Resolving structure and mechanical properties at the nanoscale of viruses with frequency modulation atomic force microscopy

Structural Biology (SB) techniques are particularly successful in solving virus structures. Taking advantage of the symmetries, a heavy averaging on the data of a large number of specimens, results in an accurate determination of the structure of the sample. However, these techniques do not provide true single molecule information of viruses in physiological conditions. To answer many fundamental questions about the quickly expanding physical virology it is important to develop techniques with the capability to reach nanometer scale resolution on both structure and physical properties of individual molecules in physiological conditions. Atomic force microscopy (AFM) fulfills these requirements providing images of individual virus particles under physiological conditions, along with the characterization of a variety of properties including local adhesion and elasticity. Using conventional AFM modes is easy to obtain molecular resolved images on flat samples, such as the purple membrane, or large viruses as the Giant Mimivirus. On the contrary, small virus particles (25-50 nm) cannot be easily imaged. In this work we present Frequency Modulation atomic force microscopy (FM-AFM) working in physiological conditions as an accurate and powerful technique to study virus particles. Our interpretation of the so called "dissipation channel" in terms of mechanical properties allows us to provide maps where the local stiffness of the virus particles are resolved with nanometer resolution. FM-AFM can be considered as a non invasive technique since, as we demonstrate in our experiments, we are able to sense forces down to 20 pN. The methodology reported here is of general interest since it can be applied to a large number of biological samples. In particular, the importance of mechanical interactions is a hot topic in different aspects of biotechnology ranging from protein folding to stem cells differentiation where conventional AFM modes are already being used.     

Dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin mediates binding and internalization of Aspergillus fumigatus conidia by dendritic cells and macrophages

Aspergillus fumigatus is responsible for a large percentage of nosocomial opportunistic fungal infections in immunocompromised hosts, especially during cytotoxic chemotherapy and after bone marrow transplantation, and is currently a major direct cause of death in leukemia patients. Dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) is a type II C-type lectin that functions as an adhesion receptor and is used by viral and bacterial pathogens to gain access to human DC. We report that DC-SIGN specifically interacts with clinical isolates of A. fumigatus. DC-SIGN-dependent binding of A. fumigatus conidia can be demonstrated with stable transfectants and monocyte-derived DC and is inhibited by anti-DC-SIGN Abs. Binding and internalization of A. fumigatus conidia correlates with DC-SIGN cell surface expression levels and is abolished in the presence of A. fumigatus-derived cell wall galactomannans. The clinical relevance of this interaction is emphasized by the presence of DC-SIGN in lung DC and alveolar macrophages, and further illustrated by the DC-SIGN-dependent attachment of A. fumigatus conidia to the cell membrane of IL-4-treated monocyte-derived macrophages. Our results suggest the involvement of DC-SIGN in the initial stages of pulmonary infection as well as in fungal spreading during invasive aspergillosis.     

A male destroying an egg in a cooperative breeding attempt in Lesser Spotted Woodpecker Dendrocopos minor

A cooperative breeding case was found in Lesser Spotted Woodpecker (LSW) in which a male and three different females were involved. The contributions of each member of the cooperative breeding team were quantified during nest excavation and incubation. This is the first such report on LSW. During daytime, the nest was mainly occupied by the male during the final excavation phase, nest guarding, and egg laying. Aggressive interactions were recorded between two of the females during nest excavation, egg laying and incubation. The male was recorded destroying an egg presumably laid by the dominant female. The highest contribution during daytime incubation was made by the dominant female (39%), followed by the male (34%) and then by a second female (27%). The third female contributed very little at any stage. A picture of the male removing the egg was analyzed to estimate the egg maximum width, ruling out a possible runt egg. Motivations behind the behaviour of the male destroying the egg remain unclear.     

Fluidity and oxidative stress in erythrocytes from very low birth weight infants during their first 7 days of life

Objective: To study the evolution of lipid peroxidation, enzymatic antioxidants response, lipid profile and membrane fluidity in erythrocytes from very low birth weight (VLBW) infants during their first 7 days of extra-uterine life.

Study design: One hundred and twenty infants were selected and divided in two groups according to their weight and gestational age. Hydroperoxides, fatty-acid profile, fluidity (DPH and TMA-DPH) and catalase, SOD and GPx activities were measured in erythrocytes.

Results: VLBW group showed higher concentration of hydroperoxides and lower membrane fluidity during the first 72 h, lower SOD activity during the first 3 h and higher GPx activity during the first 7 days of life. Also, this group showed lower n-3 polyunsaturated fatty-acids percentage with respect to the term group.

Conclusion: Erythrocytes from VLBW infants showed higher oxidative damage and lower fluidity in their membranes, at least during the first 3 days of extra-uterine life, which may cause alterations in their functions and flexibility.     

