Human TOB, an antiproliferative transcription factor, is a poly(A)-binding protein-dependent positive regulator of cytoplasmic mRNA deadenylation.

In mammalian cells, mRNA decay begins with deadenylation, which involves two consecutive phases mediated by the PAN2-PAN3 and the CCR4-CAF1 complexes, respectively. The regulation of the critical deadenylation step and its relationship with RNA-processing bodies (P-bodies), which are thought to be a site where poly(A)-shortened mRNAs get degraded, are poorly understood. Using the Tet-Off transcriptional pulsing approach to investigate mRNA decay in mouse NIH 3T3 fibroblasts, we found that TOB, an antiproliferative transcription factor, enhances mRNA deadenylation in vivo. Results from glutathione S-transferase pull-down and coimmunoprecipitation experiments indicate that TOB can simultaneously interact with the poly(A) nuclease complex CCR4-CAF1 and the cytoplasmic poly(A)-binding protein, PABPC1. Combining these findings with those from mutagenesis studies, we further identified the protein motifs on TOB and PABPC1 that are necessary for their interaction and found that interaction with PABPC1 is necessary for TOB's deadenylation-enhancing effect. Moreover, our immunofluorescence microscopy results revealed that TOB colocalizes with P-bodies, suggesting a role of TOB in linking deadenylation to the P-bodies. Our findings reveal a new mechanism by which the fate of mammalian mRNA is modulated at the deadenylation step by a protein that recruits poly(A) nuclease(s) to the 3' poly(A) tail-PABP complex.

The polarized distribution of poly(A+)-mRNA-induced functional ion channels in the Xenopus oocyte plasma membrane is prevented by anticytoskeletal drugs

Foreign mRNA was expressed in Xenopus laevis oocytes. Newly expressed ion currents localized in defined plasma membrane areas were measured using the two-electrode voltage clamp technique in combination with a specially designed chamber, that exposed only part of the surface on the oocytes to channel agonists or inhibitors. Newly expressed currents were found to be unequally distributed in the surface membrane of the oocyte. This asymmetry was most pronounced during the early phase of expression, when channels could almost exclusively be detected in the animal hemisphere of the oocyte. 4 d after injection of the mRNA, or later, channels could be found at a threefold higher density at the animal than at the vegetal pole area. The pattern of distribution was observed to be similar with various ion channels expressed from crude tissue mRNA and from cRNAs coding for rat GABAA receptor channel subunits. Electron microscopical analysis revealed very similar microvilli patterns at both oocyte pole areas. Thus, the asymmetric current distribution is not due to asymmetric surface structure. Upon incubation during the expression period in either colchicine or cytochalasin D, the current density was found to be equal in both pole areas. The inactive control substance beta-lumicolchicine had no effect on the asymmetry of distribution. Colchicine was without effect on the amplitude of the expressed whole cell current. Our measurements reveal a pathway for plasma membrane protein expression endogenous to the Xenopus oocyte, that may contribute to the formation and maintenance of polarity of this highly organized cell.

Osteo-odonto-keratoprosthesis (OOKP) and the testing of three different adhesives for bonding bovine teeth with optical poly-(methyl methacrylate) (PMMA) cylinder.

AIM Preparation of the lamina during osteo-odonto-keratoprosthesis (OOKP) design is complex, and its longevity and watertightness important. To date, only acrylic bone cements have been used for bonding the optical cylinder to the tooth dentine. Our aim was to evaluate different dental adhesives for OOKP preparation. METHODS Specimens of bovine teeth were produced by preparing 1.5-mm thick dentine slices with holes having a diameter of 3.5 mm. Each group (n=10 per group) was luted with either classic poly-(methyl methacrylate) (PMMA) bone cement, universal resin cement or glass ionomer cement. All specimens underwent force measurement using a uniaxial traction machine. RESULTS The highest mean force required to break the bond was measured for PMMA bone cement (128.2 N) followed by universal resin cement (127.9 N), with no statistically significant difference. Glass ionomer cement showed significantly lower force resistance (78.1 N). CONCLUSIONS Excellent bonding strength combined with easy application was found for universal resin cement, and thus, it is a potential alternative to acrylic bone cement in OOKP preparation.

