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Supplementary Figure 4 from Everolimus Augments the Effects of Sorafenib in a Syngeneic Orthotopic Model of Hepatocellular Carcinoma

Supplementary Figure 2 from Everolimus Augments the Effects of Sorafenib in a Syngeneic Orthotopic Model of Hepatocellular Carcinoma

Supplementary Figure Legends from Everolimus Augments the Effects of Sorafenib in a Syngeneic Orthotopic Model of Hepatocellular Carcinoma

Sorafenib targets the Raf/mitogen-activated protein kinase, VEGF, and platelet-derived growth factor pathways and prolongs survival patients in advanced hepatocellular carcinoma (HCC). Everolimus inhibits the mammalian target of rapamycin, a kinase overactive in HCC. To investigate whether the antitumor effects of these agents are additive, we compared a combined and sequential treatment regimen of everolimus and sorafenib with monotherapy. After hepatic implantation of Morris Hepatoma (MH) cells, rats were randomly allocated to everolimus (5 mg/kg, 2×/week), sorafenib (7.5 mg/kg/d), combined everolimus and sorafenib, sequential sorafenib (2 weeks) then everolimus (3 weeks), or control groups. MRI quantified tumor volumes. Erk1/2, 4E-BP1, and their phosphorylated forms were quantified by immunoblotting. Angiogenesis was assessed in vitro by aortic ring and tube formation assays, and in vivo with Vegf-a mRNA and vascular casts. After 35 days, tumor volumes were reduced by 60%, 85%, and 55%, relative to controls, in everolimus, the combination, and sequential groups, respectively (P < 0.01). Survival was longest in the combination group (P < 0.001). Phosphorylation of 4E-BP1 and Erk1/2 decreased after everolimus and sorafenib, respectively. Angiogenesis decreased after all treatments (P < 0.05), although sorafenib increased Vegf-a mRNA in liver tumors. Vessel sprouting was abundant in control tumors, lower after sorafenib, and absent after the combination. Intussusceptive angiogenic transluminal pillars failed to coalesce after the combination. Combined treatment with everolimus and sorafenib exerts a stronger antitumoral effect on MH tumors than monotherapy. Everolimus retains antitumoral properties when administered sequentially after sorafenib. This supports the clinical use of everolimus in HCC, both in combination with sorafenib or after sorafenib. Mol Cancer Ther; 10(6); 1007–17. ©2011 AACR.

Supplementary Figure 3 from Everolimus Augments the Effects of Sorafenib in a Syngeneic Orthotopic Model of Hepatocellular Carcinoma

Supplementary Figure 1 from Everolimus Augments the Effects of Sorafenib in a Syngeneic Orthotopic Model of Hepatocellular Carcinoma

In this study, polycaprolactone (PCL), as a biocompatible polymer was functionalized by addition of medicinal plant extract- Achillea millefolium L. (yarrow). Nanofiber mats were fabricated from PCL solutions containing dry yarrow extract in four concentrations (5%, 10%, 15%, and 20% relative to the weight of the polymer) by using blend electrospinning method. The nanofibers were characterized for their biological, mechanical and drug release behavior. In vitro release of yarrow polyphenols from the electrospun PCL nanofibers over a period of 5 days showed the release of up to 98% of the total loaded polyphenols. The released polyphenols retained its antioxidant activity, which was determined by DPPH assay. Electrospun PCL/yarrow nanofiber mats exhibited the antibacterial effect against Staphylococcus aureus, but had no effect on the growth of Pseudomonas aeruginosa. All PCL/yarrow nanofiber mats had improved mechanical properties compared to the neat PCL nanofibers, as evident by an increase in Young's modulus of elasticity (up to 5.7 times), the tensile strength (up to 5.5 times), and the strain at break (up to 1.45 times). Based on our results, yarrow-loaded PCL nanofiber mats appeared to be multi-functional biomaterials suitable for the production of catheter-coating materials, patches, or gauzes with antibacterial and antioxidant properties.

High performance polymers with bio-based modifiers are promising materials in terms of applications and environmental impact. In this work, raw acacia honey was used as a bio-modifier for epoxy resin, as a rich source of functional groups. The addition of honey resulted in the formation of highly stable structures that were observed in scanning electron microscopy images as separate phases at the fracture surface, which were involved in the toughening of the resin. Structural changes were investigated, revealing the formation of a new aldehyde carbonyl group. Thermal analysis confirmed the formation of products that were stable up to 600 °C, with a glass transition temperature of 228 °C. An energy-controlled impact test was performed to compare the absorbed impact energy of bio-modified epoxy containing different amounts of honey with unmodified epoxy resin. The results showed that bio-modified epoxy resin with 3 wt% of acacia honey could withstand several impacts with full recovery, while unmodified epoxy resin broke at first impact. The absorbed energy at first impact was 2.5 times higher for bio-modified epoxy resin than it was for unmodified epoxy resin. In this manner, by using simple preparation and a raw material that is abundant in nature, a novel epoxy with high thermal and impact resistance was obtained, opening a path for further research in this field.

