Publisher Correction: Cortical-like mini-columns of neuronal cells on zinc oxide nanowire surfaces.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The use of scanning electron microscopy and energy dispersive X-ray spectroscopy in a case of occupational death.

Scanning electron microscopy is a technique that provides high-resolution images at the micro- and nano-scale. The combination of scanning electron microscopy and energy dispersive X-ray spectroscopy analysis is developing fast for application in forensic science. In this work, we report a case of work-related traumatic death of a 50-year-old man. The autopsy showed cranial fractures with cerebral haemorrhage. It was more difficult to understand the accident dynamics because the body had been shifted from the accident site to mask what had really taken place. Scanning electron microscopy/energy dispersive X-ray spectroscopy was used to identify the material of the impacting tool and to establish the possible legal responsibility of the employer. In this study, we demonstrate that scanning electron microscopy/energy dispersive X-ray spectroscopy is a useful forensic tool for the analysis of biological samples. Further, for studying the lacerations on the corpse from doubtful blunt tools, scanning electron microscopy/energy dispersive X-ray spectroscopy can assist in demonstrating that the scene has been falsified, as it was in this case.

Cortical-like mini-columns of neuronal cells on zinc oxide nanowire surfaces.

A long-standing goal of neuroscience is a theory that explains the formation of the minicolumns in the cerebral cortex. Minicolumns are the elementary computational units of the mature neocortex. Here, we use zinc oxide nanowires with controlled topography as substrates for neural-cell growth. We observe that neuronal cells form networks where the networks characteristics exhibit a high sensitivity to the topography of the nanowires. For certain values of nanowires density and fractal dimension, neuronal networks express small world attributes, with enhanced information flows. We observe that neurons in these networks congregate in superclusters of approximately 200 neurons. We demonstrate that this number is not coincidental: the maximum number of cells in a supercluster is limited by the competition between the binding energy between cells, adhesion to the substrate, and the kinetic energy of the system. Since cortical minicolumns have similar size, similar anatomical and topological characteristics of neuronal superclusters on nanowires surfaces, we conjecture that the formation of cortical minicolumns is likewise guided by the interplay between energy minimization, information optimization and topology. For the first time, we provide a clear account of the mechanisms of formation of the minicolumns in the brain.

Superhydrophobic lab-on-chip measures secretome protonation state and provides a personalized risk assessment of sporadic tumour.

Secretome of primary cultures is an accessible source of biological markers compared to more complex and less decipherable mixtures such as serum or plasma. The protonation state (PS) of secretome reflects the metabolism of cells and can be used for cancer early detection. Here, we demonstrate a superhydrophobic organic electrochemical device that measures PS in a drop of secretome derived from liquid biopsies. Using data from the sensor and principal component analysis (PCA), we developed algorithms able to efficiently discriminate tumour patients from non-tumour patients. We then validated the results using mass spectrometry and biochemical analysis of samples. For the 36 patients across three independent cohorts, the method identified tumour patients with high sensitivity and identification as high as 100% (no false positives) with declared subjects at-risk, for sporadic cancer onset, by intermediate values of PS. This assay could impact on cancer risk management, individual's diagnosis and/or help clarify risk in healthy populations.

Nano-topography Enhances Communication in Neural Cells Networks.

Neural cells are the smallest building blocks of the central and peripheral nervous systems. Information in neural networks and cell-substrate interactions have been heretofore studied separately. Understanding whether surface nano-topography can direct nerve cells assembly into computational efficient networks may provide new tools and criteria for tissue engineering and regenerative medicine. In this work, we used information theory approaches and functional multi calcium imaging (fMCI) techniques to examine how information flows in neural networks cultured on surfaces with controlled topography. We found that substrate roughness S a affects networks topology. In the low nano-meter range, S a = 0-30 nm, information increases with S a . Moreover, we found that energy density of a network of cells correlates to the topology of that network. This reinforces the view that information, energy and surface nano-topography are tightly inter-connected and should not be neglected when studying cell-cell interaction in neural tissue repair and regeneration.

