Kyoto International Consensus Report on Anatomy, Pathophysiology and Clinical Significance of the Gastroesophageal Junction

Aim: to present the main statements of Kyoto International Consensus report on anatomy, pathophysiology, and clinical significance of the gastroesophageal junction.Key points. The experts reviewed and adopted 28 statements concerning (1) the definition of the gastroesophageal junction (GEJ); (2) the definition of the GEJ zone, covering the area located 1 cm proximal and 1 cm distal in relation to gastroesophageal junction; (3) the assessment of chemical and bacterial (Helicobacter pylori) factors leading to the development of inflammation, metaplasia and neoplasia of the mucosa of the GEJ; and (4) a new definition of Barrett’s esophagus.Conclusion. The new definitions of GEJ, GEJ zone and Barrett’s esophagus adopted by the International Consensus will be used in subsequent studies, which will contribute to improving the results of treatment of diseases of this area

Antimicrobial functionalized genetically engineered spider silk

Genetically engineered fusion proteins offer potential as multifunctional biomaterials for medical use. Fusion or chimeric proteins can be formed using recombinant DNA technology by combining nucleotide sequences encoding different peptides or proteins that are otherwise not found together in nature. In the present study, three new fusion proteins were designed, cloned and expressed and assessed for function, by combining the consensus sequence of dragline spider silk with three different antimicrobial peptides. The human antimicrobial peptides human neutrophil defensin 2 (HNP-2), human neutrophil defensins 4 (HNP-4) and hepcidin were fused to spider silk through bioengineering. The spider silk domain maintained its self-assembly features, a key aspect of these new polymeric protein biomaterials, allowing the formation of b-sheets to lock in structures via physical interactions without the need for chemical crosslinking. These new functional silk proteins were assessed for antimicrobial activity against Gram e Escherichia coli and Gram þ Staphylococcus aureus and microbicidal activity was demonstrated. Dynamic light scattering was used to assess protein aggregation to clarify the antimicrobial patterns observed. Attenuated-total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and circular dichroism (CD) were used to assess the secondary structure of the new recombinant proteins. In vitro cell studies with a human osteosarcoma cell line (SaOs-2) demonstrated the compatibility of these new proteins with mammalian cells.Fundação para a Ciência e a Tecnologia (FCT) - Bolsa de doutoramento (SFRH/BD/28603/2006); Chimera project (PTDC/EBB-EBI/109093/2008); NIH and Tissue Engineering Resource Center EB003210, P41 EB002520, DE017207

Development of Self-Assembling Mixed Protein Micelles with Temperature-Modulated Avidities

Elastin-like polypeptides (ELPs) are polypentapeptides that undergo hydrophobic collapse and aggregation above a specific transition temperature, Tt. ELP diblocks sharing a common “core” block (I60) but varying “outer” blocks (A80, P40) were designed, where Tt,I \u3c Tt,A \u3c Tt,P. The formation of ~55 nm diameter mixed micelles from these ELP diblocks was verified using dynamic light scattering (DLS), multiangle light scattering (MALS) and fluorescence resonance energy transfer (FRET). To confer affinity to the blood circulating protein fibrinogen, a fibrinogen-binding tetrapeptide sequence (GPRP) was fused to A80-I60, while P40-I60 was fused to a non-binding control (GPSP). The self-assembling, peptide-displaying, mixed micelles exhibit temperature-modulated avidities for immobilized and soluble fibrinogen at 32 °C and 42 °C. In this initial proof-of-concept design, the engineered mixed micelles were shown to disengage fibrinogen at elevated temperatures. The modular nature of this system can be used for developing in vivo depot systems that will only be triggered to release in situ upon specific stimuli

Optimized silk fibroin piezoresistive nanocomposites for pressure sensing applications based on natural polymers