Mechanical (hardness and adhesion) and electrical (sheet resistance) characteristics of electrolytically produced copper coatings have been investigated. Morphologies of Cu coatings produced galvanostatically at two current densities from the basic sulfate electrolyte and from an electrolyte containing levelling/brightening additives without and with application of ultrasound for the electrolyte stirring were characterized by SEM and AFM techniques. Mechanical characteristics were examined by Vickers microindentation using the Chen–Gao (C–G) composite hardness model, while electrical characteristics were examined by the four-point probe method. Application of ultrasound achieved benefits on both hardness and adhesion of the Cu coatings, thereby the use of both the larger current density and additive-free electrolyte improved these mechanical characteristics. The hardness of Cu coatings calculated according to the C–G model was in the 1.1844–1.2303 GPa range for fine-grained Cu coatings obtained from the sulfate electrolyte and in the 0.8572–1.1507 GPa range for smooth Cu coatings obtained from the electrolyte with additives. Analysis of the electrical characteristics of Cu coatings after an aging period of 4 years showed differences in the sheet resistance between the top and the bottom sides of the coating, which is attributed to the formation of a thin oxide layer on the coating surface area.

Rice husk was used to produce silica particles, which were then used to reinforce the polymer matrix. The synthesized SiO2 particles were characterized using X-ray diffraction, Fourier transforms infrared spectroscopy (FTIR) and scanning electron microscopy with EDS. In a PMMA matrix, prepared SiO2 particles in amounts of 1, 3, and 5 wt.% were used as reinforcing agents. The goal of this research was to see if SiO2 particles had any effect on the mechanical properties of polymer composite materials. The morphology of the composites was examined using a field emission scanning electron microscope (FE-SEM). Vickers microindentation hardness and impact testing were used to determine the mechanical properties of the obtained composites. The indentation creep’s behavior of a polymethylmetacrylate (PMMA) composite material with varying amounts of nanoparticles (SiO2) was investigated and analyzed. Љуска пиринча је коришћена за производњу честица силицијум диоксида, које су коришћене за ојачавање полимерне матрице. Синтетизоване честице силицијум диоксида карактерисане су дифракцијом рендгенских зрака, инфрацрвеном спектроскопијом Фуријеове трансформације (ФТИР) и скенирајућом лектронском микроскопом са ЕДС. У матрици полиметил метакриилата, припремљене честицее силицијум диоксида у количинама од 1, 3 и 5 мас. %, коришћене су као ојачање. Циљ овог истраживања био је да се утврди да ли честице силицијум диоксида утичу на механичка својства полимерних композитних материјала. Морфологија композита је испитивана коришћењем поља емисионог скенирајућег електронског микроскопа (ФЕ-СЕМ). За одређивање механичких својстава добијених композита коришћени су Вицкерс микроиндентациони тестови и испитивање на удар. Понашање пузања композитног материјала полиметилметакрилата (ПММА) са различитим количинама наночестица силицијум диоксида је испитано и анализирано.

Cellulose-fibre lifecycle is severely limited within today’s recycling methodology. Feedstock cellulose and filler abound in products considered uneconomic, unsuitable for standard deinking or too weak for original product re-use. Regeneration of cellulose from ionic liquid (IL) solution offers a novel valorisation route with potential to replace oil-based plastics, simultaneously bringing filler-containing cellulose waste into circular economy. To exemplify, composite cellulose-CaCO3 filaments were produced from cellulose pulp and waste office paper (already containing 27 w/w% precipitated CaCO3filler), without any pretreatment, using diazabicyclo[4.3.0]non-5-enium-acetate ([DHBN][OAc]) as IL solvent to form a dope. The dope suspension was extruded via dry-jet wet spinning. Surface morphology and elemental analysis were investigated using scanning electron microscopy with energy dispersive X-ray spectroscopy. X-ray diffraction was used to measure the degree of cellulose crystallinity. Static and dynamic mechanical properties were determined. Thermal stability was evaluated by thermogravimetric analysis. Filaments obtained from waste office paper showed that high filler levels and the presence of print toner enhanced both mechanical and thermal stability, while decreasing, as expected, ultimate breaking strength in comparison to filaments containing virgin cellulose alone. For comparison, 2 w/w% ground CaCO3 introduced into pure cellulose dope led to significant increase in cellulose crystallinity and resulting stiffness, while thermal properties remained unchanged at the low level filler addition. Such addition of CaCO3 during cellulose regeneration, or usage of already filled paper, could be an effective way to obtain high strength cellulose-CaCO3 composite materials, thus valorising in a circular economy renewable cellulosic wastes rejected from the current recycling stream.

In this study, a novel procedure, based on application of the Chicot––Lesage (C–L) composite hardness model, was proposed for the determination of an absolute hardness of electrolytically produced copper coatings. The Cu coatings were electrodeposited on the Si(111) substrate by the pulsating current (PC) regime with a variation of the following parameters: the pause duration, the current density amplitude and the coating thickness. The topography of produced coatings was characterized by atomic force microscope (AFM), while a hardness of the coatings was examined by Vickers microindentation test. Applying the C–L model, the critical relative indentation depth (RID)c of 0.14 was determined, which is independent of all examined parameters of the PC regime. This RID value separated the area in which the composite hardness of the Cu coating corresponded to its absolute hardness (RID < 0.14) from the area in which the application of the C–L model was necessary for a determination of the absolute coating hardness (RID ≥ 0.14). The obtained value was in a good agreement with the value already published in the literature. Предложен je нови поступак заснован на примени Chicot–Lesage (C–L) композитног модела тврдоће за одређивање апсолутне тврдоће електролитички добијених превлака бакра. Превлаке бакра су електрохемијски исталожене на силицијуму (111) оријентације режимом пулсирајуће струје варирањем следећих параметара: трајање паузе, амплитудна густина струје и дебљина превлаке. Топографија произведених превлака је окарактерисана микроскопијом атомских сила, док је тврдоћа превлака испитивана Викерсовим тестом утискивања. Применом C–L композитног модела тврдоће, одређена је критична релативна дубина утискивања (RID)c, од 0,14, која је независна од свих испитиваних параметара режима пулсирајуће струје. Ова вредност раздваја област у којој композитна тврдоћа превлаке може да се изједначи са њеном апсолутном тврдоћом (RID < 0,14) од области у којој је неопходно применити C–L модел за одређивање апсолутне тврдоће превлаке (RID ≥ 0,14). Добијена вредност RID показује добро слагање са вредностима публикованим у литератури.

