PCSK9 ablation attenuates Aß pathology, neuroinflammation and cognitive dysfunctions in 5XFAD mice.

BACKGROUND: Increasing evidence highlights the importance of novel players in Alzheimer's disease (AD) pathophysiology, including alterations of lipid metabolism and neuroinflammation. Indeed, a potential involvement of Proprotein convertase subtilisin/kexin type 9 (PCSK9) in AD has been recently postulated. Here, we first investigated the effects of PCSK9 on neuroinflammation in vitro. Then, we examined the impact of a genetic ablation of PCSK9 on cognitive performance in a severe mouse model of AD. Finally, in the same animals we evaluated the effect of PCSK9 loss on Aß pathology, neuroinflammation, and brain lipids. METHODS: For in vitro studies, U373 human astrocytoma cells were treated with Aß fibrils and human recombinant PCSK9. mRNA expression of the proinflammatory cytokines and inflammasome-related genes were evaluated by q-PCR, while MCP-1 secretion was measured by ELISA. For in vivo studies, the cognitive performance of a newly generated mouse line - obtained by crossing 5XFADHet with PCSK9KO mice - was tested by the Morris water maze test. After sacrifice, immunohistochemical analyses were performed to evaluate Aß plaque deposition, distribution and composition, BACE1 immunoreactivity, as well as microglia and astrocyte reactivity. Cholesterol and hydroxysterols levels in mouse brains were quantified by fluorometric and LC-MS/MS analyses, respectively. Statistical comparisons were performed according to one- or two-way ANOVA, two-way repeated measure ANOVA or Chi-square test. RESULTS: In vitro, PCSK9 significantly increased IL6, IL1B and TNFΑ mRNA levels in Aß fibrils-treated U373 cells, without influencing inflammasome gene expression, except for an increase in NLRC4 mRNA levels. In vivo, PCSK9 ablation in 5XFAD mice significantly improved the performance at the Morris water maze test; these changes were accompanied by a reduced corticohippocampal Aß burden without affecting plaque spatial/regional distribution and composition or global BACE1 expression. Furthermore, PCSK9 loss in 5XFAD mice induced decreased microgliosis and astrocyte reactivity in several brain regions. Conversely, knocking out PCSK9 had minimal impact on brain cholesterol and hydroxysterol levels. CONCLUSIONS: In vitro studies showed a pro-inflammatory effect of PCSK9. Consistently, in vivo data indicated a protective role of PCSK9 ablation against cognitive impairments, associated with improved Aß pathology and attenuated neuroinflammation in a severe mouse model of AD. PCSK9 may thus be considered a novel pharmacological target for the treatment of AD.

A New ABCB1 Inhibitor Enhances the Anticancer Effect of Doxorubicin in Both In Vitro and In Vivo Models of NSCLC.

In tumors, the multi drug resistance phenomenon may occur through the efflux of chemotherapeutic drugs out of cancer cells, impeding their accumulation, and eventually reducing their toxicity. This process is mediated by transporters overexpressed in the plasma membranes of tumor cells, among which is the P-glycoprotein/multidrug resistance 1/ATP-binding cassette B1 (P-gp/MDR1/ABCB1). The aim of this study was to explore the effect of a new molecule, called AIF-1, on ABCB1 activity. In a cellular model of non-small cell lung cancer (NSCLC), AIF-1 significantly inhibited ABCB1 activity, which was evaluated by the fluorimetric measurement of the intracellular accumulation of calcein. AIF-1 also significantly increased the intracellular content of doxorubicin, which was evaluated by confocal microscopy and LC-MS/MS analysis. This effect translated to higher cytotoxicity of doxorubicin and reduced cellular proliferation. Finally, in a murine xenograft model, the tumor volume increased by 267% and 148% on average in mice treated with vehicle and doxorubicin alone, respectively. After the co-administration of doxorubicin with AIF-1, tumor volume increased by only 13.4%. In conclusion, these results suggest enhancement of the efficacy of the chemotherapeutic drug doxorubicin by AIF-1, laying the basis for the future development of new ABCB1 inhibitors for tumor treatment.

3D-printed scaffold composites for the stimuli-induced local delivery of bioactive adjuncts.

