lymphoblast. He was diagnosed with pre-B ALL and treated according to high risk GBTLI-ALL2009. Twelve months after complete remission, he developed a relapse in consequence of a high central nervous system and bone marrow infiltration, and unfortunately died.

Conclusions

To our knowledge, this is the first report of a rearrangement between PAX5 and SPECC1. The presence of TCF3-PBX1 and PAX5-rearrangement at diagnosis and relapse indicates that both might have participated in the malignant transformation disease maintenance and dismal outcome.