Overexpression of miR-4669 Enhances Tumor Aggressiveness and Generates an Immunosuppressive Tumor Microenvironment in Hepatocellular Carcinoma: Its Clinical Value as a Predictive Biomarker.

Accumulating evidence suggests the involvement of tumor-derived exosomes in the development and recurrence of hepatocellular carcinoma (HCC). We previously identified miR-4669 as a highly expressed microRNA in circulating exosomes obtained from patients with post-transplant HCC recurrence. This study aimed to explore how overexpression of miR-4669 affects HCC development and recurrence. The impact of miR-4669 overexpression in Hep3B cells on tumor cell behavior and the tumor microenvironment was evaluated in vitro. In addition, the clinical value of exosomal miR-4669 for the prediction of treatment response to HCC downstaging therapies and following post-transplant HCC recurrence was explored. Overexpression of miR-4669 enhanced migration ability and led to acquired sorafenib resistance with an elevation of sirtuin 1 and long noncoding RNA associated with microvascular invasion. Active release of tumor-derived exosomes and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) contributed to generating an immunosuppressive tumor microenvironment through the induction of M2 macrophage polarization. The retrospective analysis demonstrated the clinical value of exosomal miR-4669 for predicting treatment response to HCC downstaging therapies and for risk assessment of post-transplant HCC recurrence. In summary, the present data demonstrate the impact of exosomal miR-4669 on HCC recurrence through the enhancement of tumor aggressiveness and generation of an immunosuppressive tumor microenvironment.

Hydration Dynamics and the Future of Small-Amplitude AFM Imaging in Air.

Here, we discuss the effects that the dynamics of the hydration layer and other variables, such as the tip radius, have on the availability of imaging regimes in dynamic AFM-including multifrequency AFM. Since small amplitudes are required for high-resolution imaging, we focus on these cases. It is possible to fully immerse a sharp tip under the hydration layer and image with amplitudes similar to or smaller than the height of the hydration layer, i.e., ~1 nm. When mica or HOPG surfaces are only cleaved, molecules adhere to their surfaces, and reaching a thermodynamically stable state for imaging might take hours. During these first hours, different possibilities for imaging emerge and change, implying that these conditions must be considered and reported when imaging.

Explaining doping in material research (Hf substitution in ZnO films) by directly quantifying the van der Waals force.

Non-monotonic behavior has been observed in the optoelectronic properties of ZnO thin films as doped with Hf (HZO). Here we propose that two competing mechanisms are responsible for such behaviour. Specifically, we propose that provided two crystal orientations dominate film growth, only one of them might be responsible for direct Hf substitution. Nonmonotonic behaviour is expected at once by considering that preferential growth of the crystal that allows for direct Hf substitution is inhibited by Hf concentration in the manufacturing process. This inhibition would also be a thermodynamic consequence of Hf substitution. Maxima in Hf substitution is thus reached at a point where enough crystals exhibit the preferential orientation, and where enough Hf is present on the surface for substitution. Outside this optimum scenario, Hf substitution can only decrease. We interpret the surface phenomena by discussing surface energy and the van der Waals forces as measured experimentally by means of atomic force microscopy.

Direct Measurement of the Magnitude of the van der Waals Interaction of Single and Multilayer Graphene.

Vertical stacking of monolayers via van der Waals (vdW) assembly is an emerging field that opens promising routes toward engineering physical properties of two-dimensional materials. Industrial exploitation of these engineering heterostructures as robust functional materials still requires bounding their measured properties so as to enhance theoretical tractability and assist in experimental designs. Specifically, the short-range attractive vdW forces are responsible for the adhesion of chemically inert components and are recognized to play a dominant role in the functionality of these structures. Here, we reliably quantify the strength of ambient vdW forces in terms of an effective Hamaker coefficient for chemical vapor deposition-grown graphene and show how it scales by a factor of two or three from single to multiple layers on standard supporting surfaces such as copper or silicon oxide. Furthermore, direct measurements on freestanding graphene provide the means to discern the interplay between the vdW potential of graphene and its supporting substrate. Our results demonstrated that the underlying substrates could be controllably exploited to enhance or reduce the vdW force of graphene surfaces. We interpret the physical phenomena in terms of a Lifshitz theory-based analytical model.