Polysaccharide scaffolds have been successfully employed to reconstruct environments that sustain skin tissue regeneration after injuries. Three-dimensional (3D) advanced additive manufacturing technologies allow creating scaffolds with controlled and reproducible macro- and micro-structure that improve the quality of the restored tissue to favor spontaneous repair. However, when persistent inflammation occurs, the physiological tissue healing capacity is reduced, like in the presence of pathologies like diabetes, vascular diseases, chronic infection, and others. In these circumstances, the bioavailability of therapeutic adjuncts like the growth factors in addition to the standard treatments represents undoubtedly a promising strategy to accelerate the healing of skin lesions. Precisely designed polysaccharide scaffolds obtained by 3D printing represent a robust platform that can be further implemented with the controlled delivery of bioactive adjuncts. Human elastin-like polypeptides (HELPs) are stimuli-responsive biopolymers. Their structure allows the integration of domains endowed with biological functionality, making them attractive compounds to prepare composites with smart properties. In the present study, 3D-printed alginate and chitosan scaffolds were combined with the HELP components. The HELP biopolymer was fused to the epidermal growth factor (EGF) as the bioactive domain. Different constructs were prepared and the stimuli-responsive behavior as well as the biological activity were evaluated, suggesting that these smart bioactive composites are suitable to realize multifunctional dressings that sustain the local release of therapeutic adjuncts.

Alginate Self-Crosslinking Ink for 3D Extrusion-Based Cryoprinting and Application for Epirubicin-HCl Delivery on MCF-7 Cells.

3D-printed hydrogels are particularly advantageous as drug-delivery platforms but their loading with water-soluble active compounds remains a challenge requiring the development of innovative inks. Here, we propose a new 3D extrusion-based approach that, by exploiting the internal gelation of the alginate, avoids the post-printing crosslinking process and allows the loading of epirubicin-HCl (EPI). The critical combinations of alginate, calcium carbonate and d-glucono-δ-lactone (GDL) combined with the scaffold production parameters (extrusion time, temperature, and curing time) were evaluated and discussed. The internal gelation in tandem with 3D extrusion allowed the preparation of alginate hydrogels with a complex shape and good handling properties. The dispersion of epirubicin-HCl in the hydrogel matrix confirmed the potential of this self-crosslinking alginate-based ink for the preparation of 3D-printed drug-delivery platforms. Drug release from 3D-printed hydrogels was monitored, and the cytotoxic activity was tested against MCF-7 cells. Finally, the change in the expression pattern of anti-apoptotic, pro-apoptotic, and autophagy protein markers was monitored by liquid-chromatography tandem-mass-spectrometry after exposure of MCF-7 to the EPI-loaded hydrogels.

SCREENED: A Multistage Model of Thyroid Gland Function for Screening Endocrine-Disrupting Chemicals in a Biologically Sex-Specific Manner.

Endocrine disruptors (EDs) are chemicals that contribute to health problems by interfering with the physiological production and target effects of hormones, with proven impacts on a number of endocrine systems including the thyroid gland. Exposure to EDs has also been associated with impairment of the reproductive system and incidence in occurrence of obesity, type 2 diabetes, and cardiovascular diseases during ageing. SCREENED aims at developing in vitro assays based on rodent and human thyroid cells organized in three different three-dimensional (3D) constructs. Due to different levels of anatomical complexity, each of these constructs has the potential to increasingly mimic the structure and function of the native thyroid gland, ultimately achieving relevant features of its 3D organization including: 1) a 3D organoid based on stem cell-derived thyrocytes, 2) a 3D organoid based on a decellularized thyroid lobe stromal matrix repopulated with stem cell-derived thyrocytes, and 3) a bioprinted organoid based on stem cell-derived thyrocytes able to mimic the spatial and geometrical features of a native thyroid gland. These 3D constructs will be hosted in a modular microbioreactor equipped with innovative sensing technology and enabling precise control of cell culture conditions. New superparamagnetic biocompatible and biomimetic particles will be used to produce "magnetic cells" to support precise spatiotemporal homing of the cells in the 3D decellularized and bioprinted constructs. Finally, these 3D constructs will be used to screen the effect of EDs on the thyroid function in a unique biological sex-specific manner. Their performance will be assessed individually, in comparison with each other, and against in vivo studies. The resulting 3D assays are expected to yield responses to low doses of different EDs, with sensitivity and specificity higher than that of classical 2D in vitro assays and animal models. Supporting the "Adverse Outcome Pathway" concept, proteogenomic analysis and biological computational modelling of the underlying mode of action of the tested EDs will be pursued to gain a mechanistic understanding of the chain of events from exposure to adverse toxic effects on thyroid function. For future uptake, SCREENED will engage discussion with relevant stakeholder groups, including regulatory bodies and industry, to ensure that the assays will fit with purposes of ED safety assessment. In this project review, we will briefly discuss the current state of the art in cellular assays of EDs and how our project aims at further advancing the field of cellular assays for EDs interfering with the thyroid gland.

