New insights in osteogenic differentiation revealed by mass spectrometric assessment of phosphorylated substrates in murine skin mesenchymal cells

Abstract Background Bone fractures and loss represent significant costs for the public health system and often affect the patients quality of life, therefore, understanding the molecular basis for bone regeneration is essential. Cytokines, such as IL-6, IL-10 and TNFα, secreted by inflammatory cells at the lesion site, at the very beginning of the repair process, act as chemotactic factors for mesenchymal stem cells, which proliferate and differentiate into osteoblasts through the autocrine and paracrine action of bone morphogenetic proteins (BMPs), mainly BMP-2. Although it is known that BMP-2 binds to ActRI/BMPR and activates the SMAD 1/5/8 downstream effectors, little is known about the intracellular mechanisms participating in osteoblastic differentiation. We assessed differences in the phosphorylation status of different cellular proteins upon BMP-2 osteogenic induction of isolated murine skin mesenchymal stem cells using Triplex Stable Isotope Dimethyl Labeling coupled with LC/MS. Results From 150 μg of starting material, 2,264 proteins were identified and quantified at five different time points, 235 of which are differentially phosphorylated. Kinase motif analysis showed that several substrates display phosphorylation sites for Casein Kinase, p38, CDK and JNK. Gene ontology analysis showed an increase in biological processes related with signaling and differentiation at early time points after BMP2 induction. Moreover, proteins involved in cytoskeleton rearrangement, Wnt and Ras pathways were found to be differentially phosphorylated during all timepoints studied. Conclusions Taken together, these data, allow new insights on the intracellular substrates which are phosphorylated early on during differentiation to BMP2-driven osteoblastic differentiation of skin-derived mesenchymal stem cells.

High Prevalence of Skin Disorders among HTLV-1 Infected Individuals Independent of Clinical Status

Background Human T-cell lymphotropic virus type 1 (HTLV-1) infection can increase the risk of developing skin disorders. This study evaluated the correlation between HTLV-1 proviral load and CD4+ and CD8+ T cells count among HTLV-1 infected individuals, with or without skin disorders (SD) associated with HTLV-1 infection [SD-HTLV-1: xerosis/ichthyosis, seborrheic dermatitis or infective dermatitis associated to HTLV-1 (IDH)]. Methods A total of 193 HTLV-1-infected subjects underwent an interview, dermatological examination, initial HTLV-1 proviral load assay, CD4+ and CD8+ T cells count, and lymphproliferation assay (LPA). Results A total of 147 patients had an abnormal skin condition; 116 (79%) of them also had SD-HTLV-1 and 21% had other dermatological diagnoses. The most prevalent SD-HTLV-1 was xerosis/acquired ichthyosis (48%), followed by seborrheic dermatitis (28%). Patients with SD-HTLV-1 were older (51 vs. 47 years), had a higher prevalence of myelopathy/tropical spastic paraparesis (HAM/TSP) (75%), and had an increased first HTLV-1 proviral load and basal LPA compared with patients without SD-HTLV-1. When excluding HAM/TSP patients, the first HTLV-1 proviral load of SD-HTLV-1 individuals remains higher than no SD-HTLV-1 patients. Conclusions There was a high prevalence of skin disorders (76%) among HTLV-1-infected individuals, regardless of clinical status, and 60% of these diseases are considered skin disease associated with HTLV-1 infection.

Tool wear evaluations in friction stir processing of commercial titanium Ti–6Al–4V

This research addresses the application of friction stir welding (FWS) of titanium alloy Ti–6Al–4V. Friction stir welding is a recent process, developed in the 1990s for aluminum joining; this joining process is being increasingly applied in many industries from basic materials, such as steel alloys, to high performance alloys, such as titanium. It is a process in great development and has its economic advantages when compared to conventional welding. For high performance alloys such as titanium, a major problem to overcome is the construction of tools that can withstand the extreme process environment. In the literature, the possibilities approached are only few tungsten alloys. Early experiments with tools made of cemented carbide (WC) showed optimistic results consistent with the literature. It was initially thought that WC tools may be an option to the FSW process since it is possible to improve the wear resistance of the tool. The metallographic analysis of the welds did not show primary defects of voids (tunneling) or similar internal defects due to processing, only defects related to tool wear which can cause loss of weld quality. The severe tool wear caused loss of surface quality and inclusions of fragments inside the joining, which should be corrected or mitigated by means of coating techniques on tool, or the replacement of cemented carbide with tungsten alloys, as found in the literature.

