An Intermittent Model for Intracellular Motions of Gold Nanostars by k-Space Scattering Image Correlation

Anisotropic metallic nanoparticles have been devised as powerful potential tools for in vivo imaging, photothermal therapy, and drug delivery thanks to plasmon-enhanced absorption and scattering cross sections, ease in synthesis and functionalization, and controlled cytotoxicity. The rational design of all these applications requires the characterization of the nanoparticles intracellular trafficking pathways. In this work, we exploit live-cell time-lapse confocal reflectance microscopy and image correlation in both direct and reciprocal space to investigate the intracellular transport of branched gold nanostars (GNSs). Different transport mechanisms, spanning from pure Brownian diffusion to (sub-)ballistic superdiffusion, are revealed by temporal and spatio-temporal image correlation spectroscopy on the tens-of-seconds timescale. According to these findings, combined with numerical simulations and with a Bayesian (hidden Markov model-based) analysis of single particle tracking data, we ascribe the superdiffusive, subballistic behavior characterizing the GNSs dynamics to a two-state switching between Brownian diffusion in the cytoplasm and molecular motor-mediated active transport. For the investigation of intermittent-type transport phenomena, we derive an analytical theoretical framework for Fourier-space image correlation spectroscopy (kICS). At first, we evaluate the influence of all the dynamic and kinetic parameters (the diffusion coefficient, the drift velocity, and the transition rates between the diffusive and the active transport regimes) on simulated kICS correlation functions. Then we outline a protocol for data analysis and employ it to derive whole-cell maps for each parameter underlying the GNSs intracellular dynamics. Capable of identifying even simpler transport phenomena, whether purely diffusive or ballistic, our intermittent kICS approach allows an exhaustive investigation of the dynamics of GNSs and biological macromolecules.     

D-Asp: a new player in reproductive endocrinology of the amphibian Rana esculenta

We investigated the involvement of D-Aspartic acid (D-Asp) on ovarian and testicular morphology of the green frog, Rana esculenta, and its effect on the testosterone production. The study has been performed throughout the reproductive cycle. In both ovary and testis a substantial amount of D-Asp is endogenously present and its concentration varies as function of reproduction. In the frog, D-Asp content is differently correlated with gonadal and plasmatic levels of testosterone, depending on the sex. In fact, the amount of the D-Asp is inversely linked with that of the testosterone in the ovary, while this correlation directly matched in the testis. In vivo short-term experiments, consisting of a single intra-peritoneal injection of D-Asp (2.0 μmol/g body weight), demonstrated that the enantiomer is significantly accumulated by both the ovary and testis, reaching after 3 h the highest uptake and thereafter decreasing to baseline values within 24 h. Furthermore, D-Asp influences the synthesis and/or the release of testosterone, causing a decrease of its level in the female, and an increase in the male, respectively. In vivo long-term experiments, D-Asp, chronically administered to the frogs of both sexes, enhances the maturation of both gonads, determining in the oocytes an higher accumulation of carbohydrate yolk plates in the ooplasm, and stimulating the spermatogenesis in the testis. Taken altogether, our results show that D-Asp operates differently in female and male frog gonads, indicating that it has different targets in the reproductive machinery depending on the sex.     

Molecular analysis of relapses or reinfections of Clostridium difficile-associated diarrhea

Recurrence is a major complication of Clostridium difficile-associated diarrhea and occurs in 15 to 20% of patients after discontinuation of therapy. Strains from 53 patients with Clostridium difficile recurrences were fingerprinted by PCR ribotyping. Reinfection with a different strain occurred in 15 out of 53 patients (28,3%), while 38 patients relapsed. These data suggest the need to perform molecular typing for implementation of infection control procedures and for a more appropriate therapeutic strategy.     