Environmental issues promote the development of sensors based on natural polymers which are becoming an area of increasing interest. Piezoresistive sensors based on silk fibroin with carbon nanotubes (CNTs) as fillers were produced by solvent-casting in order to tune their electrical conductivity and electromechanical responses. It is shown that the carbonaceous fillers are well dispersed in the polymer matrix and the thermal and mechanical properties are independent of the CNT content. On the other hand, the inclusion of CNTs reduces the beta-sheet content of silk fibroin and the electrical properties of the composite strongly depend on the filler content, the percolation threshold being around 1 wt% CNTs. The piezoresistive response demonstrates good reproducibility during cyclic loading without hysteresis with a piezoresistive sensitivity of similar to 4 MPa-1, regardless of the CNT content. Overall, the results confirm that polymer composites based on natural polymers exhibit excellent piezoresistive responses, also demonstrated by the implementation and testing of a pressure sensor with the corresponding readout electronics. Thus, it is shown that natural polymers such as silk fibroin will allow the development of a new generation of multifunctional force and deformation sensors.The authors acknowledge the FCT (Fundacao para a Ciencia e Tecnologia) for financial support under the framework of Strategic Funding grants UID/FIS/04650/2013, UID/EEA/04436/2013 and UID/QUI/0686/2016 and project no. PTDC/FIS-MAC/28157/2017. The authors also acknowledge the FCT for financial support under grants SFRH/BD/110622/2015 (S.G.), SFRH/BPD/112547/2015 (C.M.C.) and SFRH/BPD/110914/2015 (P.C.). Financial support from the Spanish Ministry of Economy and Competitiveness (MINECO) through project MAT2016-76039C4-3-R (AEI/FEDER, UE) (including FEDER financial support) and from the Basque Government Industry and Education Departments under the ELKARTEK, HAZITEK and PIBA (PIBA2018-06) programs is also acknowledged

Biological responses to spider silk - antibiotic fusion protein

The development of a new generation of multifunctional biomaterials is a continual goal for the field of materials science. The in vivo functional behaviour of a new fusion protein that combines the mechanical properties of spider silk with the antimicrobial properties of hepcidin was addressed in this study. This new chimeric protein, termed 6mer + hepcidin, fuses spider dragline consensus sequences (6mer) and the antimicrobial peptide hepcidin, as we have recently described, with retention of bactericidal activity and low cytotoxicity. In the present study, mouse subcutaneous implants were studied to access the in vivo biological response to 6mer + hepcidin, which were compared with controls of silk alone (6mer), polylactic–glycolic acid (PLGA) films and empty defects. Along with visual observations, flow cytometry and histology analyses were used to determine the number and type of inflammatory cells at the implantation site. The results show a mild to low inflammatory reaction to the implanted materials and no apparent differences between the 6mer + hepcidin films and the other experimental controls, demonstrating that the new fusion protein has good in vivo biocompatibility, while maintaining antibiotic function.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/28603/2006, Chimera project (No. PTDC/EBB-EBI/109093/2008), Proteo-Light (No. PTDC/FIS/68517/2006), NIH (Grant No. P41 EB002520) Tissue Engineering Resource Center; and the NIH (Grant Nos EB003210 and DE017207).Tissue Engineering Resource Center - Bolsa No. P41 EB002520, bolsa Nos EB003210 and DE017207European - Projeto EXPERTISSUES (No. NMP3- CT-2004-500283

AFM study of morphology and mechanical properties of a chimeric 2 spider silk and bone sialoprotein protein for bone regeneration

Atomic force microscopy (AFM) was used to assess a new chimeric protein consisting of a fusion protein of the consensus repeat for Nephila clavipes spider dragline protein and bone sialoprotein (6merþBSP). The elastic modulus of this protein in film form was assessed through force curves, and film surface roughness was also determined. The results showed a significant difference among the elastic modulus of the chimeric silk protein, 6merþBSP, and control films consisting of only the silk component (6mer). The behavior of the 6merþBSP and 6mer proteins in aqueous solution in the presence of calcium (Ca) ions was also assessed to determine interactions between the inorganic and organic components related to bone interactions, anchoring, and biomaterial network formation. The results demonstrated the formation of protein networks in the presence of Ca2þ ions, characteristics that may be important in the context of controlling materials assembly and properties related to bone formation with this new chimeric silk-BSP protein.Silvia Games thanks the Foundation for Science and Technology (FCT) for supporting her Ph.D. grant, SFRH/BD/28603/2006. This work was carried out under the scope of the European NoE EXPERTISSUES (NMP3-CT-2004-500283), the Chimera project (PTDC/EBB-EBI/109093/2008) funded by the FCT agency, the NIH (P41 EB002520) Tissue Engineering Resource Center, and the NIH (EB003210 and DE017207)

Recombinant human osteopontin expressed in Nicotiana benthamiana stimulates osteogenesis related genes in human periodontal ligament cells.