Friction and wear of performance of MoDTC and ester-containing lubrificants over steel surfaces under reciprocating conditions

Since the earliest developments of human history, friction has been a major issue. From the invention of the wheel and the use of the first lubricants to the studiesof coated and microtexturized surfaces, significant effort has been put on improvements that couldovercome the resistance to motion. Areview by Holmberg, Andersson and Erdemir[1] shows that, in an average passenger car, about one third of the total energy consumptionis due to friction losses. Of these, another one third is consumed in the engine system. The optimization of the lubricating oil formulation used ininternal combustion enginesis an important way to reduce friction, therefore improving energeticefficiencyand controllingemissions.Lubrication is also a way to assure the required protection to the system by maintaining wear rates in an adequate level, which helps to minimize maintenance costs.

Low temperatures reduce skin healing in the Jacaré do Pantanal (Caiman yacare, Daudin 1802)

Studies of skin wound healing in crocodilians are necessary given the frequent occurrence of cannibalism in intensive farming systems. Air temperature affects tissue recovery because crocodilians are ectothermic. Therefore, the kinetics of skin wound healing in Caiman yacare were examined at temperatures of 33°C and 23°C. Sixteen caiman were selected and divided into two groups of eight maintained at 23°C or 33°C. The studied individuals' scars were photographed after 1, 2, 3, 7, 15 and 30 days of the experimental conditions, and samples were collected for histological processing after 3, 7, 15 and 30 days. Macroscopically, the blood clot (heterophilic granuloma) noticeably remained in place covering the wound longer for the caiman kept at 23°C. Microscopically, the temperature of 23°C slowed epidermal migration and skin repair. Comparatively, new blood vessels, labeled using von Willebrand factor (vWF) antibody staining, were more frequently found in the scars of the 33°C group. The collagen fibers in the dermis were denser in the 33°C treatment. Considering the delayed healing at 23°C, producers are recommended to keep wounded animals at 33°C, especially when tanks are cold, to enable rapid wound closure and better repair of collagen fibers because such lesions tend to compromise the use of their skin as leather.

Adipose tissue-derived mesenchymal stem cells increase skin allograft survival and inhibit Th-17 immune response

Adipose tissue-derived mesenchymal stem cells (ADSC) exhibit immunosuppressive capabilities both in vitro and in vivo. Their use for therapy in the transplant field is attractive as they could render the use of immunosuppressive drugs unnecessary. The aim of this study was to investigate the effect of ADSC therapy on prolonging skin allograft survival. Animals that were treated with a single injection of donor allogeneic ADSC one day after transplantation showed an increase in donor skin graft survival by approximately one week. This improvement was associated with preserved histological morphology, an expansion of CD4(+) regulatory T cells (Treg) in draining lymph nodes, as well as heightened IL-10 expression and down-regulated IL-17 expression. In vitro, ADSC inhibit naïve CD4(+) T cell proliferation and constrain Th-1 and Th-17 polarization. In summary, infusion of ADSC one day post-transplantation dramatically increases skin allograft survival by inhibiting the Th-17 pathogenic immune response and enhancing the protective Treg immune response. Finally, these data suggest that ADSC therapy will open new opportunities for promoting drug-free allograft survival in clinical transplantation.

Computational chemistry studies of UV induced processes in human skin

This thesis presents and uses the techniques of computational chemistry to explore two different processes induced in human skin by ultraviolet light. The first is the transformation of urocanic acid into a immunosuppressing agent, and the other is the enzymatic action of the 8-oxoguanine glycosylase enzyme. The photochemistry of urocanic acid is investigated by time-dependent density functional theory. Vertical absorption spectra of the molecule in different forms and environments is assigned and candidate states for the photochemistry at different wavelengths are identified. Molecular dynamics simulations of urocanic acid in gas phase and aqueous solution reveals considerable flexibility under experimental conditions, particularly for for the cis isomer where competition between intra- and inter-molecular interactions increases flexibility. A model to explain the observed gas phase photochemistry of urocanic acid is developed and it is shown that a reinterpretation in terms of a mixture between isomers significantly enhances the agreement between theory and experiment , and resolves several peculiarities in the spectrum. A model for the photochemistry in the aqueous phase of urocanic acid is then developed, in which two excited states governs the efficiency of photoisomerization. The point of entrance into a conical intersection seam is shown to explain the wavelength dependence of photoisomerization quantum yield. Finally some mechanistic aspects of the DNA repair enzyme 8-oxoguanine glycosylase is investigated with density functional theory. It is found that the critical amino acid of the active site can provide catalytic power in several different manners, and that a recent proposal involving a SN1 type of mechanism seems the most efficient one.