Structural modification of cuminaldehyde thiosemicarbazone increases inhibition specificity toward aflatoxin biosynthesis and sclerotia development in Aspergillus flavus.

Aspergillus flavus is an opportunistic mold that represents a serious threat for human and animal health due to its ability to synthesize and release, on food and feed commodities, different toxic secondary metabolites. Among them, aflatoxin B1 is one of the most dangerous since it is provided with a strong cancerogenic and mutagenic activity. Controlling fungal contamination on the different crops that may host A. flavus is considered a priority by sanitary authorities of an increasing number of countries due also to the fact that, owing to global temperature increase, the geographic areas that are expected to be prone to experience sudden A. flavus outbreaks are widening. Among the different pre- and post-harvest strategies that may be put forward in order to prevent fungal and/or mycotoxin contamination, fungicides are still considered a prominent weapon. We have here analyzed different structural modifications of a natural-derived compound (cuminaldehyde thiosemicarbazone) for their fungistatic and anti-aflatoxigenic activity. In particular, we have focused our attention on one of the compound that presented a prominent anti-aflatoxin specificity, and performed a set of physiological and molecular analyses, taking also advantage of yeast (Saccharomyces cerevisiae) cell as an experimental model.

Mapping insulin non-covalent interactions with natural polysaccharides by hydrogen/deuterium exchange mass spectrometry.

RATIONALE: Drug development efforts involving therapeutic peptides or proteins strongly lead optimization of drug delivery, drug stability, solubility and functionality. The key feature of controlled drug delivery is the use of biocompatible polymers able to interact via non-covalent bonds with an active principle through multiple functional groups. Here amide hydrogen/deuterium exchange (HDX) mass spectrometry was employed to localize insulin dynamics induced by interactions with three natural polysaccharides, i.e. chitosan (CH), sodium alginate (ALG) and chondroitin sulfate (CS). METHODS: LTQ-Orbitap continuous-labelling mass spectra were collected by diluting insulin stock solution (10 mM in 0.1% formic acid) to a final concentration of 0.1 mM in D2 O containing 1 mM deuterated ammonium acetate (final pH .6) (insulin:polysaccharide ratio 1:2, w/w). For peptide mapping, deuterated samples were quenched after 0.5, 30, 60, 120 minutes exchange by adding HCl (pH ) and digested with pepsin before LC-MS/MS analysis. RESULTS: Differences in the insulin backbone dynamics in the presence of the three polysaccharides were highlighted by monitoring peptic peptides at different time points. No significant differences were observed in the presence of CH, whereas the negatively charged ALG and CS were able to induce significant conformational variations at the B-chain level resulting in more protection against H/D exchange. The A-chain interacted only with CS reducing the protein mobility on a long time scale (120 min). HDX data evidenced heterogeneous insulin dynamics in the presence of ALG and CS. CONCLUSIONS: The studies reported here demonstrated the capabilities of mass spectrometry techniques and HDX methods to obtain useful information toward the flexibility and the behavior of native insulin in the presence of natural polysaccharides, and could provide insights to study the behavior of pharmaceutical formulations. Copyright © 2016 John Wiley & Sons, Ltd.

Evidence for cholesterol-lowering activity by Bifidobacterium bifidum PRL2010 through gut microbiota modulation.

Bifidobacteria are members of the human gut microbiota, which are known to influence the metabolic abilities of their host. Here, we investigated the capabilities of bifidobacteria to reduce cholesterol levels in synthetic growth media, clearly demonstrating assimilation of this molecule by particular bifidobacterial strains, including Bifidobacterium bifidum PRL2010 (LMG S-28692). The transcriptomic analysis of PRL2010 cells cultivated in the presence of cholesterol revealed a significantly increased transcription level of genes encoding putative transporters and reductases, indicative of specific mechanisms for cholesterol assimilation as well as cholesterol conversion to coprostanol. Cholesterol lowering activity of B. bifidum PRL2010 cells was further evaluated by means of an in vivo murine model, showing that the fecal microbiota of mice is modified toward those bacteria involved in the metabolism of cholesterol.

