Development of a high brightness ultrafast Transmission Electron Microscope based on a laser-driven cold field emission source

We report on the development of an ultrafast Transmission Electron Microscope based on a cold field emission source which can operate in either DC or ultrafast mode. Electron emission from a tungsten nanotip is triggered by femtosecond laser pulses which are tightly focused by optical components integrated inside a cold field emission source close to the cathode. The properties of the electron probe (brightness, angular current density, stability) are quantitatively determined. The measured brightness is the largest reported so far for UTEMs. Examples of imaging, diffraction and spectroscopy using ultrashort electron pulses are given. Finally, the potential of this instrument is illustrated by performing electron holography in the off-axis configuration using ultrashort electron pulses.Comment: 23 pages, 9 figure

Evaluation of posttreatment response of hepatocellular carcinoma: comparison of ultrasonography with second-generation ultrasound contrast agent and multidetector CT

We evaluated the ability of one-month follow-up contrast- enhanced ultrasound (CEUS) with second-generation contrast agent in monitoring radio frequency ablation (RFA) and transcatheter arterial chemoembolization (TACE) treatments of hepatocellular carcinoma (HCC). One-hundred forty-eight HCCs were studied using CEUS: 110 nodules were treated with RFA [41/110 RFA were performed using a pretreatment and an immediate postablation evaluation using CEUS (group 1); 69/110 using only US guidance (group 2)] and 38 nodules treated with TACE. For statistical analysis, McNemar test was used. Overall complete response was observed in 107/148 nodules (92/110 treated with RFA and 15/38 with TACE). A better rate of complete response was found in group 1 compared to group 2 (92.7% vs. 78.3%). In RFA treatment, CEUS showed a sensitivity of 83.3% and a specificity of 100% (diagnostic accuracy of 97%) using MDCT as reference standard with no statistical difference (p > 0.05). CEUS detected all cases of incomplete response in HCC treated with TACE using angiography as reference standard (diagnostic accuracy 100%).We recommend assessing residual intratumoral flow on CEUS during RFA procedure to determine the necessity of immediate additional treatment. In case of positive CEUS results, HCC treated with TACE should be considered still viable

Nanostructured Ni-Fe-P Alloy for Alkaline Electrolyzer

In recent years, the interest towards green hydrogen has drastically increased due to the global decarbonization process. Electrochemical water splitting is considered an attractive solution to convert and store the surplus energy from renewable energy sources. However, hydrogen production by water electrolysis is not economically sustainable. To reduce the cost of produced hydrogen, it is necessary to switch from noble-metal catalyst (Pt, Pd…) to cheap alternatives with a lower per unit energy cost but at the same time able to guarantee a high electrocatalytic activity for both oxygen and hydrogen evolution reactions. Among transition metals, nickel was selected as active material for its low cost and high chemical stability in alkaline media. Currently, the most investigated transition metal catalyst includes alloy of nickel with sulfide, phosphide, and nitride. In this work, a ternary alloy of Nickel-Iron-Phosphorus with nanowires morphology was investigated and compared to the binary alloy of Nickel-Iron. Ni-Fe-P NWs electrodes were obtained by potential-controlled pulse electrochemical deposition using polycarbonate membrane as template. Electrodes morphology and structure were studied by scanning electrode microscopy (SEM), energy diffraction spectroscopy (EDS) and X-ray diffraction (XRD). Electrodes were tested both as cathodes as anodes by Quasi Steady State Polarization (QSSP) and Galvanostatic Test. All the tests were performed in 30% w/w KOH aqueous solution at room temperature. Preliminary results showed better performance of the ternary alloy compared to the binary one

Sub-Toxic Human Amylin Fragment Concentrations Promote the Survival and Proliferation of SH-SY5Y Cells via the Release of VEGF and HspB5 from Endothelial RBE4 Cells