An atlas of altered expression of deubiquitinating enzymes in human cancer

Background

Deubiquitinating enzymes (DUBs) are proteases that process ubiquitin (Ub) or ubiquitin-like gene products, remodel polyubiquitin(-like) chains on target proteins, and counteract protein ubiquitination exerted by E3 ubiquitin-ligases. A wealth of studies has established the relevance of DUBs to the control of physiological processes whose subversion is known to cause cellular transformation, including cell cycle progression, DNA repair, endocytosis and signal transduction. Altered expression of DUBs might, therefore, subvert both the proteolytic and signaling functions of the Ub system.

Methodology/Principal Findings

In this study, we report the first comprehensive screening of DUB dysregulation in human cancers by in situ hybridization on tissue microarrays (ISH-TMA). ISH-TMA has proven to be a reliable methodology to conduct this kind of study, particularly because it allows the precise identification of the cellular origin of the signals. Thus, signals associated with the tumor component can be distinguished from those associated with the tumor microenvironment. Specimens derived from various normal and malignant tumor tissues were analyzed, and the “normal” samples were derived, whenever possible, from the same patients from whom tumors were obtained. Of the ∼90 DUBs encoded by the human genome, 33 were found to be expressed in at least one of the analyzed tissues, of which 22 were altered in cancers. Selected DUBs were subjected to further validation, by analyzing their expression in large cohorts of tumor samples. This analysis unveiled significant correlations between DUB expression and relevant clinical and pathological parameters, which were in some cases indicative of aggressive disease.

Conclusions/Significance

The results presented here demonstrate that DUB dysregulation is a frequent event in cancer, and have implications for therapeutic approaches based on DUB inhibition.     

Role of the nitric oxide/cyclic GMP pathway and extracellular environment in the nitric oxide donor-induced increase in dopamine secretion from PC12 cells: a microdialysis in vitro study

In vitro microdialysis was used to investigate the mechanism of nitric oxide (NO) donor-induced changes in dopamine (DA) secretion from PC12 cells. Infusion of the NO-donor S-nitroso-N-acetylpenicillamine (SNAP, 1.0 mm) induced a long-lasting increase in DA and 3-methoxytyramine (3-MT) dialysate concentrations. SNAP-induced increases were inhibited either by pre-infusion of the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4] oxadiazolo[4,3]quinoxalin-1-one (ODQ, 0.1 mm) or by Ca2+ omission. Ca2+ re-introduction restored SNAP effects. SNAP-induced increases in DA + 3-MT were unaffected by co-infusion of the l-type Ca2+ channel inhibitor nifedipine. The NO-donor (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3, 1.0 mm) induced a short-lasting decrease in dialysate DA + 3-MT. Ascorbic acid (0.2 mm) co-infusion allowed NOR-3 to increase dialysate DA + 3-MT. ODQ pre-infusion inhibited NOR-3 + ascorbic acid-induced DA + 3-MT increases. Infusion of high K+ (75 mm) induced a 2.5-fold increase in dialysate DA + 3-MT. The increase was abolished by NOR-3 co-infusion. Conversely, co-infusion of ascorbic acid (0.2 mm) with NOR-3 + high K+ restored high K+ effects. Co-infusion of nifedipine inhibited high K+-induced DA + 3-MT increases. These results suggest that activation of the NO/sGC/cyclic GMP pathway may be the underlying mechanism of extracellular Ca2+-dependent effects of exogenous NO on DA secretion from PC12 cells. Extracellular Ca2+ entry may occur through nifedipine-insensitive channels. NO effects and DA concentrations in dialysates largely depend on both the timing of NO generation and the extracellular environment in which NO is generated.     

