Farnesoid X receptor activation by the novel agonist TC-100 (3α, 7α, 11ß-Trihydroxy-6α-ethyl-5ß-cholan-24-oic Acid) preserves the intestinal barrier integrity and promotes intestinal microbial reshaping in a mouse model of obstructed bile acid flow.

The intestinal tract hosts the gut microbiota (GM), actively shaping health. Bile acids(BAs) are both digestive and signaling molecules acting as hormones via the activation of farnesoid X receptor (FXR). Obstruction of bile flow initiates a cascade of pathological events ultimately leading to intestinal mucosal injury. Administration of BAs in models of obstructed bile flow counteracts these detrimental effects. Objective of this study was to investigate the effects of the novel FXR agonist 3α, 7α, 11ß-Trihydroxy-6α-ethyl-5ß-cholan-24-oic Acid (TC-100) on intestinal mucosa integrity and cecal microbiome composition after surgical bile duct ligation (BDL), a rodent model causing bile flow obstruction. Pharmacological FXR activation was accomplished by daily oral gavage with TC-100 for 5 days. 2 days after treatment initiation, BDL was performed. BAs measurement was carried out and the 16S rDNA (V5-V6 hyper-variable regions) extracted from the cecal content was sequenced. TC-100 activates Fxr in the gut-liver axis and this translated into a significant reduction of serum and bile BA pool size with a shift to a more hydrophilic composition, while signs of intestinal mucosal damage were prevented. Firmicutes:Bacteroidota ratio progressively increased from Sham Operated (SO) mice to TC-100-treated mice. LEfSe analysis showed that Verrucomicrobia, and particularly Akkermansia muciniphila (Amuc) increasingly recognized for improving gut homeostasis and immune functions, were strongly associated to TC-100-treated mice. Intriguingly, Amuc abundance was also negatively associated to cholic acid levels. Collectively, these data indicate that intestinal FXR activation by TC-100 prevents early signs of intestinal mucosal damage by modulating BA homeostasis and GM composition.

Nanoshaping field emitters from glassy carbon sheets: a new functionality induced by H-plasma etching.

This paper reports on the morphological and electrical characterization at the nanometer scale and the investigation of the field emission characteristics of glassy carbon (GC) plates which underwent H-induced physical/chemical processes occurring in a dual-mode MW-RF plasma reactor. Plasma treatment produced on the GC surface arrays of vertically aligned conically shaped nanostructures, with density and height depending on the plasma characteristics. Two kinds of samples obtained under two different bias regimes have been deeply analyzed using an AFM apparatus equipped with tools for electric forces and surface potential measurements. The features of electron emission via the Field Emission (FE) mechanism have been correlated with the morphology and the structure at the nanoscale of the treated glassy carbon samples. The measured current density and the characteristics of the emission, which follow the Fowler-Nordheim law, indicate that the plasma-based methodology utilized for the engineering of the GC surfaces is able to turn conventional GC plates into efficient emission devices. The outstanding properties of GC suggest the use of such nanostructured materials for the assembling of cold cathodes to be used in a harsh environment and under extreme P/T conditions.

Niosomal approach to brain delivery: Development, characterization and in vitro toxicological studies.

The majority of active agents do not readily permeate into brain due to the presence of the blood-brain barrier and blood-cerebrospinal fluid barrier. Currently, the most innovative and promising non-invasive strategy in brain delivery is the design and preparation of nanocarriers, which can move through the brain endothelium. Niosomes can perform brain delivery, in fact polysorbates, can act as an anchor for apolipoprotein E from blood plasma. The particles mimic LDL and interact with the LDL receptor leading to the endothelial cells uptake. The efficacy of niosomes for anticancer therapeutic applications was correlated to their physicochemical and drug delivery properties. Dimensions and ζ-potential were characterized using dynamic light scattering and asymmetric flow-field fractionation system. Lipid bilayer was characterized measuring the fluidity, polarity and microviscosity by fluorescent probe spectra evaluation. Morphology and homogeneity were characterized using atomic force microscopy. Physicochemical stability and serum stability (45% v/v fetal bovine and human serum) were evaluated as a function of time using dynamic light scattering. U87-MG human glioblastoma cells were used to evaluate vesicle cytotoxicity and internalisation efficiency. From the obtained data, the systems appear useful to perform a prolonged (modified) release of biological active substances to the central nervous system.

Biomedical Applications of Nanodiamonds: An Overview.