This work is licensed under a Creative Commons Attribution 4.0 International License.Human amylin is a 37-residue peptide hormone (hA1-37) secreted by β-cells of the pancreas and, along with insulin, is directly associated with type 2 diabetes mellitus (T2DM). Amyloid deposits within the islets of the pancreas represent a hallmark of T2DM. Additionally, amylin aggregates have been found in blood vessels and/or brain of patients with Alzheimer’s disease, alone or co-deposited with β-amyloid. The purpose of this study was to investigate the neuroprotective potential of human amylin in the context of endothelial-neuronal “cross-talk”. We initially performed dose-response experiments to examine cellular toxicity (quantified by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] MTT assay) of different hA17–29 concentrations in endothelial cells (RBE4). In the culture medium of these cells, we also measured heat shock protein B5 (HspB5) levels by ELISA, finding that even a sub-toxic concentration of hA17–29 (3 µM) produced an increase of HspB5. Using a cell medium of untreated and RBE4 challenged for 48 h with a sub-toxic concentration of hA17–29, we determined the potential beneficial effect of their addition to the medium of neuroblastoma SH-SY5Y cells. These cells were subsequently incubated for 48 h with a toxic concentration of hA17–29 (20 µM). We found a complete inhibition of hA17–29 toxicity, potentially related to the presence in the conditioned medium not only of HspB5, but also of vascular endothelial growth factor (VEGF). Pre-treating SH-SY5Y cells with the anti-Flk1 antibody, blocking the VEGF receptor 2 (VEGFR2), significantly decreased the protective effects of the conditioned RBE4 medium. These data, obtained by indirectly measuring VEGF activity, were strongly corroborated by the direct measurement of VEGF levels in conditioned RBE4 media as detected by ELISA. Altogether, these findings highlighted a novel role of sub-toxic concentrations of human amylin in promoting the secretion of proteic factors by endothelial cells (HspB5 and VEGF) that support the survival and proliferation of neuron-like cells.National Science Foundation (CHE-1411993)NIH COBRE P20GM103638American Heart Association-Midwest Affiliate Postdoctoral Research Fellowship (NFP0075515)Neuropsychopharmacology Research Program 2017 (RC-06-05

Microfluidics as a Novel Tool for Biological and Toxicological Assays in Drug Discovery Processes: Focus on Microchip Electrophoresis

This work is licensed under a Creative Commons Attribution 4.0 International License.The last decades of biological, toxicological, and pharmacological research have deeply changed the way researchers select the most appropriate ‘pre-clinical model’. The absence of relevant animal models for many human diseases, as well as the inaccurate prognosis coming from ‘conventional’ pre-clinical models, are among the major reasons of the failures observed in clinical trials. This evidence has pushed several research groups to move more often from a classic cellular or animal modeling approach to an alternative and broader vision that includes the involvement of microfluidic-based technologies. The use of microfluidic devices offers several benefits including fast analysis times, high sensitivity and reproducibility, the ability to quantitate multiple chemical species, and the simulation of cellular response mimicking the closest human in vivo milieu. Therefore, they represent a useful way to study drug–organ interactions and related safety and toxicity, and to model organ development and various pathologies ‘in a dish’. The present review will address the applicability of microfluidic-based technologies in different systems (2D and 3D). We will focus our attention on applications of microchip electrophoresis (ME) to biological and toxicological studies as well as in drug discovery and development processes. These include high-throughput single-cell gene expression profiling, simultaneous determination of antioxidants and reactive oxygen and nitrogen species, DNA analysis, and sensitive determination of neurotransmitters in biological fluids. We will discuss new data obtained by ME coupled to laser-induced fluorescence (ME-LIF) and electrochemical detection (ME-EC) regarding the production and degradation of nitric oxide, a fundamental signaling molecule regulating virtually every critical cellular function. Finally, the integration of microfluidics with recent innovative technologies—such as organoids, organ-on-chip, and 3D printing—for the design of new in vitro experimental devices will be presented with a specific attention to drug development applications. This ‘composite’ review highlights the potential impact of 2D and 3D microfluidic systems as a fast, inexpensive, and highly sensitive tool for high-throughput drug screening and preclinical toxicological studies.Italian Ministry of Health Research Program 2018 (2635256)American Heart Association-Midwest Affiliate Postdoctoral Research Fellowship (NFP0075515)Italian Ministry of Economic Development (F/200110/02/X45)Italian Ministry of EducationNIH COBRE P20GM103638Oasi Research Institute—IRCC

Microchip Electrophoresis with Amperometric Detection Method for Profiling Cellular Nitrosative Stress Markers