Non-invasive real-time biopsy of intracranial lesions using short time expanded circulating tumor cells on glass slide: report of two cases.

BACKGROUND: Circulating Tumor Cells (CTCs) are promising biomarkers for monitoring solid cancer and were used to monitor brain tumors. Here we report two cases in which, for the first time, CTCs were used in cytological diagnostic evaluation to discriminate a space-occupying lesion of the brain. CASE PRESENTATION: Two cases of focal intracranial lesions, unclassified for diagnosis, untreated and apparently symptomatic, were examined after high-contrast resolution Magnetic Resonance Imaging and/or Computed Tomography scans. CTCs were seeded on chamber slides and short-time expanded under the optimized conditions as we previously reported. The first case was a focal lesion localized in the parietal-occipital area in a 67-year-old woman. The second case was a 31-year-old man with an expansive intracerebral lesion localized in the left peri-trigonal area. Both patients underwent excisional biopsy. Histopathological evaluation of the biopsy confirmed the previous cytological diagnoses, and the analysis of the clinical outcomes retrospectively validated both diagnoses. CONCLUSIONS: The cases here reported illustrate the potential for using expanded CTCs as non-invasive, real-time biopsy. Moreover, non-invasive real-time biopsy can represent an alternative diagnostic tool to be used when a functional area of the brain is at risk of injury from excisional biopsy procedures.

Soluble CD54 induces human endothelial cells ex vivo expansion useful for cardiovascular regeneration and tissue engineering application.

AIM: Consistent expansion of primary human endothelial cells in vitro is critical in the development of engineered tissue. A variety of complex culture media and techniques developed from different basal media have been reported with alternate success. Incongruous results are further confounded by donor-to-donor variability and cellular source of derivation. Our results demonstrate how to overcome these limitations using soluble CD54 (sCD54) as additive to conventional culture medium. METHODS AND RESULTS: Isolated primary fragment of different vessel types was expanded in Ham's F12 DMEM, enriched with growth factors, Fetal Calf Serum and conditioned medium of Human Umbilical Vein Endothelial Cells (HUVEC) collected at different passages. Cytokine content of culture media was analyzed in order to identify the soluble factors correlating with better proliferation profile. sCD54 was found to induce the in vitro expansion of human endothelial cells (HECs) independently from the vessels source and even in the absence of HUVEC-conditioned medium. The HECs cultivated in the presence of sCD54 (50 ng/ml), resulted positive for the expression of CD146 and negative for CD45, and lower fibroblast contamination. Cells were capable to proliferate with an S phase of 25%, to produce vascular endothelial growth factor, VEGF, (10 ng/ml) and to give origin to vessel-like tubule in vitro. CONCLUSION: Our results demonstrate that sCD54 is an essential factor for the in-vitro expansion of HECs without donor and vessel-source variability. Resulting primary cultures can be useful, for tissue engineering in regenerative medicine (e.g. artificial micro tissue generation, coating artificial heart valve etc.) and bio-nanotechnology applications.

In vitro expansion of tumour cells derived from blood and tumour tissue is useful to redefine personalized treatment in non-small cell lung cancer patients.

The clinical development of locally and advanced non-small cell lung cancer (NSCLC) suffers from a lack of biomarkers as a guide in the selection of optimal prognostic prediction. Circulating Tumour Cells (CTCs) are correlated to prognosis and show efficacy in cancer monitoring in patients. However, their enumeration alone might be inadequate; it might also be critical to understand the viability, the apoptotic state and the kinetics of these cells. Here, we report what we believe to be a new and selective approach to visually detect tumour specific CTCs. Firstly, using labelled human lung cancer cells, we detected a specific density interval in which NSCL-CTCs were concentrated. Secondly, to better characterize CTCs in respect to their heterogeneous composition and tumour reference, blood and tumour biopsy were performed on specimens taken from the same patient. The approach consisted in comparing phenotype profile of CTCs, and their progenitor Tumour Stem Cells, (TSCs). Moreover, NSCL-CTCs were cultivated in short-time human cultures to provide response to drug sensitivity. Our bimodal approach allowed to reveal two items. Firstly, that one part of a tumour, proximal to the bronchial structure, displays a predominance of CD133+. Secondly, specific NSCL-CTCs Epithelial Cell Adhesion Molecule (EpCAM)+CD29+ can be used as a negative prognostic factor as well the high expression of CTCs EpCAM+. These data were confirmed by drug-sensitivity tests, in vitro, and by the survival curves, in vivo.