Fetal DNA detection in maternal plasma throughout gestation

The presence of fetal DNA in maternal plasma may represent a source of genetic material which can be obtained noninvasively. We wanted to assess whether fetal DNA is detectable in all pregnant women, to define the range and distribution of fetal DNA concentration at different gestational ages, to identify the optimal period to obtain a maternal blood sample yielding an adequate amount of fetal DNA for prenatal diagnosis, and to evaluate accuracy and predictive values of this approach. This information is crucial to develop safe and reliable non-invasive genetic testing in early pregnancy and monitoring of pregnancy complications in late gestation. Fetal DNA quantification in maternal plasma was carried out by real-time PCR on the SRY gene in male-bearing pregnancies to distinguish between maternal and fetal DNA. A cohort of 1,837 pregnant women was investigated. Fetal DNA could be detected from the sixth week and could be retrieved at any gestational week. No false-positive results were obtained in 163 women with previous embryo loss or previous male babies. Fetal DNA analysis performed blindly on a subset of 464 women displayed 99.4, 97.8 and 100% accuracy in fetal gender determination during the first, second, and third trimester of pregnancy, respectively. No SRY amplification was obtained in seven out of the 246 (2.8%) male-bearing pregnancies. Fetal DNA from maternal plasma seems to be an adequate and reliable source of genetic material for a noninvasive prenatal diagnostic approach.     

Improvement of a FRET-based indicator for cAMP by linker design and stabilization of donor-acceptor interaction

F?rster resonance energy transfer (FRET) technology has been used to develop genetically encoded fluorescent indicators for a variety of intracellular molecular events. Often, however, the poor dynamic range of such reporters prevents detection of subtle but physiologically relevant signals. Here we present a strategy for improving FRET efficiency between donor and acceptor fluorophores in a green fluorescent protein (GFP)-based protein indicator for cAMP. Such indicator is based on protein kinase A (PKA) and was generated by fusion of CFP and YFP to the regulatory and catalytic subunits of PKA, respectively. Our approach to improve FRET efficiency was to perform molecular dynamic simulations and modelling studies of the linker peptide (L11) joining the CFP moiety and the regulatory subunit in order to define its structure and use this information to design an improved linker. We found that L11 contains the X-Y-P-Y-D motif, which adopts a turn-like conformation that is stiffly conserved along the simulation time. Based on this finding, we designed a new linker, L22 in which the YPY motif was doubled in order to generate a stiffer peptide and reduce the mobility of the chromophore within the protein complex, thus favouring CFP/YFP dipole-dipole interaction and improving FRET efficiency. Molecular dynamic simulations of L22 showed, unexpectedly, that the conformational behaviour of L22 was very loose. Based on the analysis of the three principal conformational states visited by L22 during the simulation time, we modified its sequence in order to increase its rigidity. The resulting linker L20 displayed lower flexibility and higher helical content than L22. When inserted in the cAMP indicator, L20 yielded a probe showing almost doubled FRET efficiency and a substantially improved dynamic range.     

Conformational changes of murine polyomavirus capsid proteins induced by sialic acid binding

Murine polyomavirus (Py) infection initiates by the recognition of cell membrane molecules containing terminal sialic acid (SA) residues through specific binding pockets formed at the major capsid protein VP1 surface. VP1 Pockets 1, 2, and 3 bind terminal SA, Gal, and second branched SA, respectively. The consequence of recognition on viral cell entry remains elusive. In this work, we show that preincubation of Py with soluble compounds within Pocket 1 (N-acetyl or N-glycolyl neuraminic acids) increases Py cell binding and infectivity in murine 3T6 fibroblasts. In contrast, Gal does not significantly alter Py binding nor infectivity, whereas sialyllactose, in Pockets 1 and 2, decreases cell binding and infectivity. Binding experiments with Py virus-like particles confirmed the direct involvement of VP1 in this effect. To determine whether such results could reflect VP1 conformational changes induced by SA binding, protease digestion assays were performed after pretreatment of Py or virus-like particles with soluble receptor fragments. Binding of SA with the VP1 Pocket 1, but not of compounds interacting with Pocket 2, was associated with a transition of this protein from a protease-sensitive to a protease-resistant state. This effect was transmitted to the minor capsid proteins VP2 and VP3 in virus particles. Attachment of Py to cell monolayers similarly led to a VP1 trypsin-resistant pattern. Taken together, these data present evidence that initial binding of Py to terminal SA induces conformational changes in the viral capsid, which may influence subsequent virus cell entry steps.