Nanodiamonds are a novel class of nanomaterials which have raised much attention for application in biomedical field, as they combine the possibility of being produced on large scale using relatively inexpensive synthetic processes, of being fluorescent as a consequence of the presence of nitrogen vacancies, of having their surfaces functionalized, and of having good biocompatibility. Among other applications, we mainly focus on drug delivery, including cell interaction, targeting, cancer therapy, gene and protein delivery. In addition, nanodiamonds for bone and dental implants and for antibacterial use is discussed. Techniques for detection and imaging of nanodiamonds in biological tissues are also reviewed, including electron microscopy, fluorescence microscopy, Raman mapping, atomic force microscopy, thermal imaging, magnetic resonance imaging, and positron emission tomography, either in vitro, in vivo, or ex vivo. Toxicological aspects related to the use of nanodiamonds are also discussed. Finally, patents, preclinical and clinical trials based on the use of nanodiamonds for biomedical applications are reviewed.

N-terminus-modified Hec1 suppresses tumour growth by interfering with kinetochore-microtubule dynamics.

Mitotic proteins are attractive targets to develop molecular cancer therapeutics due to the intimate interdependence between cell proliferation and mitosis. In this work, we have explored the therapeutic potential of the kinetochore (KT) protein Hec1 (Highly Expressed in Cancer protein 1) as a molecular target to produce massive chromosome missegregation and cell death in cancer cells. Hec1 is a constituent of the Ndc80 complex, which mediates KT-microtubule (MT) attachments at mitosis and is upregulated in various cancer types. We expressed Hec1 fused with enhanced green fluorescent protein (EGFP) at its N-terminus MT-interaction domain in HeLa cells and showed that expression of this modified Hec1, which localized at KTs, blocked cell proliferation and promoted apoptosis in tumour cells. EGFP-Hec1 was extremely potent in tumour cell killing and more efficient than siRNA-induced Hec1 depletion. In striking contrast, normal cells showed no apparent cell proliferation defects or cell death following EGFP-Hec1 expression. Live-cell imaging demonstrated that cancer cell death was associated with massive chromosome missegregation within multipolar spindles after a prolonged mitotic arrest. Moreover, EGFP-Hec1 expression was found to increase KT-MT attachment stability, providing a molecular explanation for the abnormal spindle architecture and the cytotoxic activity of this modified protein. Consistent with cell culture data, EGFP-Hec1 expression was found to strongly inhibit tumour growth in a mouse xenograft model by disrupting mitosis and inducing multipolar spindles. Taken together, these findings demonstrate that stimulation of massive chromosome segregation defects can be used as an anti-cancer strategy through the activation of mitotic catastrophe after a multipolar mitosis. Importantly, this study represents a clear proof of concept that targeting KT proteins required for proper KT-MT attachment dynamics constitutes a powerful approach in cancer therapy.

Thickness measurement of soft thin films on periodically patterned magnetic substrates by phase difference magnetic force microscopy.

The need for accurate measurement of the thickness of soft thin films is continuously encouraging the development of techniques suitable for this purpose. We propose a method through which the thickness of the film is deduced from the quantitative measurement of the contrast in the phase images of the sample surface acquired by magnetic force microscopy, provided that the film is deposited on a periodically patterned magnetic substrate. The technique is demonstrated by means of magnetic substrates obtained from standard floppy disks. Colonies of Staphylococcus aureus adherent to such substrates were used to obtain soft layers with limited lateral (a few microns) and vertical (hundreds of nanometers) size. The technique is described and its specific merits, limitations and potentialities in terms of accuracy and measurable thickness range are discussed. These parameters depend on the characteristics of the sensing tip/cantilever as well as of the substrates, the latter in terms of spatial period and homogeneity of the magnetic domains. In particular, with the substrates used in this work we evaluated an uncertainty of about 10%, a limit of detection of 50-100 nm and an upper detection limit (maximum measurable thickness) of 1 µm, all obtained with standard lift height values (50-100 nm). Nonetheless, these parameters can be easily optimized by selecting/realizing substrates with suitable spacing and homogeneity of the magnetic domains. For example, the upper detection limit can be increased up to 25-50 µm while the limit of detection can be reduced to a few tens of nanometers or a few nanometers.

On the tip calibration for accurate modulus measurement by contact resonance atomic force microscopy.

Accurate quantitative elastic modulus measurements using contact resonance atomic force microscopy require the calibration of geometrical and mechanical properties of the tip as well as the choice of a suitable model for describing the cantilever-tip-sample system. In this work, we demonstrate with both simulations and experiments that the choice of the model influences the results of the calibration. Neglecting lateral force results in the underestimation of the tip indentation modulus and in the overestimation of the tip-sample contact radius. We propose a new approach to the calibration and data analysis, where lateral forces and cantilever inclination are neglected (which simplifies the calculations) and the tip parameters are assumed as fictitious.

