Controlled Lactonization of o-Coumaric Esters Mediated by Supramolecular Gels

Fragrances are volatile organic compounds widely used in our daily life. Unfortunately, the high volatility required to reach human receptors reduces their persistency in the air. To contrast this effect, several strategies may be used. Among them, we present here the combination of two techniques: the microencapsulation in supramolecular gels and the use of profragrances. We report a study on the controlled lactonization of four esters derived from o-coumaric acid. The ester lactonization spontaneously occurs after exposure to solar light, releasing coumarin and the corresponding alcohol. To determine the rate of fragrance release, we compared the reaction in solution and in a supramolecular gel and we demonstrated that the lactonization reaction always occurs slower in the gel. We also studied the more suitable gel for this aim, by comparing the properties of two supramolecular gels obtained with the gelator Boc-L-DOPA(Bn)(2)-OH in a 1:1 ethanol/water mixture in different gelator concentration (0.2% and 1% w/v). The gel prepared with 1% w/v gelator concentration is stronger and less transparent than the other and was used for the profragrances encapsulation. In any case, we obtained a significative reduction of lactonization reaction in gel, compared with the same reaction in solution

Use of particle counter system for the optimization of sampling ,identification and decontamination procedures for biological aerosols dispersion in confined environment

Abstract In a CBRNe (Chemical, Biological, Radiological, Nuclear and explosive) scenario, biological agents hardly allow efficient detection/identification because of the incubation time that provides a lag in symptoms outbreak following their dissemination. The detection of atmospheric dispersion of biological agents (i.e.: toxins, viruses, bacteria and so on) is a key issue for the safety of people and security of environment. Another fundamental aspect is related to the efficiency of the sampling method, which leads to the identification of the agent released, in fact an effective sampling method is needed either to identify the contamination and to check for the decontamination procedure. Environmental monitoring is one of the ways to improve fast detection of biological agents; for instance, particle counters with the ability of discriminating between biological and non-biological particles are used for a first warning when the amount of biological particles exceeds a particular threshold. Nevertheless, these systems are not able to distinguish between pathogen and non-pathogen organisms, thus, classical “laboratory” assays are still required to unambiguously identify the particle which triggered the warning signal. In this work, a combination of commercially available equipment for detection and identification of the atmospheric dispersion of biological agents was evaluated in partnership between the Italian Army, the Department of Industrial Engineering and the School of Medicine and Surgery of the University of Rome “Tor Vergata”. The aim of this work, whose results are presented here, was to conduce preliminary studies on the dynamics of biological aerosols fallout after its dispersion, to improve detection, sampling and identification techniques. This will help minimizing the impact of the release of biological agents, guarantee environmental, and people safety and securit

Identification of a family of human F-box proteins

F-box proteins are an expanding family of eukaryotic proteins characterized by an approximately 40 amino- acid motif, the F box (so named because cyclin F was one of the first proteins in which this motif was identified) [1]. Some F-box proteins have been shown to be critical for the controlled degradation of cellular regulatory proteins [2,3]. In fact, F-box proteins are one of the four subunits of ubiquitin protein ligases called SCFs. The other three subunits are the Skp1 protein; one of the cullin proteins (Cul1 in metazoans and Cdc53 or Cul A in the yeast Saccharomyces cerevisiae); and the recently identified Roc1 protein (also called Rbx1 or Hrt1). SCF ligases bring ubiquitin conjugating enzymes (either Ubc3 or Ubc4) to substrates that are specifically recruited by the different F-box proteins. The need for high substrate specificity and the large number of known F-box proteins in yeast and worms [2,4] suggest the existence of a large family of mammalian F-box proteins. Using Skp1 as a bait in a yeast two-hybrid screen and by searching DNA databases, we identified a family of 26 human F-box proteins, 25 of which were novel. Some of these proteins contained WD-40 domains or leucine-rich repeats; others contained either different protein\u2013protein interaction modules or no recognizable motifs. We have named the F-box proteins that contain WD-40 domains Fbws, those containing leucine-rich repeats Fbls, and the remaining ones Fbxs. We have further characterized representative members of these three classes of F-box proteins

Preferential Phosphorylation of R-domain Serine 768 Dampens Activation of CFTR Channels by PKA