Phenolic Constituents, Antioxidant and Preliminary Antimycoplasmic Activities of Leaf Skin and Flowers of Aloe vera (L.) Burm. f. (syn. A. barbadensis Mill.) from the Canary Islands (Spain)

[EN]The methanol extracts of leaf skins and flowers of Aloe vera from the Canary Islands were analyzed for their phenolic profiles and screened for their antioxidant and antimycoplasmic activities. The use of reversed phase high performance liquid chromatography (RP-HPLC) allowed the identification of 18 phenolic constituents. Leaf skin extracts were characterized by the abundance of catechin, sinapic acid and quercitrin. Gentisic acid, epicatechin and quercitrin were the most prominent phenolic compounds of the flowers. The in vitro antioxidant activities determined by using the 1,1-diphenyl-2- picrylhydrazyl (DPPH) and ferric antioxidant reducing power (FRAP) assays revealed that both extracts exhibited antioxidant activity, being the leaf skin extract the most active fraction. The leaf skin extract was also found to be active against the microbial strains tested. Therefore, A. vera extracts from leaf skin and flowers can be considered as good natural antioxidant sources.

Experimental Study on Friction between Saline Ice and Steel

Understanding the interaction of sea ice with offshore structures is of primary importance for the development of technology in cold climate regions. The rheological properties of sea ice (strength, creep, viscosity) as well as the roughness of the contact surface are the main factors influencing the type of interaction with a structure. A device was developed and designed and small scale laboratory experiments were carried out to study sea ice frictional interaction with steel material by means of a uniaxial compression rig. Sea-ice was artificially grown between a stainless steel piston (of circular cross section) and a hollow cylinder of the same material, coaxial to the former and of the same surface roughness. Three different values for the roughness were tested: 1.2, 10 and 30 μm Ry (maximum asperities height), chosen as representative values for typical surface conditions, from smooth to normally corroded steel. Creep tests (0.2, 0.3, 0.4 and 0.6 kN) were conducted at T = -10 ºC. By pushing the piston head towards the cylinder base, three different types of relative movement were observed: 1) the piston slid through the ice, 2) the piston slid through the ice and the ice slid on the surface of the outer cylinder, 3) the ice slid only on the cylinder surface. A cyclic stick-slip motion of the piston was detected with a representative frequency of 0.1 Hz. The ratio of the mean rate of axial displacement to the frequency of the stick-slip oscillations was found to be comparable to the roughness length (Sm). The roughness is the most influential parameter affecting the amplitude of the oscillations, while the load has a relevant influence on the their frequency. Guidelines for further investigations were recommended. Marco Nanetti - seloselo@virgilio.it

A skin surface characterization system based on capacitive image analysis

During the last few years, several methods have been proposed in order to study and to evaluate characteristic properties of the human skin by using non-invasive approaches. Mostly, these methods cover aspects related to either dermatology, to analyze skin physiology and to evaluate the effectiveness of medical treatments in skin diseases, or dermocosmetics and cosmetic science to evaluate, for example, the effectiveness of anti-aging treatments. To these purposes a routine approach must be followed. Although very accurate and high resolution measurements can be achieved by using conventional methods, such as optical or mechanical profilometry for example, their use is quite limited primarily to the high cost of the instrumentation required, which in turn is usually cumbersome, highlighting some of the limitations for a routine based analysis. This thesis aims to investigate the feasibility of a noninvasive skin characterization system based on the analysis of capacitive images of the skin surface. The system relies on a CMOS portable capacitive device which gives 50 micron/pixel resolution capacitance map of the skin micro-relief. In order to extract characteristic features of the skin topography, image analysis techniques, such as watershed segmentation and wavelet analysis, have been used to detect the main structures of interest: wrinkles and plateau of the typical micro-relief pattern. In order to validate the method, the features extracted from a dataset of skin capacitive images acquired during dermatological examinations of a healthy group of volunteers have been compared with the age of the subjects involved, showing good correlation with the skin ageing effect. Detailed analysis of the output of the capacitive sensor compared with optical profilometry of silicone replica of the same skin area has revealed potentiality and some limitations of this technology. Also, applications to follow-up studies, as needed to objectively evaluate the effectiveness of treatments in a routine manner, are discussed.