Understanding solid-state properties of triglycerides used in pharmaceutical and food microencapsulation.

Hydrophobic materials, in particular hydrogenated vegetable oils, HVO, are extensively used as coating materials in food and pharmaceutical systems. Correct application of these coatings requires an evaluation of their behaviour as a function of various parameters such as melting temperature, solubility, concentration and/or pH. The purpose of this study was to assess the physico-chemical properties of an HVO in terms of composition, crystallisation, phase transition and polymorphism using a variety of analytical techniques, such as electrospray mass spectrometry (ESI-MS), differential scanning calorimetry (DSC) and X-ray diffraction (XRD). High-resolution ESI-MS allowed establishment of the HVO main composition of long-chain triglycerides (average molecular weight 1183 Da). DSC results showed that thermal history determines the formation of at least two polymorphs of HVO, namely two different crystal forms, assigned as form α, melting point (m.p.) 48 °C, and form ß', m.p. 60 °C. A third polymorph, the more thermodynamically stable ß-form, having a melting point at 62 °C, is obtained by solution-mediated re-crystallisation. Phase transformation paths were investigated by isothermal DSC experiments, which evidenced that the α-form is kinetically stable at temperatures lower than 25 °C. These data are of particular interest in practical applications such as spray freezing or pan coating where significant heat transfer phenomena are involved.

Desorption electrospray ionization high-resolution mass spectrometry for the fast investigation of natural polysaccharide interactions with a model drug in controlled release systems.

RATIONALE: The control of drug release involves gaining an understanding of the complex interaction networks among drug-excipients-matrix-biological fluids. Thus, novel analytical methods that will lead to a better understanding of these interaction networks are urgently required. METHODS: Desorption electrospray ionization high-resolution mass spectrometry (DESI-HRMS) was used to evaluate the behaviour of four biocompatible polysaccharides (chondroitin sulfate, chitosan, sodium alginate and λ-carrageenan) in the release of atenolol (ATN) from drug tablets. An aqueous solution at three different pH values (pH 7.4, 4.5 and 1.2) was electrosprayed onto the tablets, allowing direct, fast, sensitive detection of atenolol as the protonated molecule in positive ion mode. Information about the desorption mechanism was obtained by analyzing the ATN [M+H](+) ion signal as a function of time. ATN-polymer interactions in the drug/polymer mixtures were also studied by Horizontal Attenuated Total Reflectance (HATR) Fourier transform infrared (FTIR) spectroscopy. RESULTS: The DESI-MS results revealed statistically different ATN desorption trends as a function of the polysaccharide investigated and the pH of the desorbing solution. Different release kinetics were ascribed to the drug-polymer interactions, and to the diffusion process of the drug through the hydrated polymer mesh. In particular, the alginate and λ-carrageenan matrices were able to sustain drug release from the tablet even for a highly soluble drug. The HATR results confirmed the presence of ATN-polymer interactions that, depending on the polymer-drug-solvent combination used, might affect ATN diffusion. CONCLUSIONS: These results suggest that DESI-MS has a potential role for the micro-environmental analysis of drug diffusion and surface distribution in polymeric matrices.

A bioartificial and vasculomorphic bone matrix-based organoid mimicking microanatomy of flat and short bones.

We engineered an in vitro model of bioartificial 3D bone organoid consistent with an anatomical and vascular microenvironment common to mammalian flat and short bones. To achieve this, we chose the decellularized-decalcified matrix of the adult male rat scapula, implemented with the reconstruction of its intrinsic vessels, obtained through an original intravascular perfusion with polylevolactic (PLLA), followed by coating of the PLLA-fabricated vascularization with rat tail collagen. As a result, the 3D bone and vascular geometry of the native bone cortical and cancellous compartments was reproduced, and the rat tail collagen-PLLA biomaterial could in vitro act as a surrogate of the perivascular extracellular matrix (ECM) around the wall of the biomaterial-reconstituted cancellous vessels. As a proof-of-concept of cell compatibility and site-dependent osteoinductive properties of this bioartificial 3D construct, we show that it in vitro leads to a time-dependent microtopographic positioning of rat mesenchymal stromal cells (MSCs), initiating an osteogenic fate in relation to the bone compartment. In addition, coating of PLLA-reconstructed vessels with rat tail collagen favored perivascular attachment and survival of MSC-like cells (mouse embryonic fibroblasts), confirming its potentiality as a perivascular stroma for triggering competence of seeded MSCs. Finally, in vivo radiographic topography of bone lesions in the human jaw and foot tarsus of subjects with primary osteoporosis revealed selective bone cortical versus cancellous involvement, suggesting usefulness of a human 3D bone organoid engineered with the same principles of our rat organoid, to in vitro investigate compartment-dependent activities of human MSC in flat and short bones under experimental osteoporotic challenge. We conclude that our 3D bioartificial construct offers a reliable replica of flat and short bones microanatomy, and promises to help in building a compartment-dependent mechanistic perspective of bone remodeling, including the microtopographic dysregulation of osteoporosis.