In this paper, the influence of solvents and reinforcement processing methods on the mechanical properties of nanocomposite poly-(methyl methacrylate) PMMA-Ti3C2Tx MXene was investigated. A weight fraction of reinforcement was 1% and two types of solvents were used for sample preparation: dimethylformamide (DMF) and acetone (Ac). Deionized water and DMF were used to delaminate the MXene. An additional peak at 1675 cm-1 was observed on the FTIR spectra of the obtained samples, which indicates that some amount of DMF remained in the material after drying. The results of microindentation show that samples where MXene was delaminated in deionized water and PMMA dissolved in acetone had a 74.46% higher reduced modulus of elasticity, which is supported by SEM images where better dispersion of MXenes is observed.

In this paper, the impact of wood fiber surface modification on mechanical properties of poly-(methyl methacrylate)(PMMA)-wood composite was examined. FTIR analysis has shown that the wood fiber surface modification was accomplished and that the matrix-fiber bond was established. The SEM image of the breakage surface has affirmed the improved adhesion between the fibers and the polymer in the specimen with modified wood fibers. The bond improvement between the wood fibers and the matrix results in increased hardness and a better distribution of strain within the composite. High speed impact testing results have shown that the specimen with the bonding agent can increase the absorbed energy six times when compared to the pure PMMA specimen. U ovom radu izvršeno je ispitivanje uticaja modifikacije površine drvenih vlakana na mehanička svojstva kompozita poli-(metil metakrilat)(PMMA)- drvo. FTIR analiza je pokazala da je postignuta modifikacija površine drvenih vlakana, kao i da je ostvarena veza matrica-vlakno. SEM snimak površine loma uzorka takođe potvrđuje bolju adheziju vlakana i polimera u uzorku sa modifikovanim vlaknima drveta. Poboljšanje veze između drvenih vlakana i matrice omogućava veću čvrstoću i bolji prenos opterećenja unutar kompozita. Rezultati ispitivanja udarom kontrolisane energije pokazali su da uzorak sa vezujućim agensom može da poveća apsorbovanu energiju i do šest puta u odnosu na čist PMMA.

The electrospinning of Rhodamine B (RhB) -doped poly (methyl methacrylate) (PMMA) is presented as a promising technique for processing photo luminescent nanofibers. Nanofibers with concentrations of RhB in PMMA between 0.05 wt% and 2.0 wt% were studied. The FTIR spectrum, fiber morphology, thermal properties and optical properties of the nanofibers were investigated. The smooth, bead- free and nonporous nanofibers were obtained. Differential scanning calorimetry (DSC) results revealed that the Tg increased with the increasing of Rh B content up to a 0.5 wt% and beyond that decreased. The laser induced fluorescence (LIF) technique was used to study the fluorescence properties of the nanofibers. The maximum fluorescence emission was observed at a concentration of RhB of c = 0.2 wt%. [Projekat Ministartsva nauke Republike Srbije, br. TR 34011, br. III 45019 i br. 171020]

OBJECTIVES/HYPOTHESIS: Cadherins are a superfamily of transmembrane glycoproteins, which mediate calcium-dependent intercellular adhesions. T-cadherin is an atypical member of the cadherin family in regard to its structure; it acts as a signalling receptor rather than an adhesion molecule. In this study we examine the role of T-cadherin in the mammalian cochlea. STUDY DESIGN: This study investigated the expression of T-cadherin in the inner ear under physiologic and pathologic conditions. METHODS: Expression of T-cadherin in the rat cochlea was analyzed by reverse-transcriptase polymerase chain reaction (RT-PCR), real-time RT-PCR, Western blot, and immunohistochemistry. RESULTS: We detected T-cadherin mRNA expression in three different components in the cochlea of postnatal mouse, namely the organ of Corti (OC), the spiral ganglion (SG), and the stria vascularis (SV). The SG and SV showed a higher T-cadherin mRNA level than the OC. T-cadherin protein was detected by Western blotting in the OC, SG, and SV. Immunofluorescence microscopy of adult mouse cochlea revealed the presence of T-cadherin in the apical parts of the inner and outer hair cells as well as in the SV and SG. OCs treated with gentamicin for 3, 6, or 12 hours did not show any change in T-cadherin gene expression compared to control explants maintained in culture medium alone. CONCLUSIONS: T-cadherin is expressed within the cochlea. T-cadherin seems to have a wide variety of functions in the inner ear, ranging from mechanical functions to functions in response to hair cell damage and loss.