CFTR (cystic fibrosis transmembrane conductance regulator), the protein whose dysfunction causes cystic fibrosis, is a chloride ion channel whose gating is controlled by interactions of MgATP with CFTR's two cytoplasmic nucleotide binding domains, but only after several serines in CFTR's regulatory (R) domain have been phosphorylated by cAMP-dependent protein kinase (PKA). Whereas eight R-domain serines have previously been shown to be phosphorylated in purified CFTR, it is not known how individual phosphoserines regulate channel gating, although two of them, at positions 737 and 768, have been suggested to be inhibitory. Here we show, using mass spectrometric analysis, that Ser 768 is the first site phosphorylated in purified R-domain protein, and that it and five other R-domain sites are already phosphorylated in resting Xenopus oocytes expressing wild-type (WT) human epithelial CFTR. The WT channels have lower activity than S768A channels (with Ser 768 mutated to Ala) in resting oocytes, confirming the inhibitory influence of phosphoserine 768. In excised patches exposed to a range of PKA concentrations, the open probability (Po) of mutant S768A channels exceeded that of WT CFTR channels at all [PKA], and the half-maximally activating [PKA] for WT channels was twice that for S768A channels. As the open burst duration of S768A CFTR channels was almost double that of WT channels, at both low (55 nM) and high (550 nM) [PKA], we conclude that the principal mechanism by which phosphoserine 768 inhibits WT CFTR is by hastening the termination of open channel bursts. The right-shifted Po-[PKA] curve of WT channels might explain their slower activation, compared with S768A channels, at low [PKA]. The finding that phosphorylation kinetics of WT or S768A R-domain peptides were similar provides no support for an alternative explanation, that early phosphorylation of Ser 768 in WT CFTR might also impair subsequent phosphorylation of stimulatory R-domain serines. The observed reduced sensitivity to activation by [PKA] imparted by Ser 768 might serve to ensure activation of WT CFTR by strong stimuli while dampening responses to weak signals

Xeno-free trans-differentiation of adipose tissue-derived mesenchymal stem cells into glial and neuronal cells.

Mesenchymal stem cells (MSCs) are undifferentiated cells that have the ability of self-renewal and trans-differentiation into other cell types. They hold out hope for finding a cure for many diseases. Nevertheless, there are still some obstacles that limit their clinical transplantation. One of these obstacles are the xenogeneic substances added in either proliferation or differentiation media with subsequent immunogenic and infectious transmission problems. In this study, we aimed to replace fetal bovine serum (FBS), the main nutrient source for MSC proliferation with xeno-free blood derivatives. We tested the effect of human activated pure platelet-rich plasma (P-PRP) and advanced platelet-rich fibrin (A-PRF) on the proliferation of human adipose derived-MSCs (AD-MSCs) at different concentrations. For the induction of MSC neural differentiation, we used human cerebrospinal fluid (CSF) at different concentrations in combination with P-PRP to effect xeno-free/species-specific neuronal/glial differentiation and we found that media with 10% CSF and 10% PRP promoted glial differentiation, while media with only 10% PRP induced a neuron-like phenotype

Human Olfactory Bulb Neural Stem Cells (Hu-OBNSCs) Can Be Loaded with Paclitaxel and Used to Inhibit Glioblastoma Cell Growth

Exploitation of the potential ability of human olfactory bulb (hOB) cells to carry, release, and deliver an effective, targeted anticancer therapy within the central nervous system (CNS) milieu remains elusive. Previous studies have demonstrated the marked ability of several types of stem cells (such as mesenchymal stem cells (MSCs) to carry and release different anti-cancer agents such as paclitaxel (PTX). Herein we investigate the ability of human olfactory bulb neural stem cells (Hu-OBNSCs) to carry and release paclitaxel, producing effective cytotoxic effects against cancer cells. We isolated Hu-OBNSCs from the hOB, uploaded them with PTX, and studied their potential cytotoxic effects against cancer cells in vitro. Interestingly, the Hu-OBNSCs displayed a five-fold increase in their resistance to the cytotoxicity of PTX, and the PTX-uploaded Hu-OBNSCs were able to inhibit proliferation and invasion, and to trigger marked cytotoxic effects on glioblastoma multiforme (GBM) cancer cells, and Human Caucasian fetal pancreatic adenocarcinoma 1 (CFPAC-1) in vitro. Despite their ability to resist the cytotoxic activity of PTX, the mechanism by which Hu-OBNSCs acquire resistance to PTX is not yet explained. Collectively our data indicate the ability of the Hu-OBNSCs to resist PTX, and to trigger effective cytotoxic effects against GBM cancer cells and CFPAC-1. This indicates their potential to be used as a carrier/vehicle for targeted anti-cancer therapy within the CNS

Brahma is required for cell cycle arrest and late muscle gene expression during skeletal myogenesis

Although the two catalytic subunits of the SWI/SNF chromatin-remodeling complex—Brahma (Brm) and Brg1—are almost invariably co-expressed, their mutually exclusive incorporation into distinct SWI/SNF complexes predicts that Brg1- and Brm-based SWI/SNF complexes execute specific functions. Here, we show that Brg1 and Brm have distinct functions at discrete stages of muscle differentiation. While Brg1 is required for the activation of muscle gene transcription at early stages of differentiation, Brm is required for Ccnd1 repression and cell cycle arrest prior to the activation of muscle genes. Ccnd1 knockdown rescues the ability to exit the cell cycle in Brm-deficient myoblasts, but does not recover terminal differentiation, revealing a previously unrecognized role of Brm in the activation of late muscle gene expression independent from the control of cell cycle. Consistently, Brm null mice displayed impaired muscle regeneration after injury, with aberrant proliferation of satellite cells and delayed formation of new myofibers. These data reveal stage-specific roles of Brm during skeletal myogenesis, via formation of repressive and activatory SWI/SNF complexes

