Interplay between FGFR2b-induced autophagy and phagocytosis: role of PLCγ-mediated signalling

Signalling of the epithelial splicing variant of the fibroblast growth factor receptor 2 (FGFR2b) induces both autophagy and phagocytosis in human keratinocytes. Here, we investigated, in the cell model of HaCaT keratinocytes, whether the two processes might be related and the possible involvement of PLCγ signalling. Using fluorescence and electron microscopy, we demonstrated that the FGFR2b-induced phagocytosis and autophagy involve converging autophagosomal and phagosomal compartments. Moreover, the forced expression of FGFR2b signalling mutants and the use of specific inhibitors of FGFR2b substrates showed that the receptor-triggered autophagy requires PLCγ signalling, which in turn activates JNK1 via PKCδ. Finally, we found that in primary human keratinocytes derived from light or dark pigmented skin and expressing different levels of FGFR2b, the rate of phagocytosis and autophagy and the convergence of the two intracellular pathways are dependent on the level of receptor expression, suggesting that FGFR2b signalling would control in vivo the number of melanosomes in keratinocytes, determining skin pigmentation

Role of FGFR2b expression and signaling in keratinocyte differentiation. Sequential involvement of PKCδ and PKCα

The tumor suppressor epithelial isoform of the fibroblast growth factor receptor 2 (FGFR2b) induces human keratinocyte early differentiation. Moreover, protein kinases C (PKCs) are known to regulate the differentiation program in several cellular contexts, including keratinocytes. Therefore, in this paper we propose to clarify if FGFR2b could play a role also in the late steps of keratinocyte differentiation and to assess if this receptor-induced process would sequentially involve PKCδ and PKCα isoforms. Immunofluorescence, biochemical, and molecular approaches, performed on 2D cultures or 3D organotypic rafts of human keratinocytes overexpressing FGFR2b by stable transduction, showed that receptor signaling induced the precocious onset and an accelerated progression of keratinocyte differentiation, indicating that FGFR2b is a crucial regulator of the entire program of keratinocyte differentiation. In addition, the use of specific inhibitors and gene silencing approaches through specific siRNA demonstrated that PKCδ controls the onset of FGFR2b-triggered differentiation, while PKCα plays a role restricted to the terminal stages of the process. Molecular analysis revealed that the two PKC isoforms sequentially act via induction of KLF4 and DLX3, two transcription factors linked by negative loops to p63, suggesting that p63 would represent the hub molecule at the crossroad of an intricate signaling network downstream FGFR2b, involving multiple PKC-induced transcription factors

The aberrant expression of the mesenchymal variant of FGFR2 in the epithelial context inhibits autophagy

Signaling of the epithelial splice variant of fibroblast growth factor receptor 2 (FGFR2b) triggers both differentiation and autophagy, while the aberrant expression of the mesenchymal FGFR2c isoform in epithelial cells induces impaired differentiation, epithelial mesenchymal transition (EMT) and tumorigenic features. Here we analyzed in the human keratinocyte cell line, as well as in primary cultured cells, the possible impact of FGFR2c forced expression on the autophagic process. Biochemical and quantitative immunofluorescence analysis, coupled to the use of autophagic flux sensors, specific substrate inhibitors or silencing approaches, showed that ectopic expression and the activation of FGFR2c inhibit the autophagosome formation and that AKT/MTOR is the downstream signaling mainly involved. Interestingly, the selective inhibition of AKT or MTOR substrates caused a reversion of the effects of FGFR2c on autophagy, which could also arise from the imbalance of the interplay between AKT/MTOR pathway and JNK1 signaling in favor of JNK1 activation, BCL-2 phosphorylation and possibly phagophore nucleation. Finally, silencing experiments of depletion of ESRP1, responsible for FGFR2 splicing and consequent FGFR2b expression, indicated that the switching from FGFR2b to FGFR2c isoform could represent the key event underlying the inhibition of the autophagic process in the epithelial context. Our results provide the first evidence of a negative impact of the out-of-context expression of FGFR2c on autophagy, suggesting a possible role of this receptor in the modulation of the recently proposed negative loop between autophagy and EMT during carcinogenesis

Super-Hydrophobic Multi-Walled Carbon Nanotube Coatings for Stainless Steel

We have taken advantage of the native surface roughness and the iron content of AISI 316 stainless steel to direct grow multi-walled carbon nanotube (MWCNT) random networks by chemical vapor deposition (CVD) at low-temperature ($< 1000^{\circ}$C), without the addition of any external catalysts or time-consuming pre-treatments. In this way, super-hydrophobic MWCNT films on stainless steel sheets were obtained, exhibiting high contact angle values ($154^{\circ}$) and high adhesion force (high contact angle hysteresis). Furthermore, the investigation of MWCNT films at scanning electron microscopy (SEM) reveals a two-fold hierarchical morphology of the MWCNT random networks made of hydrophilic carbonaceous nanostructures on the tip of hydrophobic MWCNTs. Owing to the Salvinia effect, the hydrophobic and hydrophilic composite surface of the MWCNT films supplies a stationary super-hydrophobic coating for conductive stainless steel. This biomimetical inspired surface not only may prevent corrosion and fouling but also could provide low-friction and drag-reduction.Comment: 6 pages, 3 figure

Exploiting the Hierarchical Morphology of Single-Walled and Multi-Walled Carbon Nanotube Films for Highly Hydrophobic Coatings