Preclinical studies identify non-apoptotic low-level caspase-3 as therapeutic target in pemphigus vulgaris

The majority of pemphigus vulgaris (PV) patients suffer from a live-threatening loss of intercellular adhesion between keratinocytes (acantholysis). The disease is caused by auto-antibodies that bind to desmosomal cadherins desmoglein (Dsg) 3 or Dsg3 and Dsg1 in mucous membranes and skin. A currently unresolved controversy in PV is whether apoptosis is involved in the pathogenic process. The objective of this study was to perform preclinical studies to investigate apoptotic pathway activation in PV pathogenesis with the goal to assess its potential for clinical therapy. For this purpose, we investigated mouse and human skin keratinocyte cultures treated with PV antibodies (the experimental Dsg3 monospecific antibody AK23 or PV patients IgG), PV mouse models (passive transfer of AK23 or PVIgG into adult and neonatal mice) as well as PV patients' biopsies (n=6). A combination of TUNEL assay, analyses of membrane integrity, early apoptotic markers such as cleaved poly-ADP-ribose polymerase (PARP) and the collapse of actin cytoskeleton failed to provide evidence for apoptosis in PV pathogenesis. However, the in vitro and in vivo PV models, allowing to monitor progression of lesion formation, revealed an early, transient and low-level caspase-3 activation. Pharmacological inhibition confirmed the functional implication of caspase-3 in major events in PV such as shedding of Dsg3, keratin retraction, proliferation including c-Myc induction, p38MAPK activation and acantholysis. Together, these data identify low-level caspase-3 activation downstream of disrupted Dsg3 trans- or cis-adhesion as a major event in PV pathogenesis that is non-synonymous with apoptosis and represents, unlike apoptotic components, a promising target for clinical therapy. At a broader level, these results posit that an impairment of adhesive functions in concert with low-level, non-lethal caspase-3 activation can evoke profound cellular changes which may be of relevance for other diseases including cancer.

Osseointegration of titanium implants functionalised with phosphoserine-tethered poly(epsilon-lysine) dendrons: a comparative study with traditional surface treatments in sheep

The aim of this study was to analyse the osseointegrative potential of phosphoserine-tethered dendrons when applied as surface functionalisation molecules on titanium implants in a sheep model after 2 and 8 weeks of implantation. Uncoated and dendron-coated implants were implanted in six sheep. Sandblasted and etched (SE) or porous additive manufactured (AM) implants with and without additional dendron functionalisation (SE-PSD; AM-PSD) were placed in the pelvic bone. Three implants per group were examined histologically and six implants were tested biomechanically. After 2 and 8 weeks the bone-to-implant contact (BIC) total values of SE implants (43.7 ± 12.2; 53.3 ± 9.0 %) and SE-PSD (46.7 ± 4.5; 61.7 ± 4.9 %) as well as AM implants (20.49 ± 5.1; 43.9 ± 9.7 %) and AM-PSD implants (19.7 ± 3.5; 48.3 ± 15.6 %) showed no statistically significant differences. For SE-PSD and AM-PSD a separate analysis of only the cancellous BIC demonstrated a statistically significant difference after 2 and 8 weeks. Biomechanical findings proved the overall increased stability of the porous implants after 8 weeks. Overall, the great effect of implant macro design on osseointegration was further supported by additional phosphoserine-tethered dendrons for SE and AM implants.

A STUDY ON THE FUNCTION OF 14-3-3SIGMA IN REGULATING CANCER ENERGY METABOLISM

Metabolic reprogramming has been shown to be a major cancer hallmark providing tumor cells with significant advantages for survival, proliferation, growth, metastasis and resistance against anti-cancer therapies. Glycolysis, glutaminolysis and mitochondrial biogenesis are among the most essential cancer metabolic alterations because these pathways provide cancer cells with not only energy but also crucial metabolites to support large-scale biosynthesis, rapid proliferation and tumorigenesis. In this study, we find that 14-3-3σ suppresses all these three metabolic processes by promoting the degradation of their main driver, c-Myc. In fact, 14-3-3s significantly enhances c-Myc poly-ubiquitination and subsequent degradation, reduces c-Myc transcriptional activity, and down-regulates c-Myc-induced metabolic target genes expression. Therefore, 14-3-3σ remarkably blocks glycolysis, decreases glutaminolysis and diminishes mitochondrial mass of cancer cells both in vitro and in vivo, thereby severely suppressing cancer bioenergetics and metabolism. As a result, a high level of 14-3-3σ in tumors is strongly associated with increased breast cancer patients’ overall and metastasis-free survival as well as better clinical outcomes. Thus, this study reveals a new role for 14-3-3s as a significant regulator of cancer bioenergetics and a promising target for the development of anti-cancer metabolism therapies.