Tissue engineering aims to utilise biologic mediators to facilitate tissue regeneration. Several recombinant proteins have potential to mediate induction of bone production, however, the high production cost of mammalian cell expression impedes patient access to such treatments. The aim of this study is to produce recombinant human osteopontin (hOPN) in plants for inducing dental bone regeneration. The expression host was Nicotiana benthamiana using a geminiviral vector for transient expression. OPN expression was confirmed by Western blot and ELISA, and OPN was purified using Ni affinity chromatography. Structural analysis indicated that plant-produced hOPN had a structure similar to commercial HEK cell-produced hOPN. Biological function of the plant-produced hOPN was also examined. Human periodontal ligament stem cells were seeded on an OPN-coated surface. The results indicated that cells could grow normally on plant-produced hOPN as compared to commercial HEK cell-produced hOPN determined by MTT assay. Interestingly, increased expression of osteogenic differentiation-related genes, including OSX, DMP1, and Wnt3a, was observed by realtime PCR. These results show the potential of plant-produced OPN to induce osteogenic differentiation of stem cells from periodontal ligament in vitro, and suggest a therapeutic strategy for bone regeneration in the future

Keratin-based particles for protection and restoration of hair properties

Objective Human hair is an element with unquestionable relevance in society both for women and men. Therefore, it is of great importance to develop new cosmetic products for hair care capable to restore and improve hair's characteristics. Here we explore the potential of keratinbased particles in the protection and recovery of hair mechanical properties and thermal stability. Methods Keratinbased particles were obtained by high pressure homogenization (HPH) using keratin and silk fibroin. The particles were characterized regarding size, superficial charge and polydispersity index. Their safety to cells was assessed using human skin keratinocytes. Virgin and overbleached Asian hair were treated with eight keratinbased formulations. The effect of particles on hair's mechanical properties was evaluated in terms of stiffness and tensile strength. The impact of treatments in hair thermal performance was studied using differential scanning calorimetry (DSC). Results Keratinbased particles were capable to recover and/or improve the mechanical properties of virgin and overbleached hair. Virgin hair treated with K80SF20P particles presented an improvement of the mechanical properties around 40%. An increase in keratin helix denaturation enthalpy and in surface smoothness for both types of hair was also verified after treatment. These particles demonstrated stability over time and proved to be safe when tested in human keratinocytes. Conclusion The keratinbased particles here presented have the potential to be incorporated in the development of new and effective hair care cosmetic formulations.This study was supported by FCT under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE2020 (POCI01-0145-FEDER-006684) and BioTecNorte operation (NORTE-010145-FEDER-000004) and Nanotechnology Based Functional Solutions (NORTE-01-0145- FEDER-000019) funded by the European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte. We also acknowledge the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER007569) funded by national funds through Fundação para a Ciência e a Tecnologia (FCT) and by the ERDF through the COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI). Artur Ribeiro and Ana Tinoco thank FCT for funding the scholarships with the references SFRH/BPD/98388/2013 and SFRH/BD/ 114035/2015, respectively.info:eu-repo/semantics/publishedVersio

A potential role for endogenous proteins as sacrificial sunscreens and antioxidants in human tissues

AbstractExcessive ultraviolet radiation (UVR) exposure of the skin is associated with adverse clinical outcomes. Although both exogenous sunscreens and endogenous tissue components (including melanins and tryptophan-derived compounds) reduce UVR penetration, the role of endogenous proteins in absorbing environmental UV wavelengths is poorly defined. Having previously demonstrated that proteins which are rich in UVR-absorbing amino acid residues are readily degraded by broadband UVB-radiation (containing UVA, UVB and UVC wavelengths) here we hypothesised that UV chromophore (Cys, Trp and Tyr) content can predict the susceptibility of structural proteins in skin and the eye to damage by physiologically relevant doses (up to 15.4J/cm2) of solar UVR (95% UVA, 5% UVB). We show that: i) purified suspensions of UV-chromophore-rich fibronectin dimers, fibrillin microfibrils and β- and γ-lens crystallins undergo solar simulated radiation (SSR)-induced aggregation and/or decomposition and ii) exposure to identical doses of SSR has minimal effect on the size or ultrastructure of UV chromophore-poor tropoelastin, collagen I, collagen VI microfibrils and α-crystallin. If UV chromophore content is a factor in determining protein stability in vivo, we would expect that the tissue distribution of Cys, Trp and Tyr-rich proteins would correlate with regional UVR exposure. From bioinformatic analysis of 244 key structural proteins we identified several biochemically distinct, yet UV chromophore-rich, protein families. The majority of these putative UV-absorbing proteins (including the late cornified envelope proteins, keratin associated proteins, elastic fibre-associated components and β- and γ-crystallins) are localised and/or particularly abundant in tissues that are exposed to the highest doses of environmental UVR, specifically the stratum corneum, hair, papillary dermis and lens. We therefore propose that UV chromophore-rich proteins are localised in regions of high UVR exposure as a consequence of an evolutionary pressure to express sacrificial protein sunscreens which reduce UVR penetration and hence mitigate tissue damage