Studies conducted over the last few years demonstrated that signaling pathways that operate in the organs of Corti (OC) play a central role in survival and death of hair cells. An important goal of molecular otology is to characterize these signaling pathways in normal inner ears and inner ears exposed to a variety of different forms of stress, such as ototoxic substances and noise overexposure. In this study, we used high-performance reverse protein microarray technology and phospho-specific antibodies to examine the activation status of defined molecules involved in cellular signaling. We demonstrate that reverse protein microarrays based on the highly sensitive planar-waveguide technology provide an effective and high-throughput means to assess the activation state of key molecules involved in apoptotic and prosurvival signaling in microdissected OC explants over time. In this study, we show that gentamicin and a specific NF-ĸB inhibitor increase the ratio of phospho-c-Jun/c-Jun in OC explants of postnatal rats soon after exposure to these drugs. In addition, we found a decrease in the phospho-Akt/Akt ratio in OC explants early after NF-ĸB inhibition. Finally, we observed an early and consistent decrease in the phospho-p38/p38 ratio in OC explants exposed to the NF-ĸB inhibitor and only a transient decrease in this ratio in OC examples after gentamicin exposure.

Neural transplantation has emerged as an attractive strategy for the replacement of neurons that have been lost in the central nervous system. Multipotent neural progenitor cells are potentially useful as donor cells to repopulate the degenerated regions. One important aspect of a transplantation strategy is whether transplanted cells are capable of fiber outgrowth with the aim of rebuilding axonal connections within the host brain. To address this issue, we expanded neuronal progenitor from the cortex of embryonic day 15 ubiquitously green fluorescent protein-expressing transgenic mice as neurospheres in vitro and grafted them into the entorhinal cortex of 8-week-old mice immediately after a perforant pathway lesion. After transplantation into a host brain with a lesion of the entorhino-hippocampal projection, the neurosphere-derived cells extended long fiber projections directed towards the dentate gyrus. Our results indicate that transplantation of neurosphere-derived cells might be a promising strategy to replace lost or damaged axonal projections.

This study was performed to examine the maintenance of blood vesselsin vitroin cortical organotypic slice cultures of mice with special emphasis on basic fibroblast growth factor (FGF-2), which is known to promote angiogenesis and to preserve the integrity of the blood–brain barrier. Slices of neonatal day 3 or 4 mouse brain were maintained for 3, 7, or 10 din vitro(DIV) under standard culture conditions or in the presence of FGF-2. Immunohistochemistry for factor VIII-related antigen or laminin revealed a relative low number of blood vessels under standard conditions. In contrast, moderate FGF-2 concentrations increased the number of vessels: with 0.5 ng/ml FGF-2 it was 1.4-fold higher after DIV 3 or 1.5-fold after DIV 7 compared with controls; with 5 ng/ml it was almost doubled in both cases. With an excess of 50 ng/ml, FGF-2 vessels were reduced after DIV 3 or similar to controls after DIV 7. FGF receptor 1 was preferentially found on endothelial cells; its immunolabeling was reduced in the presence of the ligand. Cell death detected by an ethidium bromide analog or the apoptosis marker caspase-3 was barely detectable during the 10 d culture period. Immunolabeling of the tight junction proteins ZO-1 (zonula occludens protein 1), occludin, claudin-5, and claudin-3 revealed evidence for structural integrity of the blood–brain barrier in the presence of moderate FGF-2 concentrations. In conclusion, FGF-2 maintains blood vesselsin vitroand preserves the composition of the tight junction. Hence, we propose FGF-2-treated organotypic cortical slices as a new tool for mechanistic studies of the blood–brain barrier.

The physical and mechanical properties of the six samples of polyurethane/p-aramid multiaxial fabric forms (Kolon fabrics) were analyzed by high speed impact tester. The samples were impregnated with 10 wt.% poly (vinyl butyral) (PVB)/ethanol solution and modified with γ-aminopropyltriethoxysilane (AMEO silane)/ethanol solution. Unmodified silica (SiO2) nanoparticles, tungsten disulfide (WS2) nanoparticles and multiwalled carbon nanotubes (MWCNT) were used as reinforcement for multiaxial Kolon fabrics. The SiO2/PVB mass fraction was 10 wt.%, while the WS2/PVB and MWCNT/PVB mass fractions were the same - 1 wt.%. The results revealed the increase of the maximal impact force and the total energy absorbed for all the samples compared to the unmodified Kolon fabric sample and the application possibilities of these materials for the antiballistic protection. Fizička i mehanička svojstva šest uzoraka poliuretan/p-aramidnih multiaksijalnih tkanina (Kolon lamina) su analizirana pomoću impakt tester uređaja. Uzorci su impregnisani sa 10 mas.% rastvorom poli (vinil butiral)(PVB)/etanol i modifikovani 2 mas.% rastvorom γ-aminopropiltrietoksisilana (AMEO silan) u etanolu. Nemodifikovane silika (SiO2) nanočestice, volfram disulfid (WS2) nanočestice i višeslojne ugljenične nanocevi (MWCNT) su korišćene kao ojačanja multiaksijalnih Kolon lamina. Maseni udeo ojačanja SiO2/PVB je bio 10 mas.%, dok su maseni udeli WS2/PVB i MWCNT/PVB bili 1 mas.%. Rezultati pokazuju povećanje maksimalne udarne sile i ukupne apsorbovane energije kod svih uzoraka u odnosu na nemodifikovani Kolon uzorak i mogućnosti primene ovih materijala za antibalističku zaštitu.