Self-assembled hierarchical solid surfaces are very interesting for wetting phenomena, as observed in a variety of natural and artificial surfaces. Here, we report single-walled (SWCNT) and multi-walled carbon nanotube (MWCNT) thin films realized by a simple, rapid, reproducible, and inexpensive filtration process from an aqueous dispersion, that was deposited at room temperature by a dry-transfer printing method on glass. Furthermore, the investigation of carbon nanotube films through scanning electron microscopy (SEM) reveals the multi-scale hierarchical morphology of the self-assembled carbon nanotube random networks. Moreover, contact angle measurements show that hierarchical SWCNT/MWCNT composite surfaces exhibit a higher hydrophobicity (contact angles of up to 137{\deg}) than bare SWCNT (110{\deg}) and MWCNT (97{\deg}) coatings, thereby confirming the enhancement produced by the surface hierarchical morphology.Comment: 7 pages, 5 figures, This article is part of the Thematic Series "Self-assembly of nanostructures and nanomaterials

Eco-sustainable systems based on poly(lactic acid), diatomite and coffee grounds extract for food packaging

In the food packaging sector many efforts have been (and are) devoted to the development of new materials in order to reply to an urgent market demand for green and eco-sustainable products. Particularly a lot of attention is currently devoted both to the use of compostable and biobased polymers as innovative and promising alternative to the currently used petrochemical derived polymers, and to the re-use of waste materials coming from agriculture and food industry. In this work, multifunctional eco-sustainable systems, based on poly(lactic acid) (PLA) as biopolymeric matrix, diatomaceous earth as reinforcing filler and spent coffee grounds extract as oxygen scavenger, were produced for the first time, in order to provide a simultaneous improvement of mechanical and gas barrier properties. The influence of the diatomite and the spent coffee grounds extract on the microstructural, mechanical and oxygen barrier properties of the produced films was deeply investigated by means of X-Ray diffraction (XRD), infrared spectroscopy (FT-IR, ATR), scanning electron microscopy (SEM), uniaxial tensile tests, O2 permeabilimetry measurements. An improvement of both mechanical and oxygen barrier properties was recorded for systems characterised by the co-presence of diatomite and coffee grounds extract, suggesting a possible synergic effect of the two additives

New vegetable-waste biomaterials by Lupin albus L. as cellular scaffolds for applications in biomedicine and food

The reprocessing of vegetal-waste represents a new research field in order to design novel biomaterials for potential biomedical applications and in food industry. Here we obtained a biomaterial from Lupinus albus L. hull (LH) that was characterized micro-structurally by scanning electron microscopy and for its antimicrobial and scaffolding properties. A good adhesion and proliferation of human mesenchymal stem cells (hMSCs) seeded on LH scaffold were observed. Thanks to its high content of cellulose and beneficial phytochemical substances, LH and its derivatives can represent an available source for fabrication of biocompatible and bioactive scaffolds. Therefore, a reprocessing protocol of LH was optimized for producing a new LH bioplastic named BPLH. This new biomaterial was characterized by chemico-physical analyses. The water uptake, degradability and antimicrobial properties of BPLH were evaluated, as well as the mechanical properties. A good adhesion and proliferation of both fibroblasts and hMSCs on BPLH were observed over 2 weeks, and immunofluorescence analysis of hMSCs after 3 weeks indicates an initial commitment toward muscle differentiation. Our work represents a new approach toward the recovery and valorization of the vegetal waste showing the remarkable properties of LH and BPLH as cellular waste-based scaffold with potential applications in cell-based food field as well as in medicine for topical patches in wound healing and bedsores treatment

Biomechanics of anterior cruciate ligament reconstruction using twisted doubled hamstring tendons

We studied the biomechanical properties of a twisted doubled semitendinosus and gracilis graft. We applied an un-axial load in order to reproduce the kinematics of a reconstructed anterior cruciate ligament (ACL). A modified cryo-jaw clamp system was used to minimize soft tissue slippage. The lower grip, after fixation of the free ends of the tendons, was rotated 45degrees, translated 1 cm, and bent 40degrees, simulating a knee sprain. The graft was tested to failure using a servohydraulic machine. The specimen from one knee of seven unembalmed cadavers was assigned to the untwisted (parallel) bundles group, while its pair was assigned to the twisted group. For the parallel bundles group, the mean maximum load was 1709.3+/-581.9 N, for the twisted group 2428.3+/-475.4 N (P<0.05). The mean stiffness was respectively 213.6+/-72.4 N/mm and 310.3+/-97.3 N/mm (P=0.08). Although caution should be used in extrapolating the results to clinical estimates of the strength of hamstring grafts, the results of the present study could justify the use of twisted semitendinosus and gracilis bundles in ACL reconstruction

A modified Cryo-Jaw for in vitro biomechanical testing of tendons

The purpose of this study was to develop a new device, which represents a modification of the Cryo-Jaw described by Riemersa and Schamhardt and modified by Hamner et al., for in vitro biomechanical testing of tendons which allows the lower clamp to move in every direction and thus simulate a pathological dislocation of the knee. Tendons are fixed to the device by freezing the clamped part with dry ice. After fixation of their free ends. the lower clamp was rotated 45, translated 1 em, and angled 40 to simulate a knee sprain, Various configurations of bundles were tested: parallel, twisted, and braided. Tests were performed on 10 paired bovine bifurcated digital extensor tendons and 6 paired human hamstring tendons. Grafts were then tested to failure subjected to impulsive load, using a servohydraulic machine. The highest ultimate load recorded for parallel bundles was 4662 +/- 565.71 N for bovine bifurcated digital extensor tendons, and 3057 +/- 475.44 N for human hamstring tendons. In any case, the tendons ruptured midway, well clear of the frozen part; in no case was slippage of the tendons observed. Thus the device proposed allows one to test what happens to the graft of an ACL reconstructed knee during physiological and pathological movements because it can be easily displaced in every direction

Parameters of flow in porous alluvial aquifers evaluated by tracers

Dye tracer tests, DNA biotracer, alluvial aquifer Marche, multiwell tests