Surface aging investigation by means of an AFM-based methodology and the evolution of conservative nanoscale interactions.

The divergence in physical properties of surfaces exposed to airborne contaminants in the atmosphere has been widely investigated in recent years, but agreement regarding the role that airborne hydrocarbons and water contamination have on surface property evolution remains elusive. Here we investigate the evolution of a freshly grown highly oriented pyrolytic graphite (HOPG) surface exposed to a controlled environment solely containing airborne water contamination, i.e. water vapor. Our approach combines standard electromagnetic spectrum-based spectrometry methods and atomic force microscopy based techniques to provide a holistic view of the surface properties. We provide evidence of the affect that water adsorption kinetics has on surface properties, interpreting time dependent force-distance profiles and force of adhesion maps directly obtained from the standard observables in a bimodal AFM. To demonstrate the generality of our approach we also apply it to aged calcite surfaces, proving its advantages in providing comprehensive transient characterization of surfaces with sufficient spatial resolution.

The evolution in graphitic surface wettability with first-principles quantum simulations: the counterintuitive role of water.

The surface wettability of graphite has gained a lot of interest in nanotechnology and fundamental studies alike, but the types of adsorptions that dominate its time resolved surface property variations in ambient environment are still elusive. Prediction of the intrinsic surface wettability of graphite from first-principles simulations offers an opportunity to clarify the overall evolution. In this study, by combining experimental temporal Fourier transform infrared spectroscopy, atomic force microscopy (AFM), and static contact angle measurements with density functional theory (DFT)-predicted contact angles and DFT AFM force simulations, we provide conclusive evidence to demonstrate the role played by water adsorption in the evolution of surface properties of aged graphite in ambient air. Moreover, this study has the merit of linking DFT-predicted adhesive energy at the solid/liquid interface and cohesive energy at the liquid/liquid interface with the DFT AFM-predicted force of adhesion through the Young-Dupre equation. This establishes the basis of the quantum surface wettability theory by combining two independent atomic-level quantum physics simulation methodologies.

Discerning the Contribution of Morphology and Chemistry in Wettability Studies.

The wetting behavior of homogeneous systems is now well understood at the macroscopic scale. However, this understanding offers little predictive power regarding wettability when mesoscopic chemical and morphological heterogeneities come into play. The fundamental interest in the effect of heterogeneity on wettability is derived from its high technological relevance in several industries, including the petroleum industry where wettability is recognized as a key determinant of the overall efficiency of the water-flooding-based enhanced oil recovery process. Here, we demonstrate the use of the atomic force microscopy force curve measurements to distinguish the roles of chemistry and morphology in the wetting properties of rock formations, thus providing a clear interpretation and deeper insight into the wetting behavior of heterogeneous formations. Density functional theory calculations further prove the versatility of our approach by establishing benchmarks on ideal surfaces that differ in chemistry and morphology in a predefined manner.

Integrated Nano- and Macroscale Investigation of Photoinduced Hydrophilicity in TiO2 Thin Films.

The hydrophilicity of titanium dioxide has been investigated for films, deposited on glass by e-beam evaporation, being exposed to UV radiation and subjected to thermal annealing. The wettability alteration has been showed to depend upon both treatments, and insights into how to introduce more stable hydrophilicity into these films have been presented for the sake of boosting their commercial value. Observations from multiple length scales to assess the wetting behavior of as-deposited and high-temperature annealed samples were assessed through macroscopic measurements, i.e., water contact angle measurements, showing that the annealed crystalline samples, treated at 500 °C, are much more hydrophilic (SCA ≈ 20°) than as-deposited TiO2 films (SCA ≈ 90°), and the nanoscopic experiments performed by amplitude modulation (AM) atomic force microscopy (AFM) indicated that this increased hydrophilicity is related to an enhanced adhesion force and surface energy, resulting in the partial crystallization of TiO2 and the consequent formation of crystals on its surface rather than being related to morphologic differences. XRD and Raman measurements have highlighted that the crystallinity of the TiO2 film is crucial in determining its hydrophilicity, in good agreement with the AFM study. The results also indicated that, after irradiation, the samples treated at 500 °C preserve their hydrophilicity for a significant time compared to previous studies.