The overproduction of nitric oxide (NO) in cells results in nitrosative stress due to the generation of highly reactive species such as peroxynitrite and N2O3. These species disrupt the cellular redox processes through the oxidation, nitration, and nitrosylation of important biomolecules. Microchip electrophoresis (ME) is a fast separation method that can be used to profile cellular nitrosative stress through the separation of NO and nitrite from other redox-active intracellular components such as cellular antioxidants. This paper describes a ME method with electrochemical detection (ME-EC) for the separation of intracellular nitrosative stress markers in macrophage cells. The separation of nitrite, azide (interference), iodide (internal standard), tyrosine, glutathione, and hydrogen peroxide (neutral marker) was achieved in under 40 s using a run buffer consisting of 7.5 to 10 mM NaCl, 10 mM boric acid, and 2 mM TTAC at pH 10.3 to 10.7. Initially, NO production was monitored by the detection of nitrite (NO2−) in cell lysates. There was a 2.5- to 4-fold increase in NO2− production in lipopolysaccharide (LPS)-stimulated cells. The concentration of NO2− inside a single unstimulated macrophage cell was estimatedto be 1.41 mM using the method of standard additions. ME-EC was then used for the direct detection of NO and glutathione in stimulated and native macrophage cell lysates. NO was identified in these studies based on its migration time and rapid degradation kinetics. The intracellular levels of glutathione in native and stimulated macrophages were also compared, and no significant difference was observed between the two conditions

Lung Surfactant Decreases Biochemical Alterations and Oxidative Stress Induced by a Sub-Toxic Concentration of Carbon Nanoparticles in Alveolar Epithelial and Microglial Cells

Carbon-based nanomaterials are nowadays attracting lots of attention, in particular in the biomedical field, where they find a wide spectrum of applications, including, just to name a few, the drug delivery to specific tumor cells and the improvement of non-invasive imaging methods. Nanoparticles inhaled during breathing accumulate in the lung alveoli, where they interact and are covered with lung surfactants. We recently demonstrated that an apparently non-toxic concentration of engineered carbon nanodiamonds (ECNs) is able to induce oxidative/nitrosative stress, imbalance of energy metabolism, and mitochondrial dysfunction in microglial and alveolar basal epithelial cells. Therefore, the complete understanding of their “real” biosafety, along with their possible combination with other molecules mimicking the in vivo milieu, possibly allowing the modulation of their side effects becomes of utmost importance. Based on the above, the focus of the present work was to investigate whether the cellular alterations induced by an apparently non-toxic concentration of ECNs could be counteracted by their incorporation into a synthetic lung surfactant (DPPC:POPG in 7:3 molar ratio). By using two different cell lines (alveolar (A549) and microglial (BV-2)), we were able to show that the presence of lung surfactant decreased the production of ECNs-induced nitric oxide, total reactive oxygen species, and malondialdehyde, as well as counteracted reduced glutathione depletion (A549 cells only), ameliorated cell energy status (ATP and total pool of nicotinic coenzymes), and improved mitochondrial phosphorylating capacity. Overall, our results on alveolar basal epithelial and microglial cell lines clearly depict the benefits coming from the incorporation of carbon nanoparticles into a lung surfactant (mimicking its in vivo lipid composition), creating the basis for the investigation of this combination in vivo

Hereditary Angioedema and Psychopathology: Neurobiology, Stress and Attachment Styles

Hereditary Angioedema (HAE) is considered an autosomal dominant disorder, characterized by a quantitativeand/or qualitative deficit of C1 esterase inhibitor. The aim of the study is to establish the relationshipbetween HAE, clinical events, and neurobiological and psychopathologicalparameters, which could influence the phenotype of the disease and thereforeits manifestation in terms of quality, severity and duration of symptoms. Materialsand Methods: observational study, cross-sectional, non-interventional, cohortof 46 patients with diagnosis of hereditary angioedema. ExclusionCriteria: current pharmacological treatment with ACE-inhibitors,glucocorticoids, psychotropic drugs, immunomodulators, anesthetics. A blood sampling was performed to measure cortisol,IL-6, TNF-α and catecholamines, medical examination, psychiatric examination toinvestigate the clinical characteristics of HAE and presence of life events, psychometric evaluation. Any correlation was assessed by Spearman Rho. Results the sample consists of 46 patients, including 22 women (47,8%) e 24 men (52,2%). Averageage of onset of symptoms is equal to 14,61 ± 12,46. High values ??of IL-6 (1,83 ± 3,9) and TNF-α (10,2 ± 27,5) were relatedwith severity of pathology . Conclusions levels of IL-6and TNF-α are in agreement with the increase in the number of attacks of HAE. There is a relationshipbetween increased levels of IL-6 and high scores on Hamilton Depression RatingScale and on Hamilton Anxiety Rating Scale and a higher subjective perceptionof disease severity. The higher was the perceived stress, the greater will bethe subjective perception of disease severity and the presence of pathologicalattachment styles