Direct CD32 T-cell cytotoxicity: implications for breast cancer prognosis and treatment

The FcγRII (CD32) ligands are IgFc fragments and pentraxins. The existence of additional ligands is unknown. We engineered T cells with human chimeric receptors resulting from the fusion between CD32 extracellular portion and transmembrane CD8α linked toCD28/ζ chain intracellular moiety (CD32-CR). Transduced T cells recognized three breast cancer (BC) and one colon cancer cell line among 15 tested in the absence of targeting antibodies. Sensitive BC cell conjugation with CD32-CR T cells induced CD32 polarization and down-regulation, CD107a release, mutual elimination, and proinflammatory cytokine production unaffected by human IgGs but enhanced by cetuximab. CD32-CR T cells protected immunodeficient mice from subcutaneous growth of MDA-MB-468 BC cells. RNAseq analysis identified a 42 gene fingerprint predicting BC cell sensitivity and favorable outcomes in advanced BC. ICAM1 was a major regulator of CD32-CR T cell–mediated cytotoxicity. CD32-CR T cells may help identify cell surface CD32 ligand(s) and novel prognostically relevant transcriptomic signatures and develop innovative BC treatments

Degradation of Cdc25A by \u3b2-TrCP during S phase and in response to DNA damage

The Cdc25A phosphatase is essential for cell-cycle progression because of its function in dephosphorylating cyclin-dependent kinases. In response to DNA damage or stalled replication, the ATM and ATR protein kinases activate the checkpoint kinases Chk1 and Chk2, which leads to hyperphosphorylation of Cdc25A1\u20133. These events stimulate the ubiquitin-mediated pro- teolysis of Cdc25A1,4,5 and contribute to delaying cell-cycle progression, thereby preventing genomic instability1\u20137. Here we report that b-TrCP is the F-box protein that targets phosphory- lated Cdc25A for degradation by the Skp1/Cul1/F-box protein complex. Downregulation of b-TrCP1 and b-TrCP2 expression by short interfering RNAs causes an accumulation of Cdc25A in cells progressing through S phase and prevents the degradation of Cdc25A induced by ionizing radiation, indicating that b-TrCP may function in the intra-S-phase checkpoint. Consistent with this hypothesis, suppression of b-TrCP expression results in radioresistant DNA synthesis in response to DNA damage\u2014a phenotype indicative of a defect in the intra-S-phase checkpoint that is associated with an inability to regulate Cdc25A properly. Our results show that b-TrCP has a crucial role in mediating the response to DNA damage through Cdc25A degradation

CD16-158-valine chimeric receptor T cells overcome the resistance of KRAS-mutated colorectal carcinoma cells to cetuximab

KRAS mutations hinder therapeutic efficacy of epidermal growth factor receptor (EGFR)-specific monoclonal antibodies cetuximab and panitumumab-based immunotherapy of EGFR+ cancers. Although cetuximab inhibits KRAS-mutated cancer cell growth in vitro by natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), KRAS-mutated colorectal carcinoma (CRC) cells escape NK cell immunosurveillance in vivo. To overcome this limitation, we used cetuximab and panitumumab to redirect Fcγ chimeric receptor (CR) T cells against KRAS-mutated HCT116 colorectal cancer (CRC) cells. We compared four polymorphic Fcγ-CR constructs including CD16158F-CR, CD16158V-CR, CD32131H-CR, and CD32131R-CR transduced into T cells by retroviral vectors. Percentages of transduced T cells expressing CD32131H-CR (83.5 ± 9.5) and CD32131R-CR (77.7 ± 13.2) were significantly higher than those expressing with CD16158F-CR (30.3 ± 10.2) and CD16158V-CR (51.7 ± 13.7) (p < 0.003). CD32131R-CR T cells specifically bound soluble cetuximab and panitumumab. However, only CD16158V-CR T cells released high levels of interferon gamma (IFNγ = 1,145.5 pg/ml ±16.5 pg/ml, p < 0.001) and tumor necrosis factor alpha (TNFα = 614 pg/ml ± 21 pg/ml, p < 0.001) upon incubation with cetuximab-opsonized HCT116 cells. Moreover, only CD16158V-CR T cells combined with cetuximab killed HCT116 cells and A549 KRAS-mutated cells in vitro. CD16158V-CR T cells also effectively controlled subcutaneous growth of HCT116 cells in CB17-SCID mice in vivo. Thus, CD16158V-CR T cells combined with cetuximab represent useful reagents to develop innovative EGFR+KRAS-mutated CRC immunotherapies