Reconstruction of height of sub-nanometer steps with bimodal atomic force microscopy.

Obtaining topographic images of surfaces presenting terraces with heights in the nanometer and sub-nanometer range has become routine since the advent of atomic force microscopy (AFM). There remain however several open questions regarding the validity of direct topographic measurements. Here we turn to recent advances in AFM to correct the height of nanometric terraces by exploiting the four observables of bimodal AFM operated in the non-invasive attractive regime. We first derive expressions based on the van der Waals theory and then image model terraces in air in standard bimodal AFM while simultaneously correcting and decoupling the sources of loss/gain of height.

Divergent surface properties of multidimensional sp (2) carbon allotropes: the effect of aging phenomena.

Despite the current interest in the scientific community in exploiting divergent surface properties of graphitic carbon allotropes, conclusive differentiation remains elusive even when dealing with parameters as fundamental as adhesion. Here, we set out to provide conclusive experimental evidence on the time evolution of the surface properties of highly oriented pyrolytic graphite (HOPG), graphene monolayer (GML) and multiwalled carbon nanotubes (MWCNTs) as we expose these materials to airborne contaminants, by providing (1) statistically significant results based on large datasets consisting of thousands of force measurements, and (2) errors sufficiently self-consistent to treat the comparison between datasets in atomic force microscopy (AFM) measurements. We first consider HOPG as a model system and then employ our results to draw conclusions from the GML and MWCNT samples. We find that the surface properties of aged HOPG are indistinguishable from those of aged GML and MWCNT, while being distinct from those of cleaved HOPG. Herein, we provide a sufficient body of evidence to disregard any divergence in surface properties for multidimensional sp (2) carbon allotropes that undergo similar aging processes.

Surface and Bulk Effects in Photochemical Reactions and Photomechanical Effects in Dynamic Molecular Crystals.

The increasing number of reports on photomechanical effects in molecular crystals necessitates systematic studies to understand the intrinsic and external effectors that determine and have predictive power of their type and magnitude. Differential light absorption and product gradient between the surface and the bulk of the crystal are often invoked to qualitatively explain the mechanical response of crystals to light; however, the details on how this difference in photochemical response accounts for macroscopic effects such as surface modification, deformation, or disintegration of crystals are yet to be established. Using both bulk- and surface-sensitive analytical techniques, a rare instance of benzylidenefuranone crystals is studied here, and it is capable of several distinct types of photomechanical response including surface striation and delamination, photosalient effect (ballistic disintegration and motion), and photoinduced bending by dimerization. The results provide a holistic view on these effects and set the stage for the development of overarching theoretical models to describe the photomechanics in the ordered solid state.

Cytochrome P450 in living donor liver transplantation.

Cytochrome P450 metabolizes many drugs in the liver. Three genotypes of CYP2C19 with extensive, intermediate, and poor metabolizing activity, respectively, have been identified in peripheral blood of transplant recipients and new liver grafts in living donor liver transplantation (LDLT). The expression of the final genotype in liver graft biopsies depends on the donor, whereas the expression in peripheral blood mononuclear cells depends on the recipient. The metabolizing isoenzyme of the major anti-rejection agents passes through CYP3A4, CYP3A5 and MDR1, which have also been identified to have similar biological characteristics as genotype of CYP2C19 in liver tissue. Recently, pyrosequencing has been used to investigate the expressions of different genotypes in liver grafts in LDLT. This review focuses on recent findings regarding the biological expressions of the CYP2C19, CYP3A4, CYP3A5 and MRD1 genotypes in liver grafts before and after LDLT. The application of pyrosequencing may be beneficial in further research on liver transplantation. Laser capture microdissection of hepatocytes in liver grafts may be a direction for future research.

