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Peroxiredoxins (Prxs) are ubiquitary proteins able to play multiple physiological roles, that include thiol-dependent peroxidase, chaperone holdase, sensor of H2O2, regulator of H2O2-dependent signal cascades, and modulator of the immune response. Prxs have been found in a great number of human pathogens, both eukaryotes and prokaryotes. Gene knock-out studies demonstrated that Prxs are essential for the survival and virulence of at least some of the pathogens tested, making these proteins potential drug targets. However, the multiplicity of roles played by Prxs constitutes an unexpected obstacle to drug development. Indeed, selective inhibitors of some of the functions of Prxs are known (namely of the peroxidase and holdase functions) and are here reported. However, it is often unclear which function is the most relevant in each pathogen, hence which one is most desirable to inhibit. Indeed there are evidences that the main physiological role of Prxs may not be the same in different parasites. We here review which functions of Prxs have been demonstrated to be relevant in different human parasites, finding that the peroxidase and chaperone activities figure prominently, whereas other known functions of Prxs have rarely, if ever, been observed in parasites, or have largely escaped detection thus far.

The study of the mechanical properties of biosystems and the relationship with their biochemical and structural functionality is an increasingly interesting subject of investigation. In recent years, in particular, the use of the atomic force microscopy provides the tools for understanding the molecular basis of the mechanical behaviour of the biosystems. The ageing of erythrocytes [red blood cells (RBCs)] constitutes a particularly interesting subject of study because of its fundamental role in triggering the cell turnover by promoting the removal of malfunctioning RBCs when specific ageing markers appear on their surface. Moreover, it is also interesting to study the role that the variation in the cells mechanical properties plays in the progress of the phenomenon. In this study, the ageing of RBCs, accelerated by depleting the cells of their ATP, has been investigated by two methods. The first is a recently developed nondestructive approach that correlates the roughness of the plasma membrane to the mechanical characteristics and the structural integrity of the cell membrane-skeleton. The second consists in directly measuring the nanomechanical properties by acquiring and analysing force curves on the cell membrane. The application of the two methods allowed to define, for the first time, the general scheme of alterations the cells experience during the ageing. In particular, a progressive decrease of the membrane roughness, correlated to a weakening of the membrane-skeleton support, and a complex pattern of changes in the nanomechanical properties, which drives the morphological variation and the occurrence of the specific ageing markers on the cells, have been revealed.

Schistosomiasis is a widespread tropical parasitic disease, currently treated with Praziquantel, whose precise molecular target is actually unknown. Several other drugs are known to kill the schistosomes in vivo and in vitro, but these are seldom employed because of toxicity, high cost, complex administration or other reasons. The improvement of known drugs or the development of entirely new ones is a desirable goal, in view of the fact that strains of Schistosoma mansoni with reduced sensitivity to Praziquantel have appeared. In this review, we tried to collect the information available on known or putative macromolecular targets of schistosomicidal drugs; thus we focused on the biochemistry of the parasite, rather than the clinical properties of the drugs. The rationale of this approach is that drug design may become realistic if the mechanism of action of each known drug were known at atomic detail, ideally as the 3D structure of each drug in complex with its target. Important macromolecular targets of known drugs reviewed below are: Thioredoxin Glutathione Reductase; Cyclophilin; Acetyl Cholinesterase; Proteases and Purine Nucleoside Phosphorylase. Moreover, a few enzymes of the parasite are known, or thought, to be "druggable", and therefore interesting, even though no specific drugs are available as yet: examples of such enzymes are Glutathione Peroxidase and Peroxiredoxins.

We recently developed an atomic force microscopy-based protocol to use the roughness of the plasma membrane of erythrocytes (red blood cells, RBCs) as a morphological parameter, independently from the cell shape, to investigate the membrane-skeleton integrity in healthy and pathological cells. Here we apply the method to investigate a complex physiological phenomenon, the RBCs aging, that plays a major role in the regulation of the RBCs' turnover. The aging, monitored morphologically and biochemically, has been accelerated and modulated by preventing oxidative stresses as well as the effects of proteases and divalent cations, and by artificially consuming the intracellular adenosine triphosphate. The collected data evidence that the progression of aging causes a drastic decrease of the measured roughness that is diagnostic of a progressive, adenosine triphosphate-dependent alteration of the membrane-skeleton properties. Finally, the degree of reversibility of such effects has been investigated as a function of aging time, enabling the detection of irreversible transformation in the RBCs' structure and metabolism.

Treatment of schistosomiasis, a widespread human parasitic disease caused by the helminth parasites of the genus Schistosoma, relies mainly on one chemotherapeutic agent, praziquantel, although several other compounds exert anti-parasitic effects. One such compound is the immunosuppressant cyclosporin A, which has been shown to significantly diminish worm burden in mice infected with Schistosoma mansoni. Given the well established interaction between cyclosporin A and the cyclophilin superfamily of peptidylprolyl cis-trans isomerases, we solved the structure of cyclophilin A from S. mansoni (SmCypA) by x-ray crystallography in the reduced and oxidized states at 1.5 and 1.8A of resolution, respectively. Oxidized SmCypA contains a disulfide bridge between two C-terminal cysteines (Cys-122 and Cys-126). This is the first example of a cyclophilin containing this disulfide bridge. Parallel functional studies suggest a mechanism for regulation of SmCypA activity via oxidation of its thiol groups; in fact, whereas oxidized SmCypA is inactive, reduced SmCypA is an efficient isomerase active at nanomolar levels with a kcat/Km of 1.1 × 107m–1 s–1, and it is inhibited by cyclosporin A (IC50 of 14 ± 4nm). The lack of conservation of this cysteine couple within the CypA superfamily, their close proximity to the active site, and the importance of thiol groups for peptidyl-prolyl cis-trans isomerase activity render this structural feature a challenge for the development of alternative and more effective anti-schistosomiasis inhibitors and may in addition imply an alternative function of SmCypA in the schistosome.