Rapamycin is an antifungal agent with immunosuppressive properties. Rapamycin inhibits the mammalian target of rapamycin (mTOR) by blocking the mTOR complex 1 (mTORC1). mTOR is an atypical serine/threonine protein kinase, which controls cell growth, cell proliferation, and cell metabolism. However, less is known about the mTOR pathway in the inner ear. First, we evaluated whether or not the two mTOR complexes (mTORC1 and mTORC2, resp.) are present in the mammalian cochlea. Next, tissue explants of 5-day-old rats were treated with increasing concentrations of rapamycin to explore the effects of rapamycin on auditory hair cells and spiral ganglion neurons. Auditory hair cell survival, spiral ganglion neuron number, length of neurites, and neuronal survival were analyzedin vitro. Our data indicates that both mTOR complexes are expressed in the mammalian cochlea. We observed that inhibition of mTOR by rapamycin results in a dose dependent damage of auditory hair cells. Moreover, spiral ganglion neurite number and length of neurites were significantly decreased in all concentrations used compared to control in a dose dependent manner. Our data indicate that the mTOR may play a role in the survival of hair cells and modulates spiral ganglion neuronal outgrowth and neurite formation.

The repair of axonal projections and the reconstruction of neuronal circuits after CNS lesions or during neurodegenerative disease are major challenges in restorative neuroscience. We have explored the potential of transplanted immature neurons to repair a specific axonal projection in an entorhino-hippocampal slice culture model system. When slices of immature entorhinal cortex (EC) from tau-GFP transgenic mice were cultured next to slices from postnatal hippocampus, an axonal projection from the E18 embryonic entorhinal cortex to the dentate gyrus of the postnatal hippocampus developed, which was similar to that observed in control cultures. Even more immature neuronal precursors in slices from E15 developing cerebral cortex differentiated and established an axonal projection to the hippocampal slice. This projection terminated specifically in the outer molecular layer of the dentate gyrus, the normal target area of the entorhino-hippocampal projection. When embryonic tissue from the presumptive brainstem area was used, there was still a subpopulation of fibers with a specific termination in the outer molecular layer, but few specific fibers were found in cocultures with embryonic midbrain. Our results show that very immature cortical neurons are potentially able to form an entorhino-hippocampal projection that terminates in a correct lamina-specific fashion in the dentate gyrus. These findings support the idea that immature neuronal precursor cells could be used for the reconstruction of specific neuronal circuits.

The dystrophin-associated protein complex (DAP) plays an important role in the integrity and stability of the muscle membrane. Whereas much is known about the interaction between DAP members at the sarcolemmal location, intracellular DAP assembly and trafficking is still largely unknown. In alpha-glucosidase (acid maltase) deficiency (alphaGDD), accumulation of glycogen is accompanied by cytoarchitectural abnormalities impairing normal protein metabolism. In the present study, we took advantage of this fact to examine the consequences of impaired protein handling on the formation of DAP, with the aim of gaining indirect knowledge about its sarcoplasmic trafficking and a better understanding of mechanisms leading to myopathic changes found in alphaGDD. Histological examination of alphaGDD muscle confirmed a vacuolar myopathy with glycogen accumulation both in vacuoles and within the sarcoplasm. Sarcoplasmic accumulation of sarcolemmal proteins, including dystrophin and sarcoglycans, occurred around some vacuoles and within non-vacuolated fibres. Utrophin was up-regulated and found at extra-junctional sarcolemmal locations of many fibres. AlphaGDD muscle cells developed in a fashion similar to that of controls in culture. However, vacuoles were found in 2-week-old alphaGDD myotubes, and these subsequently increased in size and number. Substantial alterations in DAP handling were found, with accumulation close to the Golgi apparatus. Utrophin was not enriched in the sarcoplasm but was up-regulated along the whole sarcolemma. Our results demonstrate a close association of dystrophin and sarcoglycans during sarcoplasmic processing. Furthermore, they suggest that the myopathy found in alphaGDD is a secondary form of DAP deficiency.

Neuron-glial-related cell adhesion molecule (NrCAM) is a neuronal cell adhesion molecule involved in neuron-neuron and neuron-glial adhesion as well as directional signaling during axonal cone growth. NrCAM has been shown to be involved in several cellular processes in the central and peripheral nervous systems, including neurite outgrowth, axonal pathfinding and myelination, fasciculation of nerve fibers, and cell migration. This includes sensory systems such as the eye and olfactory system. However, there are no reports on the expression/function of NrCAM in the auditory system. The aim of the present study was to elucidate the occurrence of NrCAM in the mammalian cochlea and its role in innervation of the auditory end organ. Our work indicates that NrCAM is highly expressed in the developing mammalian cochlea (position consistent with innervation). Moreover, we found that NrCAM, presented in stripe micropatterns, provide directional cues to neonatal rat inner ear spiral ganglion neurites in vitro. Our results are consistent with a role for NrCAM in the pathfinding of spiral ganglion dendrites toward their hair cell targets in the sensory epithelium.