Prolonged survival by combined treatment with granulocyte colony-stimulating factor and dipeptidyl peptidase IV inhibitor in a rat small-for-size liver transplantation model.

AIM: Despite the great advances and excellent outcomes of liver transplantation (LT), small-for-size (SFS) graft syndrome is a life-threatening complication that remains to be overcome. In the present study, we investigated the therapeutic effect of combined treatment with granulocyte colony-stimulating factor (G-CSF) and a dipeptidyl peptidase IV (DPP-IV) inhibitor on SFS liver graft syndrome. METHODS: The transplantation of small-sized Lewis donor livers into green fluorescent protein (GFP) transgenic Wistar rats was performed and the recipients were randomly assigned to one of four groups (without treatment, DPP-IV inhibitor treatment, G-CSF treatment and G-CSF/DPP-IV inhibitor combination). Recombinant human G-CSF was injected s.c. at a dose of 2 µg/kg per day starting 5 days prior to transplantation. G-CSF was combined with the p.o. administration of a DPP-IV inhibitor (2 mg/kg per day) after transplantation until the end of the observation period. RESULTS: The post-transplant survival and liver function of rats treated with G-CSF/DPP-IV inhibitor combination therapy were significantly improved with an increased number of recipient-derived GFP positive cells into the liver grafts. A confocal microscopy study showed cytokeratin (CK)-18 and GFP positive hepatic progenitor cells in the parenchyma of the liver allografts. Untreated rats and rats treated with either G-CSF or DPP-IV inhibitor did not exhibit the prolonged survival and had less GFP and CK-18 positive cells in the liver grafts after SFS LT. CONCLUSION: Our results suggest that combined treatment with G-CSF and DPP-IV inhibitor may synergistically induce migration and differentiation of recipient-derived stem cells into the hepatic progenitor cells, resulting in the amelioration of SFS liver graft syndrome.

Time dependent wettability of graphite upon ambient exposure: the role of water adsorption.

We report the temporal evolution of the wettability of highly ordered pyrolytic graphite (HOPG) exposed to environmental conditions. Macroscopic wettability is investigated by static and dynamic contact angles (SCA and DCA) obtaining values comparable to the ones presented in the literature. SCA increases from ∼68° to ∼90° during the first hour of exposure after cleaving, whereas DCA is characterized by longer-scale (24 h) time evolution. We interpret these results in light of Fourier transform infrared spectroscopy, which indicates that the evolution of the HOPG wettability is due to adsorption of molecules from the surrounding atmosphere. This hypothesis is further confirmed by nanoscopic observations obtained by atomic force microscope (AFM)-based force spectroscopy, which monitor the evolution of surface properties with a spatial resolution superior to macroscopic experiments. Moreover, we observe that the results of macro- and nanoscale measurements evolve in similar fashion with time and we propose a quantitative correlation between SCA and AFM measurements. Our results suggest that the cause of the transition in the wettability of HOPG is due to the adsorption of hydrocarbon contaminations and water molecules from the environment. This is corroborated by annealing the HOPG is vacuum conditions at 150°, allowing the desorption of molecules on the surface, and thus re-establishing the initial macro and nano surface properties. Our findings can be used in the interpretation of the wettability of more complicated systems derived from HOPG (i.e., graphene).

Induction of antinuclear antibodies by de novo autoimmune hepatitis regulates alloimmune responses in rat liver transplantation.

Concanavalin A (Con A) is a lectin originating from the jack-bean and well known for its ability to stimulate T cells and induce autoimmune hepatitis. We previously demonstrated the induction of immunosuppressive antinuclear autoantibody in the course of Con A-induced transient autoimmune hepatitis. This study aimed to clarify the effects of Con A-induced hepatitis on liver allograft rejection and acceptance. In this study, we observed the unique phenomenon that the induction of transient de novo autoimmune hepatitis by Con A injection paradoxically overcomes the rejection without any immunosuppressive drug and exhibits significantly prolonged survival after orthotopic liver transplantation (OLT). Significantly increased titers of anti-nuclear Abs against histone H1 and high-mobility group box 1 (HMGB1) and reduced donor specific alloantibody response were observed in Con A-injected recipients. Induction of Foxp3 and IL-10 in OLT livers of Con A-injected recipients suggested the involvement of regulatory T cells in this unique phenomenon. Our present data suggest the significance of autoimmune responses against nuclear histone H1 and HMGB1 for competing allogeneic immune responses, resulting in the acceptance of liver allografts in experimental liver transplantation.