Lactobacillus delbrueckii subsp. bulgaricus derivatives for 3D printed alginate/hyaluronic acid self-crosslinking hydrogels: Manufacturing and wound healing potential.

Derivatives [i.e. proteins and exopolysaccharides (EPS)] from Lactobacillus delbrueckii subsp. bulgaricus (LB) were extracted, characterized, and for the first time used in the production of novel self-crosslinking 3D printed alginate/hyaluronic acid (ALG/HA) hydrogels, as high-value functional biomaterials with therapeutic potentials in regenerative medicine applications. Derivatives coming from two different LB strains, LB1865 and LB1932, were tested in-vitro and compared for their cytotoxicity and effect on proliferation and migration on human fibroblast. EPS received particular attention as showing relevant dose-dependent cytocompatibility against the human fibroblast. The derivatives showed an ability to increase cell proliferation and migration, quantifiable between 10 and 20 % if compared to controls, with higher values for the derivatives obtained from the LB1932 strain. These were explained by liquid chromatography-mass spectrometry targeted protein biomarker analysis as a decrease in matrix-degrading and proapoptotic proteins, associated with an increase in collagen and antiapoptotic proteins production. LB1932 enriched hydrogel was found to be of benefit compared to control dressings, giving the more promising results as potential for in vivo skin wound healing tests.

Rapid Prototyping of 3D-Printed AgNPs- and Nano-TiO2-Embedded Hydrogels as Novel Devices with Multiresponsive Antimicrobial Capability in Wound Healing.

Two antimicrobial agents such as silver nanoparticles (AgNPs) and titanium dioxide (TiO2) have been formulated with natural polysaccharides (chitosan or alginate) to develop innovative inks for the rapid, customizable, and extremely accurate manufacturing of 3D-printed scaffolds useful as dressings in the treatment of infected skin wounds. Suitable chemical-physical properties for the applicability of these innovative devices were demonstrated through the evaluation of water content (88-93%), mechanical strength (Young's modulus 0.23-0.6 MPa), elasticity, and morphology. The antimicrobial tests performed against Staphylococcus aureus and Pseudomonas aeruginosa demonstrated the antimicrobial activities against Gram+ and Gram- bacteria of AgNPs and TiO2 agents embedded in the chitosan (CH) or alginate (ALG) macroporous 3D hydrogels (AgNPs MIC starting from 5 µg/mL). The biocompatibility of chitosan was widely demonstrated using cell viability tests and was higher than that observed for alginate. Constructs containing AgNPs at 10 µg/mL concentration level did not significantly alter cell viability as well as the presence of titanium dioxide; cytotoxicity towards human fibroblasts was observed starting with an AgNPs concentration of 100 µg/mL. In conclusions, the 3D-printed dressings developed here are cheap, highly defined, easy to manufacture and further apply in personalized antimicrobial medicine applications.

3D Printed Chitosan/Alginate Hydrogels for the Controlled Release of Silver Sulfadiazine in Wound Healing Applications: Design, Characterization and Antimicrobial Activity.

