Material properties of the cornea and sclera: a modelling approach to test experimental analysis

Material properties of cornea and sclera are important for maintaining the shape of the eye and the requisite surface curvatures for optics. They also need to withstand the forces of external and internal musculature and fluctuations in intraocular pressure (IOP). These properties are difficult to measure and variable results have been reported. A previously published experimental procedure, from which the material properties of the eyeball coats were obtained, has been modelled in this paper using Finite Element Analysis, in order to test the accuracy of the experiment. Material parameters were calculated from the model and the resulting relationships between stress and strain for the cornea and sclera compared to their experimentally obtained counterparts. The comparison between model and experiment was close for the sclera but more varied for the cornea. The pressure vessel model can be applied for measuring the material properties of the sclera but is less accurate for the cornea.     

Synthesis, structure-affinity relationships, and molecular modeling studies of novel pyrazolo[3,4-c]quinoline derivatives as adenosine receptor antagonists

This paper reports the study of new 2-phenyl- and 2-methylpyrazolo[3,4-c]quinolin-4-ones (series A) and 4-amines (series B), designed as adenosine receptor (AR) antagonists. The synthesized compounds bear at the 6-position various groups, with different lipophilicity and steric hindrance, that were thought to increase human A(1) and A(2A) AR affinities and selectivities, with respect to those of the parent 6-unsubstituted compounds. In series A, this modification was not tolerated since it reduced AR affinity, while in series B it shifted the binding towards the hA(1) subtype. To rationalize the observed structure-affinity relationships, molecular docking studies at A(2A)AR-based homology models of the A(1) and A(3) ARs and at the A(2A)AR crystal structure were carried out.     

1,4-Diazepane compounds as potent and selective CB2 agonists: optimization of metabolic stability

A high-throughput screening campaign has identified 1,4-diazepane compounds which are potent Cannabinoid receptor 2 agonists with excellent selectivity against the Cannabinoid receptor 1. This class of compounds suffered from low metabolic stability. Following various strategies, compounds with a good stability in liver microsomes and rat PK profile have been identified.     

Regulation of stereocilia length by myosin XVa and whirlin depends on the actin-regulatory protein Eps8

Myosin XVa (MyoXVa) and its cargo whirlin are implicated in deafness and vestibular dysfunction and have been shown to localize at stereocilia tips and to be essential for the elongation of these actin protrusions [1-4]. Given that whirlin has no known actin-regulatory activity, it remains unclear how these proteins work together to influence stereocilia length. Here we show that the actin-regulatory protein Eps8 [5] interacts with MyoXVa and that mice lacking Eps8 show short stereocilia compared to MyoXVa- and whirlin-deficient mice. We show that Eps8 fails to accumulate at the tips of stereocilia in the absence of MyoXVa, that overexpression of MyoXVa results in both elongation of stereocilia and increased accumulation of Eps8 at stereocilia tips, and that the exogenous expression of MyoXVa in MyoXVa-deficient hair cells rescues Eps8 tip localization. We find that Eps8 also interacts with whirlin and that the expression of both Eps8 and MyoXVa at stereocilia tips is reduced in whirlin-deficient mice. We conclude that MyoXVa, whirlin, and Eps8 are integral components of the stereocilia tip complex, where Eps8 is a central actin-regulatory element for elongation of the stereocilia actin core.     

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO₂ nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO₂ nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.     

What Story Does Geographic Separation of Insular Bats Tell? A Case Study on Sardinian Rhinolophids

Competition may lead to changes in a species’ environmental niche in areas of sympatry and shifts in the niche of weaker competitors to occupy areas where stronger ones are rarer. Although mainland Mediterranean (Rhinolophus euryale) and Mehely’s (R. mehelyi) horseshoe bats mitigate competition by habitat partitioning, this may not be true on resource-limited systems such as islands. We hypothesize that Sardinian R. euryale (SAR) have a distinct ecological niche suited to persist in the south of Sardinia where R. mehelyi is rarer. Assuming that SAR originated from other Italian populations (PES) – mostly allopatric with R. mehelyi – once on Sardinia the former may have undergone niche displacement driven by R. mehelyi. Alternatively, its niche could have been inherited from a Maghrebian source population. We: a) generated Maxent Species Distribution Models (SDM) for Sardinian populations; b) calibrated a model with PES occurrences and projected it to Sardinia to see whether PES niche would increase R. euryale’s sympatry with R. mehelyi; and c) tested for niche similarity between R. mehelyi and PES, PES and SAR, and R. mehelyi and SAR. Finally we predicted R. euryale’s range in Northern Africa both in the present and during the Last Glacial Maximum (LGM) by calibrating SDMs respectively with SAR and PES occurrences and projecting them to the Maghreb. R. mehelyi and PES showed niche similarity potentially leading to competition. According to PES’ niche, R. euryale would show a larger sympatry with R. mehelyi on Sardinia than according to SAR niche. Such niches have null similarity. The current and LGM Maghrebian ranges of R. euryale were predicted to be wide according to SAR’s niche, negligible according to PES’ niche. SAR’s niche allows R. euryale to persist where R. mehelyi is rarer and competition probably mild. Possible explanations may be competition-driven niche displacement or Maghrebian origin.     

Switching between the Alternative Structures and Functions of a 2-Cys Peroxiredoxin,by Site-Directed Mutagenesis

Members of the typical 2-Cys peroxiredoxin (Prx) subfamily represent an intriguing example of protein moonlighting behavior since this enzyme shifts function: indeed, upon chemical stimuli, such as oxidative stress, Prx undergoes a switch from peroxidase to molecular chaperone, associated to a change in quaternary structure from dimers/decamers to higher-molecular-weight (HMW) species. In order to detail the structural mechanism of this switch at molecular level, we have designed and expressed mutants of peroxiredoxin I from Schistosoma mansoni (SmPrxI) with constitutive HMW assembly and molecular chaperone activity. By a combination of X-ray crystallography, transmission electron microscopy and functional experiments, we defined the structural events responsible for the moonlighting behavior of 2-Cys Prx and we demonstrated that acidification is coupled to local structural variations localized at the active site and a change in oligomerization to HMW forms, similar to those induced by oxidative stress. Moreover, we suggest that the binding site of the unfolded polypeptide is at least in part contributed by the hydrophobic surface exposed by the unfolding of the active site. We also find an inverse correlation between the extent of ring stacking and molecular chaperone activity that is explained assuming that the binding occurs at the extremities of the nanotube, and the longer the nanotube is, the lesser the ratio binding sites/molecular mass is.     

Stem cell therapy in ocular pathologies in the past 20 years

Stem cell therapies are successfully used in various fields of medicine. This new approach of research is also expanding in ophthalmology. Huge investments, resources and important clinical trials have been performed in stem cell research and in potential therapies. In recent years, great strides have been made in genetic research, which permitted and enhanced the differentiation of stem cells. Moreover, the possibility of exploiting stem cells from other districts (such as adipose, dental pulp, bone marrow stem cells, etc.) for the treatment of ophthalmic diseases, renders this topic fascinating. Furthermore, great strides have been made in biomedical engineering, which have proposed new materials and three-dimensional structures useful for cell therapy of the eye. The encouraging results obtained on clinical trials conducted on animals have given a significant boost in the creation of study protocols also in humans. Results are limited to date, but clinical trials continue to evolve. Our attention is centered on the literature reported over the past 20 years, considering animal (the most represented in literature) and human clinical trials, which are limiting. The aim of our review is to present a brief overview of the main types of treatments based on stem cells in the field of ophthalmic pathologies.