Protein-carbohydrate complex reveals circulating metastatic cells in a microfluidic assay.

Advances in carbohydrate sequencing technologies reveal the tremendous complexity of the glycome and the role that glycomics might have to bring insight into the biological functions. Carbohydrate-protein interactions, in particular, are known to be crucial to most mammalian physiological processes as mediators of cell adhesion and metastasis, signal transducers, and organizers of protein interactions. An assay is developed here to mimic the multivalency of biological complexes that selectively and sensitively detect carbohydrate-protein interactions. The binding of ß-galactosides and galectin-3--a protein that is correlated to the progress of tumor and metastasis--is examined. The efficiency of the assay is related to the expression of the receptor while anchoring to the interaction's strength. Comparative binding experiments reveal molecular binding preferences. This study establishes that the assay is robust to isolate metastatic cells from colon affected patients and paves the way to personalized medicine.

Interleukin-6 receptor superantagonist Sant7 inhibits TGF-beta-induced proliferation of human lung fibroblasts.

OBJECTIVES: Both interleukin-6 (IL-6) and transforming growth factor-beta (TGF-beta) are crucially involved in fibrotic events that characterize interstitial lung diseases (ILD). Therefore, the aim of this study was to investigate in primary cultures of normal and fibrotic human lung fibroblasts (HLF), exposed to either IL-6 or TGF-beta1, the effects on phosphorylation of mitogen-activated protein kinases (MAPK) and cell growth of IL-6 signalling inhibition, performed by the IL-6 receptor superantagonist Sant7. MATERIALS AND METHODS: MAPK phosphorylation was detected by Western blotting, HLF viability and proliferation were evaluated using the trypan blue staining and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, respectively. RESULTS: Sant7, at a concentration of 1 microg/mL, was capable of significantly inhibiting HLF proliferation and MAPK phosphorylation induced by cell exposure to IL-6 (100 ng/mL) or TGF-beta1 (10 ng/mL), whose actions were more evident in fibrotic cells. CONCLUSIONS: These findings suggest that, in HLFs derived from patients with ILDs, the proliferative mechanisms activated by TGF-beta1 are at least in part mediated by an increased release of IL-6, leading to phosphorylation-dependent MAPK activation. Such preliminary findings may thus open new therapeutic perspectives for fibrogenic ILDs, based on inhibition of signal transduction pathways stimulated by the IL-6 receptor.

Ageing, hormonal behaviour and cyclin D1 in ductal breast carcinomas.

Owing to the gradual modification of breast tissue in postmenopausal women, there can be differential effects on local oestrogen receptor (ER) expression, with potential impingement on the biological behaviour of cancer cells in the ageing. A series of 45 ductal carcinoma (DC) cases were selected in postmenopausal women who were not being treated with HRT. Immunohistochemical analyses were performed for hormone receptors and Ki67 expression. Fluorescence in situ hybridisation (FISH) analysis was carried out to study CCND1 amplification. The selected population was subdivided into three groups by age and was subjected to statistical studies: linear model analysis, estimation of relative incidence (RI), multivariate analysis, and nonparametric tests were performed to investigate whether there were any links between age and molecular variables in DCs. The results show a low rate of proliferation and high ER expression in the oldest age group. In the same group a close correlation was found between high ER expression and CCN in the older age group D1 amplification (P=0.000), as was a more advanced phenotype in terms of tumour size and presence of positive lymph nodes than in the other age groups considered. The results suggest that ductal breast cancer has a favourable molecular prognosis, especially in extreme old age. In particular, there is an inverse correlation between ageing and proliferation rate despite the presence of an accentuated proliferation stimulus (high ER with CCD1 amplifications) in the oldest group relative to the other groups considered.