The growing demand for personalized medicine requires innovation in drug manufacturing to combine versatility with automation. Here, three-dimensional (3D) printing was explored for the production of chitosan (CH)/alginate (ALG)-based hydrogels intended as active dressings for wound healing. ALG hydrogels were loaded with 0.75% w/v silver sulfadiazine (SSD), selected as a drug model commonly used for the therapeutic treatment of infected burn wounds, and four different 3D CH/ALG architectures were designed to modulate the release of this active compound. CH/ALG constructs were characterized by their water content, elasticity and porosity. ALG hydrogels (Young's modulus 0.582 ± 0.019 Mpa) were statistically different in terms of elasticity compared to CH (Young's modulus 0.365 ± 0.015 Mpa) but very similar in terms of swelling properties (water content in ALG: 93.18 ± 0.88% and in CH: 92.76 ± 1.17%). In vitro SSD release tests were performed by using vertical diffusion Franz cells, and statistically significant different behaviors in terms of the amount and kinetics of drugs released were observed as a function of the construct. Moreover, strong antimicrobial potency (100% of growth inhibition) against Staphylococcus aureus and Pseudomonas aeruginosa was demonstrated depending on the type of construct, offering a proof of concept that 3D printing techniques could be efficiently applied to the production of hydrogels for controlled drug delivery.

Woven bone formation and mineralization by rat mesenchymal stromal cells imply increased expression of the intermediate filament desmin.

Background: Disordered and hypomineralized woven bone formation by dysfunctional mesenchymal stromal cells (MSCs) characterize delayed fracture healing and endocrine -metabolic bone disorders like fibrous dysplasia and Paget disease of bone. To shed light on molecular players in osteoblast differentiation, woven bone formation, and mineralization by MSCs we looked at the intermediate filament desmin (DES) during the skeletogenic commitment of rat bone marrow MSCs (rBMSCs), where its bone-related action remains elusive. Results: Monolayer cultures of immunophenotypically- and morphologically - characterized, adult male rBMSCs showed co-localization of desmin (DES) with vimentin, F-actin, and runx2 in all cell morphotypes, each contributing to sparse and dense colonies. Proteomic analysis of these cells revealed a topologically-relevant interactome, focused on cytoskeletal and related enzymes//chaperone/signalling molecules linking DES to runx2 and alkaline phosphatase (ALP). Osteogenic differentiation led to mineralized woven bone nodules confined to dense colonies, significantly smaller and more circular with respect to controls. It significantly increased also colony-forming efficiency and the number of DES-immunoreactive dense colonies, and immunostaining of co-localized DES/runx-2 and DES/ALP. These data confirmed pre-osteoblastic and osteoblastic differentiation, woven bone formation, and mineralization, supporting DES as a player in the molecular pathway leading to the osteogenic fate of rBMSCs. Conclusion: Immunocytochemical and morphometric studies coupled with proteomic and bioinformatic analysis support the concept that DES may act as an upstream signal for the skeletogenic commitment of rBMSCs. Thus, we suggest that altered metabolism of osteoblasts, woven bone, and mineralization by dysfunctional BMSCs might early be revealed by changes in DES expression//levels. Non-union fractures and endocrine - metabolic bone disorders like fibrous dysplasia and Paget disease of bone might take advantage of this molecular evidence for their early diagnosis and follow-up.

Anti-Fibronectin Aptamer Modifies Blood Clot Pattern and Stimulates Osteogenesis: An Ex Vivo Study.

BACKGROUND: Scaffold (SCA) functionalization with aptamers (APT) provides adsorption of specific bioactive molecules on biomaterial surfaces. The aim of this study was to observe if SCA enriched with anti-fibronectin APT can favor coagulum (PhC) and osteoblasts (OSB) differentiation. METHODS: 20 µg of APT was functionalized on SCA by simple adsorption. For PhC formation, SCAs were inserted into rat calvaria defects for 17 h. Following proper transportation (buffer solution PB), OSBs (UMR-106 lineage) were seeded over PhC + SCAs with and without APT. Cells and PhC morphology, PhC cell population, protein labeling and gene expression were observed in different time points. RESULTS: The APT induced higher alkaline phosphatase and bone sialoprotein immunolabeling in OSB. Mesenchymal stem cells, leukocytes and lymphocytes cells were detected more in the APT group than when scaffolds were not functionalized. Additionally, an enriched and dense fibrin network and different cell types were observed, with more OSB and white blood cells in PhC formed on SCA with APT. The gene expression showed higher transforming growth factor beta 1 (TGF-b1) detection in SCA with APT. CONCLUSIONS: The SCA functionalization with fibronectin aptamers may alter key morphological and functional features of blood clot formation, and provides a selective expression of proteins related to osteo differentiation. Additionally, aptamers increase TGF-b1 gene expression, which is highly associated with improvements in regenerative therapies.

