An Automated Live Imaging Platform for Studying Merozoite Egress-Invasion in Malaria Cultures

Most cases of severe and fatal malaria are caused by the intraerythrocytic asexual reproduction cycle of Plasmodium falciparum. One of the most intriguing and least understood stages in this cycle is the brief preinvasion period during which dynamic merozoite–red-cell interactions align the merozoite apex in preparation for penetration. Studies of the molecular mechanisms involved in this process face formidable technical challenges, requiring multiple observations of merozoite egress-invasion sequences in live cultures under controlled experimental conditions, using high-resolution microscopy and a variety of fluorescent imaging tools. Here we describe a first successful step in the development of a fully automated, robotic imaging platform to enable such studies. Schizont-enriched live cultures of P. falciparum were set up on an inverted stage microscope with software-controlled motorized functions. By applying a variety of imaging filters and selection criteria, we identified infected red cells that were likely to rupture imminently, and recorded their coordinates. We developed a video-image analysis to detect and automatically record merozoite egress events in 100% of the 40 egress-invasion sequences recorded in this study. We observed a substantial polymorphism of the dynamic condition of pre-egress infected cells, probably reflecting asynchronies in the diversity of confluent processes leading to merozoite release.     

Energetic diversity of DNA minor-groove recognition by small molecules displayed through some model ligand-DNA systems

Energetics of interactions occurring in the model ligand-DNA systems constituted from distamycin A (DST), netropsin (NET) and the oligomeric duplexes d(GCAAGTTGCGATATACG)d(CGTATATCGCAACTTGC)=D#1 and d(GCAAGTTGCGAAAAACG)d(CGTTTTTCGCAACTTGC)=D#2 was studied by spectropolarimetry, UV-absorption spectroscopy and isothermal titration calorimetry. Model analysis of the measured signals was applied to describe individual and competitive binding in terms of populations of various species in the solution. Our results reveal several unprecedented ligand-DNA binding features. DST binds to the neighboring 5'-AAGTT-3' and 5'-ATATA-3' sites of D#1 statistically in a 2:1 binding mode. By contrast, its association to D#2 appears to be a 2:1 binding event only at the DST/D#2 molar ratios between 0 and 2 while its further binding to D#2 may be considered as a step-by-step binding to the unoccupied 5'-AAAAA-3' sites resulting first in DST3D#2 and finally in DST4D#2 complex formation. Competition between DST and NET binding shows that for the most part DST displaces NET from its complexes with D#1 and D#2. In contrast to the obligatory 1:1 binding of DST to the ligand-free 5'-AAAAA-3' sites observed at DST/5'-AAAAA-3' <1 the displacement of NET bound to the 5'-AAAAA-3' sites by added DST occurs even at the smallest additions of DST in a 2:1 manner. NET can also displace DST molecules but only those bound monomerically to the 5'-AAAAA-3' sites of DST3D#2. Actually, only half of these molecules can be displaced due to the simultaneous rebinding of the displaced DST to the unreacted 5'-AAAAA-3' sites in DST3D#2. Binding of DST and NET to D#1 and D#2 is an enthalpy driven process accompanied by large unfavorable (DST), small (NET) or large favorable (NET binding to 5'-AAAAA-3') entropy contributions and negative deltaCP degrees that are reasonably close to deltaCP degrees predicted from the calculated changes in solvent-accessible surface areas that accompany complex formation. Although various modes of DST and NET binding within D#1 and D#2 are characterized by significant energetic differences they seem to be governed by the same driving forces; the hydrophobic transfer of ligand from the solution into the duplex binding site and the accompanying specific non-covalent ligand-DNA and ligand-ligand interactions occurring within the DNA minor groove.     

What drives the binding of minor groove-directed ligands to DNA hairpins?

Understanding the molecular basis of ligand-DNA-binding events, and its application to the rational design of novel drugs, requires knowledge of the structural features and forces that drive the corresponding recognition processes. Existing structural evidence on DNA complexation with classical minor groove-directed ligands and the corresponding studies of binding energetics have suggested that this type of binding can be described as a rigid-body association. In contrast, we show here that the binding-coupled conformational changes may be crucial for the interpretation of DNA (hairpin) association with a classical minor groove binder (netropsin). We found that, although the hairpin form is the only accessible state of ligand-free DNA, its association with the ligand may lead to its transition into a duplex conformation. It appears that formation of the fully ligated duplex from the ligand-free hairpin, occurring via two pathways, is enthalpically driven and accompanied by a significant contribution of the hydrophobic effect. Our thermodynamic and structure-based analysis, together with corresponding theoretical studies, shows that none of the predicted binding steps can be considered as a rigid-body association. In this light we anticipate our thermodynamic approach to be the basis of more sophisticated nucleic acid recognition mechanisms, which take into account the dynamic nature of both the nucleic acid and the ligand molecule.     