Analytical techniques to study microbial biofilm on abiotic surfaces: pros and cons of the main techniques currently in use.

Biofilm is a bacterial lifestyle widespread in microbial world and represents a concern in health care. Despite the great life expectancy related to advanced health care, the increasing numbers of biofilm-mediated infections remain a significant public health challenge. Moreover, the problem of biofilm-mediated infections becomes much more severe when biofilm colonizes medical devices and biomaterials. The public health risk due to microbial biofilm-related infections is a concern that requires full attention. However, the complexity of biofilm makes difficult its exhaustive analysis. Although biofilm represents a major challenge in both microbiological and hygiene areas, at now methods aimed to analyse biofilm formation and development are not standardized yet. Different methods have been employed to qualitatively and quantitatively evaluate biofilm each of which is useful to estimate a peculiar aspect of biofilm lifestyle. In the present review, fifteen assays for the qualitative and quantitative evaluation of bacterial biofilm colonizing abiotic substrates, such as medical devices, prosthesis or surfaces for food production together with advantages and limitations of each method were described and compared. Some methods are suited to quantify biofilm matrix while others are capable to evaluate both living and dead cells or quantify exclusively viable cells in biofilm. In particular, colorimetric methods to evaluate biofilm matrix (crystal violet; 1,9-dimethyl methylen blue and fluorescein-di-acetate methods) or viable cells (LIVE/DEAD BacLight, BioTimer Assay, resazurin, tetrazolium hydroxide salt methods) and genetic methods to estimate the bacterial population (PCR and FISH) are reported. Moreover, a section is dedicated to examine the performances of advanced microscopic techniques employed to study microbial biofilms (mass spectrometry; confocal laser scanning microscopy; Raman spectroscopy and electron microscopy). Because of its complexity, an exhaustive study of biofilm requires a combination of different experimental approaches as biochemical, genetic or physical ones.

Photoacoustic cell for ultrasound contrast agent characterization.

Photoacoustics has emerged as a tool for the study of liquid gel suspension behavior and has been recently employed in a number of new biomedical applications. In this paper, a photoacoustic sensor is presented which was designed and realized for analyzing photothermal signals from solutions filled with microbubbles, commonly used as ultrasound contrast agents in echographic imaging techniques. It is a closed cell device, where photothermal volume variation of an aqueous solution produces the periodic deflection of a thin membrane closing the cell at the end of a short pipe. The cell then acts as a Helmholtz resonator, where the displacement of the membrane is measured through a laser probe interferometer, whereas photoacoustic signal is generated by a laser chopped light beam impinging onto the solution through a glass window. Particularly, the microbubble shell has been modeled through an effective surface tension parameter, which has been then evaluated from experimental data through the shift of the resonance frequencies of the photoacoustic sensor. This shift of the resonance frequencies of the photoacoustic sensor caused by microbubble solutions is high enough for making such a cell a reliable tool for testing ultrasound contrast agent, particularly for bubble shell characterization.

PARP1 is activated at telomeres upon G4 stabilization: possible target for telomere-based therapy.

New anti-telomere strategies represent important goals for the development of selective cancer therapies. In this study, we reported that uncapped telomeres, resulting from pharmacological stabilization of quadruplex DNA by RHPS4 (3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate), trigger specific recruitment and activation of poly-adenosine diphosphate (ADP) ribose polymerase I (PARP1) at the telomeres, forming several ADP-ribose polymers that co-localize with the telomeric repeat binding factor 1 protein and are inhibited by selective PARP(s) inhibitors or PARP1-specific small interfering RNAs. The knockdown of PARP1 prevents repairing of RHPS4-induced telomere DNA breaks, leading to increases in chromosome abnormalities and eventually to the inhibition of tumor cell growth both in vitro and in xenografts. More interestingly, the integration of a TOPO1 inhibitor on the combination treatment proved to have a high therapeutic efficacy ensuing a complete regression of the tumor as well as a significant increase in overall survival and cure of mice even when treatments started at a very late stage of tumor growth. Overall, this work reveals the unexplored link between the PARP1 and G-quadruplex ligands and demonstrates the excellent efficacy of a multi-component strategy based on the use of PARP inhibitors in telomere-based therapy.

In vitro and in vivo antitumor efficacy of docetaxel and sorafenib combination in human pancreatic cancer cells.