Biological characterization of central and peripheral primary non small cell lung cancers (NSCLC).

BACKGROUND: Non Small Cell Lung Carcinomas (NSCLC) comprise 90% of all lung carcinomas. Studies have demonstrated a preferential central (bronchus-derived) localization for squamous cells, whereas adenocarcinomas are frequently peripheral (bronchiolo-alveolus derived). It has been suggested that exposure to carcinogenic insults including cigarette smoke, may induce different types of tumors in different locations. MATERIALS AND METHODS: Forty one NSCLC patients staged according to WHO and TNM were considered for localization and biological parameters (p53 expression, cell ploidy and S-phase). RESULTS: p53 overexpression was found more frequently in central than in peripheral tumors (69% vs 39%) (p = 0.074). Central tumors were more aneuploid (69%) than peripheral ones (46%) (p = 0.03) No difference in smoking habit was observed in the two groups. CONCLUSIONS: Our results suggest that there is no apparent biological difference between these two groups of NSCLCs, and that the smoking does not play a role in either histotype determination or biological behavior.

Immunohistochemical analysis of 4-aminobiphenyl-DNA adducts in oral mucosal cells of smokers and nonsmokers.

BACKGROUND: The "biologically effective dose markers", DNA and protein adducts, are a direct index of carcinogen induced cell damage and an indirect one of genetic susceptibility. This study aimed to examine the dose-response relationship for 4-Aminobiphenyl-DNA adducts in oral cells of smokers and non smokers. MATERIALS AND METHODS: An immunoperoxidase method with the monoclonal 3C8 antibody, which recognizes 4-Aminobiphenyl-DNA, has been used for detecting DNA damage in oral cells of 12 smokers and 12 non smokers. RESULTS: Higher staining for 4-Aminobiphenyl-DNA was detected in the cells of smokers (187 +/- 42) vs. non smokers (135 +/- 35) (p = 0.004), with a twofold range in relative staining for both groups, suggesting individual differences relevance in metabolizing carcinogens and/or repairing DNA damage. CONCLUSIONS: This non invasive method requiring small cell amounts is a tool for monitoring large groups of subjects at risk in primary prevention programs.

Screening of gestational diabetes in Tuscany: results in 2000 cases.

According to the guidelines of the "Third international workshop conference on GDM", we have examined 2000 pregnant women. The glucose challenge test (GCT) was positive in 408 cases (20.4%) and negative in 1592 (79.6%). The OGTT (Carpenter and Coustan's criteria) was performed in 647 pregnant women. GDM and IGGT prevalence was of 6.25% and 5.5% respectively and normal glucose tolerance (NGT) 88.25%. The GCT effectiveness for GDM and IGGT diagnosis is: sensibility 75.1%, specificity 44%, positive predictive value 46.4% and negative predictive value 74%. GDM and IGGT compared with NGT women were significantly older (p < 0.05) and prepregnancy BMI was higher (p < 0.01); the prevalence of previous macrosomia (p < 0.01), previous gestational diabetes (p < 0.01) and family history for diabetes mellitus (p < 0.05) was greater in GDM and IGCT. The prevalence of preterm delivery was higher in both GDM and IGCT (GDM 12.5% and IGGT 15.4% vs NGT 6%; p < 0.01), as well as the prevalence of cesarean sections (GDM 31.6% vs IGGT 23.5% and NGT 20.3%; p < 0.02), and the occurrence of macrosomia (GDM 27.6%, IGGT 16.6% and NGT 16.2%). In addition a higher prevalence (p < 0.01) of hyperbilirubinaemia, hypoglycemia and hypertrophy cardiomyopathy was observed in newborns from GDM women. Our data show that: GCT has a good specificity for GDM diagnosis, prevalence of GDM in our population is about 6%, GDM is still correlated to an elevated maternal and neonatal morbility.