Fermented Soybean Meal Increases Lactic Acid Bacteria in Gut Microbiota of Atlantic Salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>)

The main goal of the present study was to address the effect of feeding fermented soybean meal-based diet to Atlantic salmon on gut microbiota. Further, expression of genes of interest, including cathelicidin antimicrobial peptide (cath), mucin 2 (muc2), aquaporin (aqp8ab), and proliferating cell nuclear antigen (pcna), in proximal intestine of fish fed either experimental diet was analyzed. Three experimental diets, including a control fishmeal (30% FM), soybean meal (30% SBM), or fermented soybean meal diet (30% FSBM) were randomly assigned to triplicate tanks during a 50-day trial. The PCR-TTGE showed microbiota composition was influenced by experimental diets. Bands corresponding to genus Lactobacillus and Pediococcus were characteristic in fish fed the FSBM-based diet. On the other hand, bands corresponding to Isoptericola, Cellulomonas, and Clostridium sensu stricto were only observed in fish FM-based diet, while Acinetobacter and Altererythrobacter were detected in fish fed SBM-based diet. The expression of muc2 and aqp8ab were significantly greater in fish fed the FSBM-based diet compared with the control group. Our results suggest feeding FSBM to Atlantic salmon may (1) boost health and growth physiology in fish by promoting intestinal lactic acid bacteria growth, having a prebiotic-like effect, (2) promote proximal intestine health by increasing mucin production, and (3) boost intestinal trans-cellular uptake of water. Further research to better understands the effects of bioactive compounds derived from the fermentation process of plant feedstuff on gut microbiota and the effects on health and growth in fish is required.     

Open-Boundary Molecular Dynamics of a DNA Molecule in a Hybrid Explicit/Implicit Salt Solution

The composition and electrolyte concentration of the aqueous bathing environment have important consequences for many biological processes and can profoundly affect the behavior of biomolecules. Nevertheless, because of computational limitations, many molecular simulations of biophysical systems can be performed only at specific ionic conditions: either at nominally zero salt concentration, i.e., including only counterions enforcing the system’s electroneutrality, or at excessive salt concentrations. Here, we introduce an efficient molecular dynamics simulation approach for an atomistic DNA molecule at realistic physiological ionic conditions. The simulations are performed by employing the open-boundary molecular dynamics method that allows for simulation of open systems that can exchange mass and linear momentum with the environment. In our open-boundary molecular dynamics approach, the computational burden is drastically alleviated by embedding the DNA molecule in a mixed explicit/implicit salt-bathing solution. In the explicit domain, the water molecules and ions are both overtly present in the system, whereas in the implicit water domain, only the ions are explicitly present and the water is described as a continuous dielectric medium. Water molecules are inserted and deleted into/from the system in the intermediate buffer domain that acts as a water reservoir to the explicit domain, with both water molecules and ions free to enter or leave the explicit domain. Our approach is general and allows for efficient molecular simulations of biomolecules solvated in bathing salt solutions at any ionic strength condition.     

Evidence against a Role of Elevated Intracellular Ca2+ during Plasmodium falciparum Preinvasion

Severe malaria is primarily caused by Plasmodium falciparum parasites during their asexual reproduction cycle within red blood cells. One of the least understood stages in this cycle is the brief preinvasion period during which merozoite-red cell contacts lead to apical alignment of the merozoite in readiness for penetration, a stage of major relevance in the control of invasion efficiency. Red blood cell deformations associated with this process were suggested to be active plasma membrane responses mediated by transients of elevated intracellular calcium. Few studies have addressed this hypothesis because of technical challenges, and the results remained inconclusive. Here, Fluo-4 was used as a fluorescent calcium indicator with optimized protocols to investigate the distribution of the dye in red blood cell populations used as P. falciparum invasion targets in egress-invasion assays. Preinvasion dynamics was observed simultaneously under bright-field and fluorescence microscopy by recording egress-invasion events. All the egress-invasion sequences showed red blood cell deformations of varied intensities during the preinvasion period and the echinocytic changes that follow during invasion. Intraerythrocytic calcium signals were absent throughout this interval in over half the records and totally absent during the preinvasion period, regardless of deformation strength. When present, calcium signals were of a punctate modality, initiated within merozoites already poised for invasion. These results argue against a role of elevated intracellular calcium during the preinvasion stage. We suggest an alternative mechanism of merozoite-induced preinvasion deformations based on passive red cell responses to transient agonist-receptor interactions associated with the formation of adhesive coat filaments.     

Gap regeneration and replacement patterns in an old-growth <Emphasis Type="Italic">Fagus</Emphasis>–<Emphasis Type="Italic">Abies</Emphasis> forest of Bosnia–Herzegovina

We examined the influence of small-scale gap disturbances on stand development and tree species coexistence in an old-growth Fagus sylvatica–Abies alba forest in the Dinaric Mountains of Bosnia–Herzegovina. The structure and composition of tree regeneration in gaps were compared to the forest as a whole, and the influence of gap size on the density and composition of regeneration was assessed. Transition probabilities were also calculated from gapfillers in different life stages to examine canopy replacement patterns. The structure and composition of tree regeneration were similar between gaps and the forest as a whole, and there was no relationship between overall regeneration density and gap size, indicating most individuals established prior to gap formation. Likewise, there was no strong evidence of gap-size partitioning for shade tolerant F. sylvatica and A. alba, although less tolerant Acer pseudoplatanus only recruited to taller life stages in larger gaps. Transition probabilities calculated from the seedling and sapling data suggest that most gaps will be captured by F. sylvatica, while probabilities based on pole-sized gapmakers indicate both A. alba and F. sylvatica will be maintained in the canopy. We suggest that gaps primarily play a role in reorganizing advance regeneration, and that coexistence of shade tolerant F. sylvatica and A. alba is more likely related to their differential ability to tolerate shaded understory conditions, particularly during larger life stages, rather than gap-size partitioning.