In search of a vaccine for mouse allergy: significant reduction of Mus m 1 allergenicity by structure-guided single-point mutations.

BACKGROUND: Mouse urinary proteins are relevant allergens from mice urine. We used the recombinant protein Mus m 1 as an allergen model to identify if, by altering Mus m 1 architecture via single-point mutations, we could effectively modify its allergenicity. METHODS: Based on structural considerations, we synthesized two single-point mutants, Mus m 1-Y120A and Mus m 1-Y120F, which were expected to harbor large structural alterations. Circular dichroism and fluorescence analysis showed significant conformational rearrangements of the aromatic side chains in the internal cavity of Mus m 1-Y120A when compared to Mus m 1-Y120F and Mus m 1. Evaluation of the allergenic potential of the recombinant molecules was performed in vitro with both immunochemical approaches and assays based on the measurement of basophil degranulation. Moreover, to assess the integrity of the T cell epitopes and as an in vitro measure of immunogenicity, we tested the reactivity of T lymphocytes from subjects allergic to mouse urine against proteins and synthetic peptides encompassing the immunodominant linear epitope containing the mutation. RESULTS: We found that the selected point mutation was able to modulate the protein allergenicity, and to severely impair the recognition of Mus m 1 by IgE, while T cell reactivity was fully maintained. CONCLUSIONS: In silico predicted, minimum selected structural modifications allowed to design one protein with reduced allergenicity and preserved immunogenicity. Structurally guided mutations can direct the design of proteins with reduced allergenicity which can be used as vaccines for a safer and more effective immunotherapy of allergic disorders.

Decellularization Detergents As Methodological Variables in Mass Spectrometry of Stromal Matrices.

Collagens, elastin, fibrillin, decorin, and laminin are key constituents of the extracellular matrix and basement membrane of mammalian organs. Thus, changes in their quantities may influence the mechanochemical regulation of resident cells. Since maintenance of a native stromal composition is a requirement for three-dimensional (3D) matrix-based recellularization techniques in tissue engineering, we studied the influence of the decellularization detergents on these proteins in porcine kidney, liver, pancreas, and skin. Using a quick thawing/quick microwave-assisted decellularization protocol and two different detergents, sodium dodecyl sulfate (SDS) vs Triton X-100 (TX100), at identical concentration, variations in matrix conservation of stromal proteins were detected by liquid chromatography-mass spectrometry coupled to light and scanning electron microscopies, in dependence on each detergent. In all organs tested except pancreas, collagens were retained to a statistically significant level using the TX100-based protocol. In contrast fibrillin, elastin (except in kidney), and decorin (only in liver) were better preserved with the SDS-dependent protocol. Irrespective of the detergent used, laminin always remained at an irrelevant level. Our results prompt attention to the type of detergent in organ decellularization, suggesting that its choice may influence morphoregulatory inputs peculiar to the type of 3D bioartificial mammalian organ to be reconstructed. Impact statement Simple change of the protocol's main detergent leads to a very substantial difference in the panel of the stromal proteins detected by qualitative and semiquantitative mass spectrometry in acellular porcine matrices. This remarkable methodological variable promises to yield proteomic reference panels in a number of different species-specific acellular matrices allowing for selective retainment of peculiar mechanochemical inputs, to differently address the development of the seeded cells in relation to the type of organ to be bioartificially reconstructed.

Development and single laboratory validation of a targeted liquid chromatography-triple quadrupole mass spectrometry-based method for the determination of insulin like growth factor-1 in different types of milk samples.

A simple and reliable targeted liquid chromatography-electrospray-tandem mass spectrometry (LC-MS/MS) method was developed and validated through the selection of two biomarker peptides for the identification and determination of bovine insulin like growth factor-1 (IGF-1) in milk samples. Two urea-based sample extraction procedures were tested. The validation results provided detection limits at the 1-5 ng IGF-1/mL level as a function of the milk matrix, precision ranged from 3 to 8% and the method accuracy in the different milk matrices was assured. Finally, IGF-1 was measured in milk samples obtained by treatment with eleven different technological processes: IGF-1 concentrations were spread over a wide range from 11.2 ± 0.3 ng/mL to 346 ± 8 ng/mL with a median of 57.0 ± 0.2 ng/mL. The highest amount of IGF-1 was found in fresh whole milk samples and no significant correlation was found between the total milk protein content and the IGF-1 concentration level.