Chemical Composition and Sulfur Speciation in Bulk Tissue by X-Ray Spectroscopy and X-Ray Microscopy: Corneal Development during Embryogenesis

The chemical composition and sulfur (S) speciation of developing chick corneas at embryonic days 12, 14, and 16 were investigated using synchrotron scanning x-ray fluorescence microscopy and x-ray absorption near-edge structure spectroscopy. The aim was to develop techniques for the analysis of bulk tissue and identify critical physicochemical variations that correlate with changes in corneal structure-function relationships. Derived data were subjected to principal component analysis and linear discriminant analysis, which highlighted differences in the elemental and S species composition at different stages of embryonic growth. Notably, distinct elemental compositions of chlorine, potassium, calcium, phosphorus, and S altered with development during the transition of the immature opaque cornea to a mature transparent tissue. S  structure spectroscopy revealed developmentally regulated alterations in thiols, organic monosulfides, ester sulfate, and inorganic sulfate species. The transient molecular structures and compositional changes reported here provide a deeper understanding of the underlying basis of corneal development during the acquisition of transparency. The experimental and analytical approach is new, to our knowledge, and has wide potential applicability in the life sciences.     

Predicting potentially pathogenic effects of hRPE65 missense mutations: a computational strategy based on molecular dynamics simulations

The human retinal pigment epithelium-specific 65-kDa protein (hRPE65) plays a crucial role within the retinoid visual cycle and several mutations affecting either its expression level or its enzymatic function are associated with inherited retinal diseases such as Retinitis Pigmentosa. The gene therapy product voretigene neparvovec (Luxturna) has been recently approved for treating hereditary retinal dystrophies; however, the treatment is currently accessible only to patients presenting confirmed biallelic mutations that severely impair hRPE65 function, and many reported hRPE65 missense mutations lack sufficient evidences for proving their pathogenicity. In this context, we developed a computational approach aimed at evaluating the potential pathogenic effect of hRPE65 missense variants located on the dimerisation domain of the protein. The protocol evaluates how mutations may affect folding and conformation stability of this protein region, potentially helping clinicians to evaluate the eligibility for gene therapy of patients diagnosed with this type of hRPE65 variant of uncertain significance.     

The autonomic innervation of rat jugular vein

Summary The autonomic innervation of rat jugular vein was studied using glyoxylic acid fluorescence and acetylcholinesterase histochemical methods. The rat jugular vein is provided with both adrenergic and cholinergic nerve fibers organized in plexuses located at the adventitial-medial border. The existence of these nerve plexuses does not seem to support biochemical findings that suggest a lack of innervation in the rat jugular vein and which propose this blood vessel as a model for the analysis of drug-smooth muscle cell interaction without the interference of neuronal uptake mechanisms.     

A truncated SV40 large T antigen lacking the p53 binding domain overcomes p53-induced growth arrest and immortalizes primary mesencephalic cells

As an alternative to primary fetal tissue, immortalized central nervous system (CNS)-derived cell lines are useful for in vitro CNS model systems and for gene manipulation with potential clinical use in neural transplantation. However, obtaining immortalized cells with a desired phenotype is unpredictable, because the molecular mechanisms of growth and differentiation of CNS cells are poorly understood. The SV40 large T antigen is commonly used to immortalize mammalian cells, but it interferes with multiple cell-cycle components, including p53, p300, and retinoblastoma protein, and usually produces cells with undifferentiated phenotypes. In order to increase the phenotypic repertoire of immortalized CNS cells and to address the molecular mechanisms underlying immortalization and differentiation, we constructed an expression vector containing a truncated SV40 large T gene that encodes only the amino-terminal 155 amino acids (T155), which lacks the p53-binding domain. Constructs were first transfected into a p53-temperature-sensitive cell line, T64-7B. Colonies expressing T155 proliferated at the growth-restrictive temperature. T155 was then transfected into primary cultures from embryonic day-14 rat mesencephalon. Two clonal cell lines were derived, AF-5 and AC-10, which co-expressed T155 and mature neuronal and astrocytic markers. Thus, the amino-terminal portion of SV40 large T is sufficient to: (1) overcome p53-mediated growth arrest despite the absence of a p53-binding region, and (2) immortalize primary CNS cells expressing mature markers while actively dividing. T155 and T155-transfectants may be useful for further studies of cell-cycle mechanisms and phenotyic expression in CNS cells or for further gene manipulation to produce cells with specific properties.     

Computing diversity from dated phylogenies and taxonomic hierarchies: does it make a difference to the conclusions?