Influence of the molecular architecture on the secondary relaxations of Poly(styrene-co-methylmethacrylate) copolymers

The processes of adsorption of grafted copolymers onto negatively charged surfaces were studied using a dissipative quartz crystal microbalance (D-QCM) and ellipsometry. The control parameters in the study of the adsorption are the existence or absence on the molecular architecture of grafted polyethyleneglycol (PEG) chains with different lengths and the chemical nature of the main chain, poly(allylamine) (PAH) or poly(L-lysine) (PLL). It was found out that the adsorption kinetics of the polymers showed a complex behavior. The total adsorbed amount depends on the architecture of the polymer chains (length of the PEG chains), on the polymer concentration and on the chemical nature of the main chain. The comparison of the thicknesses of the adsorbed layers obtained from D-QCM and from ellipsometry allowed calculation of the water content of the layers that is intimately related to the grafting length. The analysis of D-QCM results also provides information about the shear modulus of the layers, whose values have been found to be typical of a rubber-like polymer system. It is shown that the adsorption of polymers with a charged backbone is not driven exclusively by the electrostatic interactions, but the entropic contributions as a result of the trapping of water in the layer structure are of fundamental importance.

Confined crystallization of nanolayered poly(ethylene terephthalate) using X-ray diffraction methods

The development of crystalline lamellae in ultra-thin layers of poly(ethylene terephthalate) PET confined between polycarbonate (PC) layers in an alternating assembly is investigated as a function of layer thickness by means of X-ray diffraction methods. Isothermal crystallization from the glassy state is in-situ followed by means of small-angle X-ray diffraction. It is found that the reduced size of the PET layers influences the lamellar nanostructure and induces a preferential lamellar orientation. Two lamellar populations, flat-on and edge-on, are found to coexist in a wide range of crystallization temperatures (Tc = 117–150 °C) and within layer thicknesses down to 35 nm. Flat-on lamellae appear at a reduced crystallization rate with respect to bulk PET giving rise to crystals of similar dimensions separated by larger amorphous regions. In addition, a narrower distribution of lamellar orientations develops when the layer thickness is reduced or the crystallization temperature is raised. In case of edge-on lamellae, crystallization conditions also influence the development of lamellar orientation; however, the latter is little affected by the reduced size of the layers. Results suggest that flat-on lamellae arise as a consequence of spatial confinement and edge-on lamellae could be generated due to the interactions with the PC interface. En este trabajo se investiga mediante difracción de rayos X a ángulos bajos (SAXS) y a ángulos altos (WAXS), la cristalización de láminas delgadas de Polietilén tereftalato (PET) confinadas entre láminas de Policarbonato (PC), tomando como referencia PET sin confinar. El espesor de las capas de PET varía entre 35nm y 115 nm. Se realizaron medidas de difracción a tres temperaturas de cristalización (117ºC, 132ºC y 150ºC) encontrándose que el reducido espesor de las capas de PET influye en la estructura lamelar que se desarrolla, induciendo una orientación preferente de las láminas. Se integró la intensidad difractada alrededor del máximo en SAXS para obtener una representación de la intensidad en función del ángulo acimutal. Mediante análisis de mínimos cuadrados se separó la curva experimental obtenida en tres contribuciones diferentes: una función Gausiana que describe la distribución de las orientaciones de las lamelas, una función lorenziana asociada a los máximos meridionales (asociados a las interfases PET-PC) y un background constante. Por otra parte la cantidad de material cristalizado se estimó asumiendo que la intensidad del background en el barrido acimutal, una vez restado el background del primer difractograma (sin máximos en SAXS) se asocia con la contribución del material isotrópico que resta en la muestra cristalizada. Se observa la coexistencia de dos poblaciones de lamelas: flat-on y edge-on. A medida que el espesor de las láminas de PET disminuye la población de las lamelas flat-on experimenta los siguientes cambios: 1) la distribución de orientación se estrecha, 2) la fracción de material cristalizado orientado aumenta, 3) la cinética de cristalización se ralentiza y 4) el largo espaciado aumenta es decir las regiones amorfas entre lamelas aumentan su tamaño. Parece demostrarse que es en las primeras etapas del crecimiento lamelar cuando la restricción espacial fuerza a las lamelas a esta orientación tipo flat-on frente a la orientación edge-on.