Activin, a member of the TGF-F superfamily, was found to play an important role in the development, repair and apoptosis of different tissues and organs. Accordingly, activin signaling is involved in the development of the cochlea. Activin binds to its receptor ActRII, then dimerizes with ActRI and induces a signaling pathway resulting in gene expression. A study reported a case of fibrodysplasia ossificans progressiva with an unusual mutation in the ActRI gene leading to sensorineural hearing loss. This draws attention to the role of activin and its receptors in the developed cochlea. To date, only the expression of ActRII is known in the adult mammalian cochlea. In this study, we present for the first time the presence of activin A and ActRIB in the adult cochlea. Transgenic mice with postnatal dominant-negative ActRIB expression causing disruption of activin signaling in vivo were used for assessing cochlear morphology and hearing ability through the auditory brainstem response (ABR) threshold. Nonfunctioning ActRIB did not affect the ABR thresholds and did not alter the microscopic anatomy of the cochlea. We conclude, therefore, that activin signaling is not necessary for hearing in adult mice under physiological conditions but may be important during and after damaging events in the inner ear. i 2014 S. Karger AG, Basel

Based on learned helplessness theory, thisresearch evaluated a model in which helplessness andperceived control (internality) were hypothesized tomediate the effects of pain and disability(quality-of-well-being)on depression and pain behavior in 122participants with fibromyalgia. Path analytic proceduresrevealed that helplessness partially mediated theeffects of pain and disability on depression, and fullymediated the effects ofpain on self-reported painbehavior. Disability was an independent predictor ofboth self-reported and observed pain behavior.Beliefs ininternality were unrelated to depression and painbehavior measures, and thus did not serve in amediational capacity. The findings underscore theimportance of the helplessness construct infibromyalgia, a chronic pain syndrome of unknownetiology.

Somatostatin (SST) is a peptide hormone that exerts inhibitory effects mediated through binding to specific cell surface G protein-coupled receptors, of which five distinct subtypes (SSTR1-SSTR5) have been characterized. Our study performed on mouse cochlear hair cells shows the expression and localization of the three receptors (SSTR3-SSTR5) in wild-type (WT), single-knockout (SSTR1 KO) and double-knockout SSTR1/SSTR2 (DKO) mice. Similar SSTRs expression were observed in the inner hair cells (IHC), outer hair cells (OHC) and supporting cells of cultivated P7 mouse organ of Corti (OC) explants as well as in cultivated cochlear neuroepithelial supporting cells (NEsc). We found differences in the expression of SSTR3-5 in WT, SSTR1 KO and DKO mouse cochlea, which might be explained as a compensatory effect in the cochlea after the loss of SSTR1 and/or SSTR2.

The neuropeptide somatostatin (SST) is an important modulator of neurotransmission in the central nervous system (CNS) and binds to G-protein-coupled receptors (SSTR1-5) on target cells. Little is known about the expression and function of the somatostatinergic system in the mammalian cochlea. We analyzed the expression of SSTR1-SSTR5 in the immature mammalian cochlea. The peak in the expression of SSTR1 and SSTR2 at mRNA and protein level is around the onset of hearing to airborne sound, at postnatal day (P)14. This suggests their involvement in the maturation of the mammalian cochlea. We demonstrated that all five receptors are expressed in the inner hair cells (IHC) and outer hear cells (OHC) as well as in defined supporting cells of the organ of Corti (OC) in the adult mouse cochlea. A similar expression of the SSTRs in the IHC and OHC was found in cultivated P6 mouse OC explants as well as in neuroepithelial cell culture. In order to learn more about the regulation of SSTRs, we used mice with either a deletion of SSTR1, SSTR2 or SSTR1/SSTR2 double knock out (DKO). In DKO mice, SSTR5 was up-regulated and SSTR3 and SSTR4 were down regulated. These findings provide evidence of a compensatory regulation in the mammalian cochlea as a consequence of a receptor subtype deletion. In addition, we observed reduced levels of phospho-Akt and total-Akt in SSTR1 KO and DKO mice as compared to wild type (WT) mice. Akt is likely to be involved in hair cell survival. Most importantly, we found improved hair cell survival in somatostatin and octreotide treated OC explants that had been exposed to gentamicin compared to those explants exposed to gentamicin alone. These findings propose that the somatostatinergic system within the cochlea may have neuroprotective properties.

Depression in systemic lupus erythematosus (SLE) is a common yet poorly understood phenomenon. This research adopted a biopsychosocial model in which pain and family support were hypothesized to predict depressive symptoms directly, and indirectly, through perceived helplessness. A total of 44 confirmed SLE patients recruited through the community completed a set of questionnaires in a cross-sectional, correlation design. The results confirmed our model's applicability, demonstrating that high pain and high helplessness independently contributed to depression; however, the contribution of family support was not significant. While mixed support was found for a pain-learned helplessness link, helplessness mediated the effects of patients' socio-economic status on depression such that patients of lower socio-economic status reported greater helplessness in the face of their illness. In summary, the data indicate that pain and helplessness are important concomitants of SLE-related depression. Longitudinal research is recommended to clarify the relationships among these variables over time in clinical samples.

This research evaluated a model for examining the role of family cohesion and the pain-coping process in predicting depressive symptoms in fibromyalgia, a chronic pain disorder of unknown etiology. Depressive symptoms were highly prevalent in this patient group. Fifty-nine percent of the sample met or exceeded the cutoff score of 16 for depression on the Center for Epidemiological Studies Depression Scale (CES-D), while slightly greater than 50% exceeded the cutoff score of 19, a figure that is suggested for evaluating depression in chronic pain populations. Multiple regression analyses, controlling for demographic factors and medication use, revealed that low family cohesion (either reported by the patient or the patient's spouse), high pain, high helplessness, and high passive coping contributed independently to greater CES-D scores. Pain also was related to higher depression scores indirectly through its association with greater helplessness and passive coping. In contrast, no indirect effects of family cohesion were found on depressive symptoms through pain, helplessness, and passive coping. Structural equation modeling procedures provided confirmatory evidence of the significance of these relationships, indicating a high degree of goodness-of-fit with the model examined. The data illustrate the import of a multidimensionalframeworkfor conceptualizing physical, psychological, and social determinants of depressive disturbance in fibromyalgia.