Modulation of Pro-Oxidant and Pro-Inflammatory Activities of M1 Macrophages by the Natural Dipeptide Carnosine

This work is licensed under a Creative Commons Attribution 4.0 International License.Carnosine is a natural endogenous dipeptide widely distributed in mammalian tissues, existing at particularly high concentrations in the muscles and brain and possesses well-characterized antioxidant and anti-inflammatory activities. In an in vitro model of macrophage activation, induced by lipopolysaccharide + interferon-gamma (LPS + IFN-γ), we here report the ability of carnosine to modulate pro-oxidant and pro-inflammatory activities of macrophages, representing the primary cell type that is activated as a part of the immune response. An ample set of parameters aimed to evaluate cytotoxicity (MTT assay), energy metabolism (HPLC), gene expressions (high-throughput real-time PCR (qRT-PCR)), protein expressions (western blot) and nitric oxide production (qRT-PCR and HPLC), was used to assess the effects of carnosine on activated macrophages challenged with a non cytotoxic LPS (100 ng/mL) + IFN-γ (600 U/mL) concentration. In our experimental model, main carnosine beneficial effects were: (1) the modulation of nitric oxide production and metabolism; (2) the amelioration of the macrophage energy state; (3) the decrease of the expressions of pro-oxidant enzymes (Nox-2, Cox-2) and of the lipid peroxidation product malondialdehyde; (4) the restoration and/or increase of the expressions of antioxidant enzymes (Gpx1, SOD-2 and Cat); (5) the increase of the transforming growth factor-β1 (TGF-β1) and the down-regulation of the expressions of interleukins 1β and 6 (IL-1β and IL-6) and 6) the increase of the expressions of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase-1 (HO-1). According to these results carnosine is worth being tested in the treatment of diseases characterized by elevated levels of oxidative stress and inflammation (atherosclerosis, cancer, depression, metabolic syndrome, and neurodegenerative diseases)

Indirect detection of superoxide in RAW 264.7 macrophage cells using microchip electrophoresis coupled to laser-induced fluorescence

The final publication is available at Springer via http://dx.doi.org/10.1007/s00216-015-8865-1Superoxide is a naturally produced reactive oxygen species (ROS) in the human body and is involved in many pathological and physiological signaling processes. However, if superoxide formation is left unregulated, overproduction can lead to oxidative damage to important biomolecules, such as DNA, lipids, and proteins. Superoxide can also lead to the formation of peroxynitrite, an extremely hazardous substance, through its reaction with endogenously produced nitric oxide. Despite its importance, quantitative information regarding superoxide production is difficult to obtain due to its high reactivity and low concentrations in vivo. MitoHE, a fluorescent probe that specifically reacts with superoxide, was used in conjunction with microchip electrophoresis (ME) and laser-induced fluorescence detection to investigate changes in superoxide production by RAW 264.7 macrophage cells following stimulation with phorbol 12-myristate 13-acetate (PMA). Stimulation was performed in the presence and absence of the superoxide dismutase (SOD) inhibitors, diethyldithiocarbamate (DDC) and 2-metoxyestradiol (2-ME). The addition of these inhibitors resulted in an increase in the amount of superoxide specific product (2-OH-MitoE+) from 0.08 ± 0.01 fmol (0.17 ± 0.03 mM) in native cells to 1.26 ± 0.06 fmol (2.5 ± 0.1 mM) after PMA treatment. This corresponds to an approximately 15-fold increase in intracellular concentration per cell. Furthermore, the addition of 3-morpholino-sydnonimine (SIN-1) to the cells during incubation resulted in 0.061 ± 0.006 fmol (0.12 ± 0.01 mM) of 2-OH-MitoE+ per cell on average. These results demonstrate that indirect superoxide detection coupled with the use of SOD inhibitors and a separation method is a viable method to discriminate the 2-OH-MitoE+ signal from possible interferences