We used air operating atomic force microscopy (AFM) to study several morphological modifications of human erythrocytes, artificially produced by addition of exogenous agents including phospholipids, low ionic strength media and drugs. Most experiments were performed on unfixed samples to avoid treating red blood cells (RBCs) with chemical agents that can, in principle, induce morphological alteration. After detailed quantitative AFM characterization, the artificially produced abnormally shaped RBCs were compared with cells that occur with high incidence in blood pathologies. This morphological approach suggests a new strategy to describe and understand the biochemical and/or mechanical modifications responsible for the underlying pathologically induced changes and prove AFM to be a suitable tool to study erythrocyte deformation.

The red blood cells (RBCs) are among the most simple and less expensive cells to purify; for this reason and for their physiological relevance, they have been extensively studied with a variety of techniques. The picture that results is that these cells have several peculiarities including extreme mechanical performances, relatively simple architecture, biological relevance and predictable behavior that make them a perfect laboratory of testing for novel techniques, methodologies and ideas. These include the re-evaluation of old concepts, such as the relationship between structure and function (which is one of the guideline of this report) but considered at the cellular level. The studies reported on this paper, indeed, exploit the full potential of an high resolution quantitative microscopy such as the atomic force microscopy (AFM) to investigate different aspect of the erythrocytes' life, death and interaction with the environment. Indeed, the erythrocytes have a special relationship with the environment that is able to deeply influence their morphology as consequence of alteration of their biochemical or biophysical status. In this context the conditions under which the erythrocytes can be considered as biochemically programmable systems have been investigated by analyzing different environmentally induced alteration of the cell's morphology and comparing the results with naturally occurring pathological morphologies. This class of studies takes great advantage by the additional consideration of the nanomechanical properties of the cells. These latter are particularly important for the cell functionality and are shown to be of practical usefulness to discriminate and partition environmental effects charging different cellular structure (e.g. membrane or membrane-skeleton). Moreover, the development of novel morphological parameter can be important to push the level of investigation on the RBCs' status towards the molecular level. In particular, we describe the introduction and use of the plasma membrane roughness as a morphometric parameter of simple derivation from the AFM images and that results sensitive to the structural integrity of the cells' membrane-skeleton. This offer a remarkable opportunity to investigate the relationship between structure and function in normal and pathological cells by using a morphometric parameter that probes the cell surface at the nanoscale level. At last, a complex but physio-pathologically important phenomenon such as the erythrocytes aging was considered. To properly analyze the many variation that the cells experience during the whole aging path we used all the parameters that the AFM can provides: quantitative imaging, analysis of the membrane roughness and local measure of the nanomechanical properties analyzed together with biochemical parameter such as the ATP content. The picture that emerged is that the aging path is triggered by the ATP intracellular concentration that influence the membrane-skeleton structure and the support exerted on the plasma membrane. The consequences of the membrane-skeleton involvement can be monitored by AFM and showed the occurrence of peculiar morphologies and morphological defects that appear in the very place where the membrane-skeleton contact with the membrane became loose. As a whole, the collected data enable to describe the entire phenomenon as a sequence of morphological intermediates following one another along the aging path.

Human erythrocytes (RBCs), stored at 4 degrees C under nominal absence of external energy sources and calcium ions, show a gradual decrease in membrane roughness (R(rms)) at the end of which the appearance of morphological phenomena (spicules, vesicles and spherocytes) is observed on the cell membrane, phenomena that can mainly be ascribed to the ATP-dependent disconnection of the cortical cytoskeleton from the lipid bilayer. After depletion of the intracellular energy sources obtained under the extreme conditions chosen, treatment with a minimal rejuvenation solution makes the following remarks possible: (i) RBCs are able to regenerate adenosine triphosphate (ATP) and 2,3-bisphosphoglycerate only up to 4 days of storage at 4 degrees C, whereas from the eighth day energy stocks cannot be replenished because of a disorder in the transmembrane mechanisms of transport; (ii) the RBCs' roughness may be restored to the initial value (i.e. that observed in fresh RBCs) only in samples stored up to 4-5 days, whereas after the eighth day of storage the rejuvenation procedure appears to be inefficient; (iii) membrane physical properties - as measured by R(rms) - are actually controlled by the metabolic production of ATP, necessary to perform the RBCs' basic functions; (iv) once energy stores cannot be replenished, a regulated sequence of the morphological events (represented by local buckles that lead to formation of spicules and vesicles of the lipid bilayer with generation of spherocytes) is reminiscent of the RBCs' apoptotic final stages; (v) the morphological phenomenology of the final apoptotic stages is passive (i.e. determined by simple mechanical forces) and encoded in the mechanical properties of the membrane-skeleton; and (vi) necrotic aspects (e.g. disruption of cell membrane integrity, so that intracellular protein content is easily released) ensue when RBCs are almost totally (or =90%) depleted in an irreversible way of the energetic stores.