Item does not contain fulltext The neuropeptide somatostatin (SST) exerts several important physiological actions in the adult central nervous system through interactions with membrane-bound receptors. Transient expression of SST and its receptors has been described in several brain areas during early ontogeny. It is therefore believed that SST may play a role in neural maturation. The present study provides the first evidence for the developmental expression of SST receptors in the mammalian cochlea, emphasizing their possible roles in cochlear maturation. In the developing mouse cochlea, cells immunoreactive to somatostatin receptor 1 (SSTR1) and somatostatin receptor 2 (SSTR2) were located in the embryonic cochlear duct on Kolliker's organ as early as embryonic day (E) 14 (E14). At E17, the expression of both receptors was high and already located at the hair cells and supporting cells along the length of the cochlear duct, which have become arranged into the characteristic pattern for the organ of Corti (OC) at this stage. At birth, SSTR1- and SSTR2-containing cells were only localized in the OC. In general, immunoreactivity for both receptors increased in the mouse cochlea from postnatal day (P) 0 (P0) to P10; the majority of immunostained cells were inner hair cells, outer hair cells, and supporting cells. Finally, a peak in the mRNA and protein expression of both receptors is present near the time when they respond to physiological hearing (i.e., hearing of airborne sound) at P14. At P21, SSTR1 and SSTR2 levels decrease dramatically. A similar developmental pattern was observed for SSTR1 and SSTR2 mRNA, suggesting that the expression of the SSTR1 and SSTR2 genes is controlled at the transcriptional level throughout development. In addition, we observed reduced levels of phospho-Akt and total Akt in SSTR1 knockout and SSTR1/SSTR2 double-knockout mice compared with wild-type mice. We know from previous studies that Akt is involved in hair cell survival. Taken together, the dynamic nature of SSTR1 and SSTR2 expression at a time of major developmental changes in the cochlea suggests that SSTR1 and SSTR2 (and possibly other members of this family) are involved in the maturation of the mammalian cochlea.

Background Little is known about expression and function of the somatostatinergic system in the mammalian cochlea. We have previously shown that somatostatin administration may have a protective effect on gentamicin-induced hair cell loss. In this study, we have analyzed the cochlear expression of somatostatin receptor 1 (SST1) and somatostatin receptor 2 (SST2) at both the mRNA and the protein level in wild-type mice, as well as in SST1 and SST2 knock-out (KO) mice and in cultivated neurosensory cells. Results We demonstrate that the somatostatin receptors SST1 and SST2 are specifically expressed in outer and inner hair cells (HCs) of the organ of Corti (OC), as well as in defined supporting cells. The expression of SST1 and SST2 receptors in cultivated P5 mouse OC explants was similar to their expression in inner and outer hair cells. Somatostatin itself was not expressed in the mammalian cochlea, suggesting that somatostatin reaches its receptors either through the blood-labyrinthine barrier from the systemic circulation or via the endolymphatic duct from the endolymphatic sac. We used mice with a deletion of either SST1 or SST2 to learn more about the regulation of SST1 and SST2 receptor expression. We demonstrate that in SST1 KO mice, SST2 was expressed in outer HCs and Deiters' cells, but not in pillar cells or inner HCs, as compared with wild-type mice. In contrast, in SST2 KO mice, the expression pattern of the SST1 receptor was not altered relative to wild-type mice. Conclusions These findings reveal that somatostatin receptors demonstrate specific expression in HCs and supporting cells of the mouse cochlea, and that absence of SST1 alters the expression of SST2. This specific expression pattern suggests that somatostatin receptors may have important functional roles in the inner ear.

Sorafenib targets the Raf/mitogen-activated protein kinase, VEGF, and platelet-derived growth factor pathways and prolongs survival patients in advanced hepatocellular carcinoma (HCC). Everolimus inhibits the mammalian target of rapamycin, a kinase overactive in HCC. To investigate whether the antitumor effects of these agents are additive, we compared a combined and sequential treatment regimen of everolimus and sorafenib with monotherapy. After hepatic implantation of Morris Hepatoma (MH) cells, rats were randomly allocated to everolimus (5 mg/kg, 2×/week), sorafenib (7.5 mg/kg/d), combined everolimus and sorafenib, sequential sorafenib (2 weeks) then everolimus (3 weeks), or control groups. MRI quantified tumor volumes. Erk1/2, 4E-BP1, and their phosphorylated forms were quantified by immunoblotting. Angiogenesis was assessed in vitro by aortic ring and tube formation assays, and in vivo with Vegf-a mRNA and vascular casts. After 35 days, tumor volumes were reduced by 60%, 85%, and 55%, relative to controls, in everolimus, the combination, and sequential groups, respectively (P < 0.01). Survival was longest in the combination group (P < 0.001). Phosphorylation of 4E-BP1 and Erk1/2 decreased after everolimus and sorafenib, respectively. Angiogenesis decreased after all treatments (P < 0.05), although sorafenib increased Vegf-a mRNA in liver tumors. Vessel sprouting was abundant in control tumors, lower after sorafenib, and absent after the combination. Intussusceptive angiogenic transluminal pillars failed to coalesce after the combination. Combined treatment with everolimus and sorafenib exerts a stronger antitumoral effect on MH tumors than monotherapy. Everolimus retains antitumoral properties when administered sequentially after sorafenib. This supports the clinical use of everolimus in HCC, both in combination with sorafenib or after sorafenib. Mol Cancer Ther; 10(6); 1007–17. ©2011 AACR.