Microbial degradation of palm (Elaeis guineensis) biodiesel

The kinetics of biodegradation of palm-derived fatty methyl and ethyl esters (Elaeis guineensis biodiesel) by a wild-type aerobic bacterial population was measured at 20 degrees C, as the rate of oxygen uptake by a manometric technique. The methyl and ethyl biodiesels were obtained by potassium-hydroxide catalysed trans-esterification of palm oil, respectively. The bacterial flora included the genera Bacillus, Proteus, Pseudomonas, Citrobacter and Enterobacter. The rate of oxygen uptake for palm biodiesel is similar to the quantity observed in the biodegradation of 1.0 mM solutions of simple substrates such as carbohydrates or amino acids. Palm methyl or ethyl biodiesel is subjected to facile aerobic biodegradation by wild-type bacteria commonly present in natural open environments. This result should lessen any environmental concern for its use as alternative fuel, solvent or lubricant.     

Seed predation by birds and small mammals in semiarid Chile

We studied spatial and temporal patterns in foraging activity among diurnal birds and nocturnal mammals at a semiarid site in northern Chile using artificial foraging trays. Small mammals foraged more extensively under shrubs than in open microhabitats, but birds showed no such selection. Moreover, avian foraging was more extensive than that by small mammals in all seasons and both microhabitats. Avian foraging was highly seasonal, as many birds at our site migrate to the Andean prepuna or to Patagonia during the austral summer. Birds have tended to be overshadowed by small mammals and ants in studies of granivory, but this study suggests that their importance may be underestimated in some systems.     

Dehydroepiandrosterone decreases while cortisol increases in vitro growth and viability of Entamoeba histolytica

In vitro exposure of Entamoeba histolytica trophozoites to the sex steroids 17beta-estradiol, progesterone, and dehydrotestosterone had little effect on parasite viability or proliferation. However, treatment with the adrenal steroid dehydroepiandrosterone (DHEA) markedly inhibited parasite proliferation, adherence and motility, and at a certain dose it induced trophozoite lysis. The opposite effect on proliferation was found when the trophozoites were exposed to cortisol. Moreover, DHEA decreased while cortisol increased the parasite's DNA synthesis determined by 3H-thymidine incorporation. Trophozoite lysis by DHEA appeared to be caused by a necrotic rather than an apoptotic process, as observed in propidium iodide and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling assays. A possible mechanisms of action was derived from experiments demonstrating that the activity of a putative 3-hydroxy-3-methyl glutaryl CoA reductase detected in trophozoite extracts was inhibited in the presence of DHEA. Contrary to its in vitro inhibitory effect, in vivo administration of DHEA to infected hamsters resulted in exacerbation of the amebic liver abscesses. These results demonstrated that androgen steroids act directly upon E. histolytica growth and viability, and may shed new light on some age and gender differences in disease progression, as well as finding application in the drug treatment of human amebiasis.     

Late Hauterivian coralline algae (Rhodophyta,Corallinales) from the Iberian Chain (E Spain). Taxonomy and the evolution of multisporangial reproductive structures

Upper Hauterivian reefal carbonates of the Llàcova Formation (Maestrat Basin, Iberian Chain, E Spain) contain Sporolithon phylloideum (Bucur and Dragastan) Tomás, Aguirre, Braga and Martín-Closas comb. nov. and Sporolithon rude (Lemoine) Ghosh and Maithy (1996). Moussavian et al. (1993) identified them as Parakymalithon phylloideum (Bucur and Dragastan) Moussavian 1987 and Archaeolithothamnium rude Lemoine 1925. The re-assessment of the type of P. phylloideum and additional material indicate that the diagnostic characters of the genus do not warrant separation from Sporolithon and the new combination Sporolithon phylloideum is proposed. The lectotype of Sporolithon rude presents sporangial cavities grouped in sori that can be merged originating a structure that resembles the multiporate tetrasporangial conceptacles of the Hapalidiaceae. We hypothesize that multiporate tetrasporangial conceptacles could have originated from the fusion of several sporangial cavities, suggesting a phylogenetic linkage between Sporolithaceae and Hapalidiaceae supported by other anatomical features, molecular phylogeny and the fossil record.     

How sugars convey information on protein conformation in the endoplasmic reticulum

The N-glycan-dependent quality control of glycoprotein folding prevents endoplasmic reticulum to Golgi exit of folding intermediates, irreparably misfolded glycoproteins and not completely assembled multimeric complexes. It also enhances folding efficiency by preventing aggregation and facilitating formation of proper disulfide bonds. The control mechanism essentially involves four components, resident lectin-chaperones that recognize monoglucosylated polymannose glycans, a lectin-associated oxidoreductase acting on monoglucosylated glycoproteins, a glucosyltransferase and a glucosidase that creates monoglucosylated epitopes in glycans transferred in protein N-glycosylation or removes the glucose units added by the glucosyltransferase. This last enzyme is the only mechanism component sensing glycoprotein conformations as it creates monoglucosylated glycans exclusively in not properly folded species or in not completely assembled complexes. The purpose of the review is to describe the most significant recent findings on the mechanism of glycoprotein folding and assembly quality control and to discuss the main still unanswered questions.