Search

Understanding the molecular mechanism by which the signal of the presence of an antibiotic is transduced from outside to inside the bacterial cell is of fundamental interest for the beta-lactam antibiotic resistance problem, but remains difficult to accomplish. No approach has ever addressed entire penicillin receptors in a membrane environment. Here we describe a method to investigate the purified Bacillus licheniformis BlaR1 receptor -a membrane-bound penicillin receptor involved in beta-lactam resistance- embedded into a lipid bilayer in absence or presence of penicillin. By selecting a mutated receptor blocked in its signal transduction pathway just after its activation by penicillin, we revealed the very first step of receptor signalling by unfolding the receptor from its C-terminal end by AFM-based single-molecule force spectroscopy. We showed that the presence of the antibiotic entails significant conformational changes within the receptor. Our approach opens an avenue to study signal-transduction pathways mediated by membrane-bound proteins in a membrane environment.

Human bone marrow-derived mesenchymal stem cells (hBMSCs) and their derivative enhanced green fluorescent protein (eGFP)-hBMSCs were employed to evaluate an innovative hybrid scaffold composed of granular hydroxylapatite and collagen hemostat (Coll/HA). The cellular morphology/cytoskeleton organization and cell viability were investigated by immunohistochemistry (IHC) and AlamarBlue metabolic assay, respectively. The expression of osteopontin and osteocalcin proteins was analyzed by IHC and ELISA, whereas osteogenic genes were investigated by quantitative PCR (Q-PCR). Cell morphology of eGFP-hBMSCs was indistinguishable from that of parental hBMSCs. The cytoskeleton architecture of hBMSCs grown on the scaffold appeared to be well organized, whereas its integrity remained uninfluenced by the scaffold during the time course. Metabolic activity measured in hBMSCs grown on a biomaterial was increased during the experiments, up to day 21 (p < 0.05). The biomaterial induced the matrix mineralization in hBMSCs. The scaffold favored the expression of osteogenic proteins, such as osteocalcin and osteopontin. In hBMSC cultures, the scaffold induced up-regulation in specific genes that are involved in ossification process (BMP2/3, SPP1, SMAD3, and SP7), whereas they showed an up-regulation of MMP9 and MMP10, which play a central role during the skeletal development. hBMSCs were induced to chondrogenic differentiation through up-regulation of COL2A1 gene. Our experiments suggest that the innovative scaffold tested herein provides a good microenvironment for hBMSC adhesion, viability, and osteoinduction. hBMSCs are an excellent in vitro cellular model to assay scaffolds, which can be employed for bone repair and bone tissue engineering.

Stainless steel, widely present in the food industry, is frequently exposed to bacterial colonization with possible consequences on consumers&rsquo, health. 288 stainless steel disks with different roughness (0.25, 0.5 and 1 &mu, m) were challenged with four Gram-negative (Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 1402, Yersinia enterocolitica ATCC 9610 and Pseudomonas aeruginosa ATCC 27588) and four Gram-positive bacteria (Staphylococcus aureus ATCC 6538, Enterococcus faecalis ATCC 29212, Bacillus cereus ATCC 14579 and Listeria monocytogenes NCTT 10888) and underwent three different sanitizing treatments (UVC, alcohol 70% v/v and Gold lotion). Moreover, the same procedure was carried out onto the same surfaces after a nanotechnological surface coating (nanoXHAM&reg, D). A significant bactericidal effect was exerted by all of the sanitizing treatments against all bacterial strains regardless of roughness and surface coating. The nanoXHAM&reg, D coating itself induced an overall bactericidal effect as well as in synergy with all sanitizing treatments regardless of roughness. Stainless steel surface roughness is poorly correlated with bacterial adhesion and only sanitizing treatments can exert significant bactericidal effects. Most of sanitizing treatments are toxic and corrosive causing the onset of crevices that are able to facilitate bacterial nesting and growth. This nanotechnological coating can reduce surface adhesion with consequent reduction of bacterial adhesion, nesting, and growth.

One of the main concerns of the food industry is microbial adhesion to food contact surfaces and consequent contamination. We evaluated the potential bacteriostatic/bactericidal efficacy of aluminum surfaces with different large-scale roughness (0.25, 0.5 and 1 &mu, m) before and after the surface treatment with a special anodizing based on titanium oxide nanotechnology (DURALTI&reg, ) and after 3 different sanitizing treatments, e.g., UV, alcohol and a natural product named Gold lotion. Four Gram-negative (Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 1402, Yersinia enterocolitica ATCC 9610 and Pseudomonas aeruginosa ATCC 27588) and four Gram-positive (Staphylococcus aureus ATCC 6538, Enterococcus faecalis ATCC 29212, Bacillus cereus ATCC 14579 and Listeria monocytogenes NCTT 10888) bacteria were screened. As far as concerns aluminum surfaces without nanotechnology surface treatment, an overall bacteriostatic effect was observed for all strains with respect to the initial inoculum that was 106 CFU/mL. Conversely, an overall bactericidal effect was observed both for Gram-negative and -positive bacteria on DURALTI&reg, treated aluminum disks, regardless of roughness and sanitizing treatment. These results are innovative in terms of the great potential of the antibacterial activity of nanotechnologically treated food contact surfaces and their combination with some sanitizing agents that might be exploited in the food industry.