Tactile Perception - Perception of tactile distance on a single skin surface changes with stimulus orientation: a neuronal network modelling study

L’interazione che abbiamo con l’ambiente che ci circonda dipende sia da diverse tipologie di stimoli esterni che percepiamo (tattili, visivi, acustici, ecc.) sia dalla loro elaborazione per opera del nostro sistema nervoso. A volte però, l’integrazione e l’elaborazione di tali input possono causare effetti d’illusione. Ciò si presenta, ad esempio, nella percezione tattile. Infatti, la percezione di distanze tattili varia al variare della regione corporea considerata. Il concetto che distanze sulla cute siano frequentemente erroneamente percepite, è stato scoperto circa un secolo fa da Weber. In particolare, una determinata distanza fisica, è percepita maggiore su parti del corpo che presentano una più alta densità di meccanocettori rispetto a distanze applicate su parti del corpo con inferiore densità. Oltre a questa illusione, un importante fenomeno osservato in vivo è rappresentato dal fatto che la percezione della distanza tattile dipende dall’orientazione degli stimoli applicati sulla cute. In sostanza, la distanza percepita su una regione cutanea varia al variare dell’orientazione degli stimoli applicati. Recentemente, Longo e Haggard (Longo & Haggard, J.Exp.Psychol. Hum Percept Perform 37: 720-726, 2011), allo scopo di investigare come sia rappresentato il nostro corpo all’interno del nostro cervello, hanno messo a confronto distanze tattili a diverse orientazioni sulla mano deducendo che la distanza fra due stimoli puntuali è percepita maggiore se applicata trasversalmente sulla mano anziché longitudinalmente. Tale illusione è nota con il nome di Illusione Tattile Orientazione-Dipendente e diversi risultati riportati in letteratura dimostrano che tale illusione dipende dalla distanza che intercorre fra i due stimoli puntuali sulla cute. Infatti, Green riporta in un suo articolo (Green, Percpept Pshycophys 31, 315-323, 1982) il fatto che maggiore sia la distanza applicata e maggiore risulterà l’effetto illusivo che si presenta. L’illusione di Weber e l’illusione tattile orientazione-dipendente sono spiegate in letteratura considerando differenze riguardanti la densità di recettori, gli effetti di magnificazione corticale a livello della corteccia primaria somatosensoriale (regioni della corteccia somatosensoriale, di dimensioni differenti, sono adibite a diverse regioni corporee) e differenze nella dimensione e forma dei campi recettivi. Tuttavia tali effetti di illusione risultano molto meno rilevanti rispetto a quelli che ci si aspetta semplicemente considerando i meccanismi fisiologici, elencati in precedenza, che li causano. Ciò suggerisce che l’informazione tattile elaborata a livello della corteccia primaria somatosensoriale, riceva successivi step di elaborazione in aree corticali di più alto livello. Esse agiscono allo scopo di ridurre il divario fra distanza percepita trasversalmente e distanza percepita longitudinalmente, rendendole più simili tra loro. Tale processo assume il nome di “Rescaling Process”. I meccanismi neurali che operano nel cervello allo scopo di garantire Rescaling Process restano ancora largamente sconosciuti. Perciò, lo scopo del mio progetto di tesi è stato quello di realizzare un modello di rete neurale che simulasse gli aspetti riguardanti la percezione tattile, l’illusione orientazione-dipendente e il processo di rescaling avanzando possibili ipotesi circa i meccanismi neurali che concorrono alla loro realizzazione. Il modello computazionale si compone di due diversi layers neurali che processano l’informazione tattile. Uno di questi rappresenta un’area corticale di più basso livello (chiamata Area1) nella quale una prima e distorta rappresentazione tattile è realizzata. Per questo, tale layer potrebbe rappresentare un’area della corteccia primaria somatosensoriale, dove la rappresentazione della distanza tattile è significativamente distorta a causa dell’anisotropia dei campi recettivi e della magnificazione corticale. Il secondo layer (chiamato Area2) rappresenta un’area di più alto livello che riceve le informazioni tattili dal primo e ne riduce la loro distorsione mediante Rescaling Process. Questo layer potrebbe rappresentare aree corticali superiori (ad esempio la corteccia parietale o quella temporale) adibite anch’esse alla percezione di distanze tattili ed implicate nel Rescaling Process. Nel modello, i neuroni in Area1 ricevono informazioni dagli stimoli esterni (applicati sulla cute) inviando quindi informazioni ai neuroni in Area2 mediante sinapsi Feed-forward eccitatorie. Di fatto, neuroni appartenenti ad uno stesso layer comunicano fra loro attraverso sinapsi laterali aventi una forma a cappello Messicano. E’ importante affermare che la rete neurale implementata è principalmente un modello concettuale che non si preme di fornire un’accurata riproduzione delle strutture fisiologiche ed anatomiche. Per questo occorre considerare un livello astratto di implementazione senza specificare un’esatta corrispondenza tra layers nel modello e regioni anatomiche presenti nel cervello. Tuttavia, i meccanismi inclusi nel modello sono biologicamente plausibili. Dunque la rete neurale può essere utile per una migliore comprensione dei molteplici meccanismi agenti nel nostro cervello, allo scopo di elaborare diversi input tattili. Infatti, il modello è in grado di riprodurre diversi risultati riportati negli articoli di Green e Longo & Haggard.