Cadherins are a superfamily of transmembrane glycoproteins, which mediate calcium-dependent intercellular adhesions. T-cadherin is an atypical member of the cadherin family in regard to its structure; it acts as a signalling receptor rather than an adhesion molecule. In this study we examine the role of T-cadherin in the mammalian cochlea.This study investigated the expression of T-cadherin in the inner ear under physiologic and pathologic conditions.Expression of T-cadherin in the rat cochlea was analyzed by reverse-transcriptase polymerase chain reaction (RT-PCR), real-time RT-PCR, Western blot, and immunohistochemistry.We detected T-cadherin mRNA expression in three different components in the cochlea of postnatal mouse, namely the organ of Corti (OC), the spiral ganglion (SG), and the stria vascularis (SV). The SG and SV showed a higher T-cadherin mRNA level than the OC. T-cadherin protein was detected by Western blotting in the OC, SG, and SV. Immunofluorescence microscopy of adult mouse cochlea revealed the presence of T-cadherin in the apical parts of the inner and outer hair cells as well as in the SV and SG. OCs treated with gentamicin for 3, 6, or 12 hours did not show any change in T-cadherin gene expression compared to control explants maintained in culture medium alone.T-cadherin is expressed within the cochlea. T-cadherin seems to have a wide variety of functions in the inner ear, ranging from mechanical functions to functions in response to hair cell damage and loss.

This paper analyzes the relationship between family functioning and chronic pain, and evaluates a model which integrates the role of family variables with the pain-coping process in patients with rheumatoid arthritis (RA) and fibromyalgia (FM). Family variables, assessed by subscales of the Family Environment Scale (FES), and different components of the pain-coping process varied significantly in their contribution to pain, psychological functioning, and disability in these two chronic pain disorders. High system maintenance control and low independence contributed significantly to pain in RA and FM, respectively, while low family cohesiveness contributed to psychological disturbances in FM subjects. Lack of promotion of activity and recreation in the family was associated with greater disability in both groups. In addition to family variables, helplessness proved to be a potent predictor of pain and psychological functioning in both groups, and disability in FM subjects. Pain-coping had its predominant influence on pain. As an exploratory test of the model, the data confirm the importance of examining this integrated framework in longitudinal, prospective studies, and its potential utility in clinical assessment and intervention.

The matrix metalloproteinases (MMPs) are a family of proteins involved in the remodelling and homeostasis of the extracellular matrix. These proteases have been well studied in the retina and the brain, marking their importance in neuronal cell survival and death [Chintala (2006) Exp Eye Res 82:5-12; Candelario-Jalil et al. (2009) Neuroscience 158:983-994]. The neuroepithelia of the eye and the inner ear share common characteristics. Therefore, we hypothesized that MMPs could play a similar role in the cochlea as described in the retina. We focused on the localization and function of MMP-2 and MMP-9 in the cochlea, by determining their expression and activity under normal conditions and after cochlear damage via aminoglycoside exposition. We examined their expression in 5-day-old Wistar rat cochleas by RT-PCR, real-time PCR, and Western blot. We used immunohistochemistry to investigate their location in the cochleas of adult C57BL/6 mice. We also determined whether or not the exposure of the organs of Corti to aminoglycosides would change MMP-2 and MMP-9 expression patterns. Western blotting identified MMP-2 and MMP-9 in neonatal spiral ganglion, stria vascularis, and to a lesser extent the organ of Corti. Neonatal mRNA expression of MMP-2 was approximately equivalent in all three tissues, while MMP-9 mRNA was highest in spiral ganglion. Immunohistochemistry showed MMP-2 primarily in adult spiral ganglion neurons and inner hair cells, while MMP-9 was found mainly in spiral ganglion neurons, inner hair cells and supporting cells. Organs of Corti treated with gentamicin for 24 h showed an upregulation of MMP-2 and MMP-9 proteins, but did not show a significant upregulation of mRNA expression 3, 6, 12, 24, and 36 h after gentamicin exposure. Inhibition of MMP activity in organs of Corti incubated with an MMP inhibitor in organotypic cultures resulted in hair cell death-suggesting that a basal level of MMP activity is required for hair cell survival.

Advanced glycation end products (AGE) have recently been implicated in aging changes within different tissues of the body. The role of AGEs and their receptors in the mammalian inner ear is largely unknown. In this study we analyzed for the expression of two AGE receptors, namely RAGE and Ddost (AGE-R1).Controlled animal study. Controlled animal study. This study confirmed the expression of the AGEs receptors RAGE and Ddost (AGE-R1) both on mRNA and protein level in the cochlea. Furthermore, we were able to localize these two receptors in the organ of Corti (OC).Expression of RAGE and Ddost (AGE-R1) receptors in the rat cochlea were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot. Specific localization of these two AGE receptors was also obtained within the mouse OC using immunohistochemistry.We detected RAGE and Ddost (AGE-R1) at the mRNA and protein level in the OC, spiral ganglion, and stria vascularis. Moreover, RAGE and Ddost (AGE-R1) could specifically be identified within the immature and mature OC.The AGE receptors RAGE and Ddost (AGE-R1) could be identified within the cochlea. Different expression patterns of these two receptors were observed in the immature and mature inner ear.