multiplication of the alpha-synuclein-encoding gene is frequent cause of early onset Parkinson’s disease. Recent evidences point to the pathogenic role of excess alpha-synuclein also in sporadic Parkinson’s disease. Alpha-synuclein is a cytosolic protein which has been show to be released from neurons. Here we provide evidence that extracellular alpha-synuclein induces an increase in surface-exposed glucose-related protein of 78 kDa (GRP78), which becomes clustered in microdomains of the neuronal plasma membrane. Upon interacting with alpha-synuclein, GRP78 activates a signalling cascade leading to cofilin 1 inactivation and stabilization of microfilaments, thus affecting morphology and dynamics of actin cytoskeleton in cultured neurons. Down-regulation of GRP78 abolishes the activity of exogenous alpha-synuclein, indicating that it is the primary target of alpha-synuclein. Inactivation of cofilin 1 and stabilization of actin cytoskeleton are present also in fibroblasts derived from genetic Parkinson’s disease patients, which show a dramatic increase in stress fibers. Similar changes are displayed by control cells incubated with the medium of Parkinson’s disease fibroblasts, only when alpha-synuclein is present. The accumulation of alpha-synuclein in the extracellular milieu, its interaction with the plasma membrane, and alpha-synuclein-driven clustering of GRP78 appear therefore responsible for the dysregulation of actin turnover, leading to early deficits in synaptic function that precede neurodegeneration.

Glioblastoma multiforme (GBM) is the most aggressive malignant tumor of the central nervous system, with poor survival in both treated and untreated patients. Recent studies began to explain the molecular pathway, comprising the dynamic structural and mechanical changes involved in GBM. In this context, some studies showed that the human glioblastoma cells release high levels of glutamate, which regulates the proliferation and survival of neuronal progenitor cells. Considering that cancer cells possess properties in common with neural progenitor cells, it is likely that the functions of glutamate receptors may affect the growth of cancer cells and, therefore, open the road to new and more targeted therapies.

Single-cell force spectroscopy is an emerging technique in the field of biomedicine because it has proved to be a unique tool to obtain mechanical and functional information on living cells, with force resolution up to single molecular bonds. This technique was applied to the study of the cytoskeleton organisation of neuroblastoma cells, a life-threatening cancer typically developing during childhood, and the results were interpreted on the basis of reference experiments on human embryonic kidney cell line. An intimate connection emerges among cellular state, cytoskeleton organisation and experimental outcome that can be potentially exploited towards a new method for cancer stadiation of neuroblastoma cells. Copyright © 2012 John Wiley & Sons, Ltd.

// Ilaria Penna 1, 3, * , Arianna Gigoni 1, 3, * , Delfina Costa 1, 3, * , Serena Vella 4, * , Debora Russo 3 , Alessandro Poggi 3 , Federico Villa 1, 3 , Antonella Brizzolara 1, 3 , Claudio Canale 5 , Andrea Mescola 5 , Antonio Daga 3 , Claudio Russo 6 , Mario Nizzari 2 , Tullio Florio 2, 7 , Paola Menichini 3 , Aldo Pagano 1, 3 1 Department of Experimental Medicine (DIMES), University of Genova, Genova, Italy 2 Department of Internal Medicine (DIMI), University of Genova, Genova, Italy 3 IRCCS-AOU San Martino-IST, Genova, Italy 4 Department of Laboratory Medicine and Advanced Biotechnologies, IRCCS-ISMETT (Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione), Palermo, Italy 5 Nanophysics Unit, Italian Institute of Technology, Morego, Genova, Italy 6 Department of Health Sciences, University of Molise, Campobasso, Italy 7 Center of Excellence for Biomedical Research (CEBR), University of Genova, Genova, Italy * These authors have contributed equally to this work Correspondence to: Aldo Pagano, email: aldo.pagano@unige.it Keywords: neuroblastoma, non-coding RNA, GTSE1, tumorigenesis, metastasis Received: March 04, 2016 Accepted: November 23, 2016 Published: December 24, 2016 ABSTRACT We recently reported the in vitro over-expression of 45A, a RNA polymerase III-transcribed non-coding (nc)RNA, that perturbs the intracellular content of FE65L1 affecting cell proliferation rate, short-term response to genotoxic stress, substrate adhesion capacity and, ultimately, increasing the tumorigenic potential of human neuroblastoma cells. In this work, to deeply explore the mechanism by which 45A ncRNA contributes to cancer development, we targeted in vitro and in vivo 45A levels by the stable overexpression of antisense 45A RNA. 45A downregulation leads to deep modifications of cytoskeleton organization, adhesion and migration of neuroblastoma cells. These effects are correlated with alterations in the expression of several genes including GTSE1 (G2 and S phase-expressed-1), a crucial regulator of tumor cell migration and metastatic potential. Interestingly, the downregulation of 45A ncRNA strongly affects the in vivo tumorigenic potential of SKNBE2 neuroblastoma cells, increasing tumor nodule compactness and reducing GTSE1 protein expression in a subcutaneous neuroblastoma mouse model. Moreover, intracardiac injection of neuroblastoma cells showed that downregulation of 45A ncRNA also influences tumor metastatic ability. In conclusion, our data highlight a key role of 45A ncRNA in cancer development and suggest that its modulation might represent a possible novel anticancer therapeutic approach.