Recently, dated phylogenies have been increasingly used for ecological studies on community structure and conservation planning. There is, however, a major impediment to a systematic application of phylogenetic methods in ecology: reliable phylogenies with time-calibrated branch lengths are lacking for a large number of taxonomic groups and this condition is likely to continue for a long time. A solution for this problem consists in using undated phylogenies or taxonomic hierarchies as proxies for dated phylogenies. Nonetheless, little is known on the potential loss of information of these approaches compared to studies using dated phylogenies with time-calibrated branch lengths. The aim of this study is to ask how the use of undated phylogenies and taxonomic hierarchies biases a very simple measure of diversity, the mean pairwise phylogenetic distance between community species, compared to the diversity of dated phylogenies derived from the freely available software Phylomatic. This is illustrated with three sets of data on plant species sampled at different scales. Our results show that: (1) surprisingly, the diversity computed from dated phylogenies derived from Phylomatic is more strongly related to the diversity computed from taxonomic hierarchies than to the diversity computed from undated phylogenies, while (2) less surprisingly, the strength of this relationship increases if we consider only angiosperm species.     

Association of dystrobrevin and regulatory subunit of protein kinase A: a new role for dystrobrevin as a scaffold for signaling proteins

The dystrophin-related and -associated protein dystrobrevin is a component of the dystrophin-associated protein complex, which directly links the cytoskeleton to the extracellular matrix. It is now thought that this complex also serves as a dynamic scaffold for signaling proteins, and dystrobrevin may play a role in this context. Since dystrobrevin involvement in signaling pathways seems to be dependent on its interaction with other proteins, we sought new insights and performed a two-hybrid screen of a mouse brain cDNA library using beta-dystrobrevin, the isoform expressed in non-muscle tissues, as bait. Among the positive clones characterized after the screen, one encodes the regulatory subunit RIalpha of the cAMP-dependent protein kinase A (PKA). We confirmed the interaction by in vitro and in vivo association assays, and mapped the binding site of beta-dystrobrevin on RIalpha to the amino-terminal region encompassing the dimerization/docking domain of PKA regulatory subunit. We also found that the domain of interaction for RIalpha is contained in the amino-terminal region of beta-dystrobrevin. We obtained evidence that beta-dystrobrevin also interacts directly with RIIbeta, and that not only beta-dystrobrevin but also alpha-dystrobrevin interacts with PKA regulatory subunits. We show that both alpha and beta-dystrobrevin are specific phosphorylation substrates for PKA and that protein phosphatase 2A (PP2A) is associated with dystrobrevins. Our results suggest a new role for dystrobrevin as a scaffold protein that may play a role in different cellular processes involving PKA signaling.     

Development of a homogeneous high-throughput live-cell G-protein-coupled receptor binding assay

The measurement of ligand receptor binding parameters for G-protein-coupled receptors is indispensable in the drug discovery process. Traditional ligand receptor binding assays require scale-up of cells and membrane preparations, which is an expensive and time-consuming process. In this report, the authors describe the development of a homogeneous live-cell binding assay for GPCRs using a fluorophore-labeled nonpeptide ligand. The model assay used Cy3B-labeled telenzepine and Chinese hamster ovary cells expressing M1 muscarinic acetylcholine receptors. This homogeneous live-cell fluorescence binding assay format is superior to the traditional binding methods because it measures binding of a ligand to intact receptors on living cells. The assay requires no washing or separation steps, thereby allowing a real-time kinetic readout for the determination of ligand association and dissociation from the intact receptors. The results also suggest that miniaturization is feasible without compromising the data quality.     

High‐Performance and Stable All‐Polymer Solar Cells Using Donor and Acceptor Polymers with Complementary Absorption

To explore the advantages of emerging all‐polymer solar cells (all‐PSCs), growing efforts have been devoted to developing matched donor and acceptor polymers to outperform fullerene‐based PSCs. In this work, a detailed characterization and comparison of all‐PSCs using a set of donor and acceptor polymers with both conventional and inverted device structures is performed. A simple method to quantify the actual composition and light harvesting contributions from the individual donor and acceptor is described. Detailed study on the exciton dissociation and charge recombination is carried out by a set of measurements to understand the photocurrent loss. It is unraveled that fine‐tuned crystallinity of the acceptor, matched donor and acceptor with complementary absorption and desired energy levels, and device architecture engineering can synergistically boost the performance of all‐PSCs. As expected, the PBDTTS‐FTAZ:PNDI‐T10 all‐PSC attains a high and stable power conversion efficiency of 6.9% without obvious efficiency decay in 60 d. This work demonstrates that PNDI‐T10 can be a potential alternative acceptor polymer to the widely used acceptor N2200 for high‐performance and stable all‐PSCs.     

Paleoenvironmental analysis

New analysis has been carried out concerning the palaeoenvironmental reconstruction of some Italian sites dating from the Middle Pleistocene to the Bronze Age. Different aspects have been investigated on each site considering the data collected. The following sites have been analyzed: Isernia La Pineta (Molise); Visogliano and Caverna degli Orsi (Tieste); Toirano Caves (Liguria); Grotta Paglicci (Gargano); Riparo del Molare (Salerno); Grotta del Cavallo (Lecce); Castellaro Lagusello (Monzambano, Mantova).