Tuning the properties of carbon fiber-reinforced poly (phenylene sulphide) laminates via incorporation of inorganic fullerene-like WS2 nanoparticles

Novel carbon fiber (CF)-reinforced poly(phenylene sulphide) (PPS) laminates incorporating inorganic fullerene-like tungsten disulfide (IF-WS2) nanoparticles were prepared via melt-blending and hot-press processing. The influence of the IF-WS2 on the morphology, thermal, mechanical and tribological properties of PPS/CF composites was investigated. Efficient nanoparticle dispersion within the matrix was attained without using surfactants. A progressive rise in thermal stability was found with increasing IF-WS2 loading, as revealed by thermogravimetric analysis. The addition of low nanoparticle contents retarded the crystallization of the matrix, whereas concentrations equal or higher than 1.0 wt% increased both the crystallization temperature and degree of crystallinity compared to those of PPS/CF. Mechanical tests indicated that with only 1.0 wt% IF-WS2 the flexural modulus and strength of PPS/CF improved by 17 and 14%, respectively, without loss in toughness, ascribed to a synergistic effect between the two fillers. A significant enhancement in the storage modulus and glass transition temperature was also observed. Moreover, the wear rate and coefficient of friction strongly decreased, attributed to the lubricant role of the IF-WS2 combined with their reinforcing effect. These inorganic nanoparticles show great potential to improve the mechanical and tribological properties of conventional thermoplastic/CF composites for structural applications.

Rheological and tribological properties of poly(phenylene sulphide) and poly(ether ether ketone) composites incorporating carbon nanotubes and inorganic nanoparticles: a comparative study

The rheological and tribological properties of single-walled carbon nanotube (SWCNT)-reinforced poly(phenylene sulphide) (PPS) and poly(ether ether ketone) (PEEK) nanocomposites prepared via melt-extrusion were investigated. The effectiveness of employing a dual-nanofiller strategy combining polyetherimide (PEI)-wrapped SWCNTs with inorganic fullerene-like tungsten disulfide (IF-WS2) nanoparticles for property enhancement of the resulting hybrid composites was evaluated. Viscoelastic measurements revealed that the complex viscosity ?, storage modulus G?, and loss modulus G? increased with SWCNT content. In the low-frequency region, G? and G? became almost independent of frequency at higher SWCNT loadings, suggesting a transition from liquid-like to solid-like behavior. The incorporation of increasing IF-WS2 contents led to a progressive drop in ? and G? due to a lubricant effect. PEEK nanocomposites showed lower percolation threshold than those based on PPS, ascribed to an improved SWCNT dispersion due to the higher affinity between PEI and PEEK. The SWCNTs significantly lowered the wear rate but only slightly reduced the coefficient of friction. Composites with both nanofillers exhibited improved wear behavior, attributed to the outstanding tribological properties of these nanoparticles and a synergistic reinforcement effect. The combination of SWCNTs with IF-WS2 is a promising route for improving the tribological and rheological performance of thermoplastic nanocomposites.

Towards the development of poly (phenylene sulphide) based nanocomposites with enhanced mechanical, electrical and tribological properties

Novel poly(phenylene sulphide) (PPS) nanocomposites reinforced with an aminated derivative (PPS-NH2) covalently attached to acid-treated single-walled carbon nanotubes (SWCNTs) were prepared via simple melt-blending technique. Their morphology, viscoelastic behaviour, electrical conductivity, mechanical and tribological properties were investigated. Scanning electron microscopy revealed that the grafting process was effective in uniformly dispersing the SWCNTs within the matrix. The storage and loss moduli as a function of frequency increased with the SWCNT content, tending to a plateau in the low-frequency regime. The electrical conductivity of the nanocomposites was considerably enhanced in the range 0.1?0.5 wt% SWCNTs; electrical and rheological percolation thresholds occurred at similar nanotube concentrations. Mechanical tests demonstrated that with only 1.0 wt% SWCNTs the Young's modulus and tensile strength of the matrix improved by 51 and 37%, respectively, without decrement in toughness, ascribed to a very efficient load transfer. A moderate decrease in the friction coefficient and a 75% reduction in wear rate were found for the abovementioned nanotube loading, indicating that PPS-NH2-g-SWCNTs are good tribological additives for thermoplastic polymers. Based on the promising results obtained in this work, it is expected that these nanofillers will be used to develop high-performance thermoplastic/CNT nanocomposites for structural applications.