The response of pancreatic cancer to treatments remains unsatisfactory, highlighting the need for more effective therapeutic regimens. Sorafenib, an orally available multikinase inhibitor, is active against different tumors, including pancreatic cancer. We studied the antitumor efficacy of sorafenib in combination with different antitumor drugs currently used in clinical practice in in vitro and in vivo experimental models of human pancreatic cancer. The cytotoxic effect of sorafenib and conventional antitumor drug combinations was evaluated in vitro in human pancreatic cancer cell lines and the efficacy of the most active combination was tested on tumor-bearing mice. Flow cytometric, Western blot and immunohistochemistry analyses were performed to investigate the mechanisms involved in the activity of single drugs and in their interaction when used in combination. Sorafenib showed a strong sequence-dependent synergistic interaction in vitro with docetaxel, which was highly dependent on the drug sequence employed. In vivo, human pancreatic cancer-xenografted mice treated with docetaxel followed by sorafenib reduced and delayed tumor growth, with complete tumor regression observed in half of the mice. This marked antitumor effect resulted in an overall increase in mouse survival of about 70% and in a complete cure in 3 of the 8 treated mice. The strong activity was also accompanied by marked apoptosis induction, inhibition of tumor angiogenesis and downregulation of ERK signalling. Our results show that the docetaxel and sorafenib combination exerts high therapeutic efficacy in experimental models of human pancreatic cancer, indicating a promising antitumor strategy for clinical use.

Water temperature dependence of single bubble sonoluminescence threshold.

Water temperature dependence of single bubble sonoluminescence (SBSL) threshold has been experimentally measured to perform measurements at different temperatures on the very same bubble. Results show lower thresholds, i.e. an easier prime of mechanism, of sonoluminescence at lower water temperatures. Dependence is almost linear at lower temperatures while between 14 degrees C and about 20 degrees C the curve changes its slope reaching soon a virtual independence from water temperature above about 20 degrees C.

Indentation modulus and hardness of viscoelastic thin films by atomic force microscopy: A case study.

We propose a nanoindentation technique based on atomic force microscopy (AFM) that allows one to deduce both indentation modulus and hardness of viscoelastic materials from the force versus penetration depth dependence, obtained by recording the AFM cantilever deflection as a function of the sample vertical displacement when the tip is pressed against (loading phase) and then removed from (unloading phase) the surface of the sample. Reliable quantitative measurements of both indentation modulus and hardness of the investigated sample are obtained by calibrating the technique through a set of different polymeric samples, used as reference materials, whose mechanical properties have been previously determined by standard indentation tests. By analyzing the dependence of the cantilever deflection versus time, the proposed technique allows one to evaluate and correct the effect of viscoelastic properties of the investigated materials, by adapting a post-experiment data processing procedure well-established for standard depth sensing indentation tests. The technique is described in the case of the measurement of indentation modulus and hardness of a thin film of poly(3,4-ethylenedioxythiophene) doped with poly(4-styrenesulfonate), deposited by chronoamperometry on an indium tin oxide (ITO) substrate.

Quantitative measurement of indentation hardness and modulus of compliant materials by atomic force microscopy.

An atomic force microscopy (AFM) based technique is proposed for the characterization of both indentation modulus and hardness of compliant materials. A standard AFM tip is used as an indenter to record force versus indentation curves analogous to those obtained in standard indentation tests. In order to overcome the lack of information about the apex geometry, the proposed technique requires calibration using a set of reference samples whose mechanical properties have been previously characterized by means of an independent technique, such as standard indentation. Due to the selected reference samples, the technique has been demonstrated to allow reliable measurements of indentation modulus and hardness in the range of 0.3-4.0 GPa and 15-250 MPa, respectively.

Harmonic and subharmonic acoustic wave generation in finite structures.

The generation of harmonic and subharmonic vibrations is considered in a finite monodimensional structure, as it is produced by the nonlinear acoustic characteristics of the medium. The equation of motion is considered, where a general function of the displacement and its derivatives acts as the forcing term for (sub)harmonic generation and a series of 'selection rules' is found, depending on the sample constrains. The localization of the nonlinear term is also considered that mimics the presence of defects or cracks in the structure, together with the spatial distribution of subharmonic modes. Experimental evidence is given relative to the power law dependence of the harmonic modes vs. the fundamental mode displacement amplitude, and subharmonic mode distribution with hysteretic effects is also reported in a cylindrical sample of piezoelectric material.

Interaction between leukocytic elastase and Kunitz-type inhibitors from bovine spleen.

The four Kunitz-type protease inhibitors purified from bovine spleen, which include the basic pancreatic trypsin inhibitor (BPTI), form stable complexes with human leukocytic elastase. The values of the affinity constants of these complexes are similar, in agreement with the great structural similarity of the four inhibitors, but are lower than those measured for the complexes with other serine proteases. Two main factors appear to be responsible for the stability of these complexes, i.e., hydrophobic interactions and ionization phenomena that take place during complex formation. These two factors have been analyzed in terms of the general model previously used for describing the interaction between the serine proteases and their natural inhibitors.