The effect of exogenous histone H1 on rat adipose-derived stem cell proliferation, migration, and osteogenic differentiation in vitro.

Adipose-derived stem cells (ASCs) are of great interest for the development of novel cell therapies due to their ease of isolation and expansion, immunosuppressive activity, and multilineage differentiation potential. However, the mechanisms underlying the therapeutic potential of ASCs remain to be elucidated. Others and we have shown that nuclear proteins such as histone H1 and high mobility group box 1 (HMGB1) play important roles in the maturation of dendritic cells (DCs). Furthermore, we previously demonstrated translocation of histone H1 from the nucleus to the cytoplasm and activation of mitogen-activated protein kinases (MAPKs) in DCs. In the present study, we confirmed that histone H1 does not alter the immunophenotype and immunosuppression potential of ASCs, but that histone H1 enhanced wound healing and increased interleukin (IL)-6 expression. Moreover, histone H1 treated-ASCs showed up-regulation of MAPKs extracellular-regulated kinase 1/2 (ERK1/2) and sequential NF-κB translocation. Finally, we found that culture in differentiation media supplemented with histone H1 enhanced ASC osteogenesis. In contrast, inhibition of histone H1 by small interfering RNA (siRNA) reduced osteogenic differentiation markers including ALP. These results suggest that histone H1 may be useful for induction of mesenchymal stem cells in tissue engineering and future potential ASC therapies.

Impact of artificial sunlight therapy on the progress of non-alcoholic fatty liver disease in rats.

BACKGROUND & AIMS: Non-alcoholic steatohepatitis (NASH) is recognized as the most severe form of non-alcoholic fatty liver disease, with likely progression to liver cirrhosis and hepatocellular carcinoma. However, there is no unified standard for diagnosis and therapeutics. This study aimed to characterize lipid transfer/metabolic proteins as non-invasive diagnostic markers, and to evaluate the therapeutic effects of phototherapy on the progression of NASH in rats. METHODS: Lewis rats given a choline-deficient and iron-supplemented l-amino acid-defined (CDAA) diet and Zucker fa/fa rats were used as a diet-induced and an obesity-related NASH models, respectively, with or without phototherapy. RESULTS: Serum apolipoprotein E and low molecular weight-adiponectin levels were gradually reduced and reached the lowest level at fatty liver/NASH stage both in CDAA diet-induced NASH model and in genetically obese model. Total-adiponectin levels were dramatically elevated after NASH was established in CDAA diet-induced NASH model. Phototherapy ameliorated hepatocyte apoptosis, inflammation, fibrosis, and insulin/leptin resistance caused by CDAA diet with alteration of the levels of lipid transfer/metabolic proteins and elevation of the circulating active form of vitamin D(3). Vitamin D(3) supplementation ameliorated NASH progression in CDAA diet-induced NASH model. However, phototherapy failed to ameliorate the obesity and steatosis, suggesting that phototherapy may possess anti-inflammatory/fibrotic activity rather than anti-obesity/steatotic activity. CONCLUSIONS: These results suggest that serum lipid transfer/metabolic proteins and vitamin D(3) status may be effective biomarkers for non-invasive diagnosis of NASH progression, and that phototherapy may be a good complementary therapy for NASH because of its regulation of lipid transfer/metabolic proteins and vitamin D(3).

Adaptor protein Shc acts as an immune-regulator for the LPS-stimulated maturation of bone marrow-derived dendritic cells.