Enhancing the thermomechanical behaviour of poly(phenylene sulphide) based composites via incorporation of covalently grafted carbon nanotubes

The thermal and thermomechanical properties of poly(phenylene sulphide) (PPS) based nanocomposites incorporating a polymer derivative covalently anchored onto single-walled carbon nanotubes (SWCNTs) were investigated. The grafted fillers acted as nucleating agents, increasing the crystallization temperature and degree of crystallinity of the matrix. They also enhanced its thermal stability, flame retardancy, glass transition (Tg) and heat deflection temperatures while reduced the coefficient of thermal expansion at temperatures below Tg. A strong rise in the thermal conductivity, Young?s modulus and tensile strength was found with increasing filler loading both in the glassy and rubbery states. All these outstanding improvements are ascribed to strong matrix-filler interfacial interactions combined with a compatibilization effect that results in very homogeneous SWCNT dispersion. The results herein offer useful insights towards the development of engineering thermoplastic/CNT nanocomposites for high-temperature applications.

Inorganic Nanoparticle-Modified Poly(Phenylene Sulphide)/ Carbon Fiber Laminates: Thermomechanical Behaviour

Carbon fiber (CF)-reinforced high-temperature thermoplastics such as poly(phenylene sulphide) (PPS) are widely used in structural composites for aerospace and automotive applications. The porosity of CF-reinforced polymers is a very important topic for practical applications since there is a direct correlation between void content and mechanical properties. In this study, inorganic fullerene-like tungsten disulphide (IF-WS2) lubricant nanoparticles were used to manufacture PPS/IF-WS2/CF laminates via melt-blending and hot-press processing, and the effect of IF-WS2 loading on the quality, thermal and mechanical behaviour of the hybrid composites was investigated. The addition of IF-WS2 improved fiber impregnation, resulting in lower degree of porosity and increased delamination resistance, compression and flexural properties; their reinforcement effect was greater at temperatures above the glass transition (Tg). IF-WS2 contents higher than 0.5 wt % increased Tg and the heat deflection temperature while reduced the coefficient of thermal expansion. The multiscale laminates exhibited higher ignition point and notably reduced peak heat release rate compared to PPS/CF. The coexistence of micro- and nano-scale fillers resulted in synergistic effects that enhanced the stiffness, strength, thermal conductivity and flame retardancy of the matrix. The results presented herein demonstrate that the IF-WS2 are very promising nanofillers to improve the thermomechanical properties of conventional thermoplastic/CF composites.

Development of novel melt-processable biopolymer nanocomposites based on poly(L-lactic acid) and WS2 inorganic nanotubes.

The use of tungsten disulphide inorganic nanotubes (INT-WS2) offers the opportunity to produce novel and advanced biopolymer-based nanocomposite materials with excellent nanoparticle dispersion without the need for modifiers or surfactants via conventional melt blending. The study of the non-isothermal melt-crystallization kinetics provides a clear picture of the transformation of poly(L-lactic acid) (PLLA) molecules from the non-ordered to the ordered state. The overall crystallization rate, final crystallinity and subsequent melting behaviour of PLLA were controlled by both the incorporation of INT-WS2 and the variation of the cooling rate. In particular, it was shown that INT-WS2 exhibits much more prominent nucleation activity on the crystallization of PLLA than other specific nucleating agents or nano-sized fillers. These features may be advantageous for the enhancement of mechanical properties and process-ability of PLLA-based materials. PLLA/INT-WS2 nanocomposites can be employed as low cost biodegradable materials for many eco-friendly and medical applications, and the exceptional crystallization behaviour observed opens new perspectives for scale-up and broader applications.