BACKGROUND: The Shc isoforms is known to mediate immune responses and has been indicated as a negative regulator of autoimmunity and lymphocyte activation. We aimed to evaluate the immune-regulatory role of Shc in rat bone marrow-derived DCs in the maturation process triggered by LPS. RESULTS: We found that, in response to LPS, expression of Shc proteins was induced and that neutralization of Shc inhibited the LPS-induced transient phosphorylation of p52Shc on pTyr239/240 in DCs of Lewis (LEW; RT1(l)) rats. Moreover, the significantly enhanced expression of IL-10 and the surface level of costimulatory molecule CD80, as well as suppressed expression of IL-6 and IL-12 in the Shc-silenced DCs were also observed. Similar IκB phosphorylation occurred in Shc-silenced DCs primed by LPS, indicating Shc is not associated with NF-κB pathway. We further demonstrate that Shc blockade on LPS-treated DCs results in significant increase of the overall STAT3 phosphorylation and the relative levels of phospho-STAT3 in the nuclear fraction. STAT3 activation by LPS with or without Shc blockade was totally abolished by SU6656, a selective Src family kinases inhibitor, underscoring the critical role of Src-mediated activation. CONCLUSIONS: We conclude that Shc blockade in LPS-primed DC leads to the development of tolerogenic DC via Src-dependent STAT3 activation and that adaptor protein Shc might play a pivotal role in mediating immunogenic and tolerogenic properties of DCs.

A novel peptide mimotope identified as a potential immunosuppressive vaccine for organ transplantation.

We reported that anti-histone H1 autoantibody is one of the main immunosuppressive factors in serum that is induced after orthotopic liver transplantation in a rat tolerogenic model. We generated a novel anti-histone H1 IgM mAb produced by hybridoma 16G9 (16G9 mAb) that shows MLR-inhibitory activity. Identification of a functional epitope responsible for the immunosuppressive activity of 16G9 mAb may lead to the establishment of a novel therapeutic strategy. We used a combinatorial phage display peptide library to screen for peptides that bind to 16G9 mAb. Consequently, two peptides that bind to 16G9 mAb, SSV and LPQ, were selected from the library. The binding of 16G9 mAb to histone H1 was inhibited by SSV. SSV was recognized by rat tolerogenic post-orthotopic liver transplantation serum and the binding to SSV was inhibited by histone H1. Mice were immunized with keyhole limpet hemocyanin-conjugated SSV and LPQ. Abs induced by SSV immunization inhibited Con A-stimulated splenocyte proliferation, and the inhibition was neutralized by preincubation with SSV. Splenocytes stimulated by anti-CD3 Ab were inhibited by SSV-induced Abs using CFSE labeling. SSV immunization in rats before heterotopic heart transplantation resulted in significant prolonged allograft survival. These findings suggested that SSV is a functional histone H1-binding epitope for 16G9 mAb. SSV is capable of determining serum immunoreactivity against histone H1 as an index marker for tolerance. The inhibitory activity of SSV-induced Abs on blast cell proliferation and the prolonged graft survival that results from SSV immunization imply a potential for the development of an immunosuppressive vaccine.

Daily full spectrum light exposure prevents food allergy-like allergic diarrhea by modulating vitamin D3 and microbiota composition.

The importance of sun exposure on human health is well recognized, and a recent trend in the avoidance of sun exposure has led to the risk of missing the beneficial effects such as vitamin D3 biogenesis. Vitamin D3 insufficiency is one of the risk factors for the development of food allergies (FAs), and vitamin D3 status controls gut homeostasis by modulating the microbiota. This study aimed to explore the impact of daily full spectrum light exposure (phototherapy) on the pathogenesis of FAs. Phototherapy ameliorated allergic diarrhea and improved FA-associated vitamin D3 insufficiency and dysbiosis. Fecal microbiota transplantation (FMT) of FA donor feces induced allergic diarrhea with OVA-specific IgE elevation in naïve mice. In contrast, FMT of naïve donor feces ameliorated allergic diarrhea in established FA mice, suggesting the involvement of the microbiota composition in FA. Phototherapy is an alternative approach for the prevention of FA-like allergic diarrhea through the modulation of vitamin D3 status and microbiota composition.