Synthesis and Antiproliferative Evaluation of d-Glucuronamide-Based Nucleosides and (Triazolyl)methyl Amide-Linked Pseudodisaccharide Nucleosides.

The synthesis and antiproliferative evaluation of novel d-glucopyranuronamide-containing nucleosides is described. Based on our previously reported anticancer d-glucuronamide-based nucleosides, new analogues comprising N/O-dodecyl or N-propargyl substituents at the glucuronamide unit and anomerically-N-linked 2-acetamido-6-chloropurine, 6-chloropurine or 4-(6-chloropurinyl)methyl triazole motifs were synthesized in 4-6 steps starting from acetonide-protected glucofuranurono-6,3-lactone. The methodologies were based on the access to N-substituted glycopyranuronamide precursors, namely 1,2-O-acetyl derivatives or glucuronoamidyl azides for further nucleobase N-glycosylation or 1,3-dipolar cycloaddition with N9 - and N7 -propargyl-6-chloropurines, respectively. N-Propargyl glucuronamide-based N9 -purine nucleosides were converted into (triazolyl)methyl amide-6,6-linked pseudodisaccharide nucleosides via cycloaddition with methyl 6-azido-glucopyranoside. A CuI/Amberlyst A-21 catalytic system employed in the cycloaddition reactions also effected conversion into 6-dimethylaminopurine nucleosides. Antiproliferative evaluation in chronic myeloid leukemia (K562) and breast cancer (MCF-7) cells revealed significant effects exhibited by the synthesized monododecylated purine-containing nucleosides. A N-propargyl 3-O-dodecyl glucuronamide derivative comprising a N9 -ß-linked 6-chloropurine moiety was the most active compound against MCF-7 cells (GI50 =11.9 µM) while a related α-(purinyl)methyltriazole nucleoside comprising a N7 -linked 6-chloropurine moiety exhibited the highest activity against K562 cells (GI50 =8.0 µM). Flow cytometry and immunoblotting analysis of apoptosis-related proteins in K562 cells treated with the N-propargyl 3-O-dodecyl glucuronamide-based N9 -linked 6-chloropurine nucleoside indicated that it acts via apoptosis induction.

Cinnamaldehyde modulates host immunoinflammatory responses in rat ligature-induced periodontitis and peripheral blood mononuclear cell models.

Cinnamaldehyde is a natural product with anti-inflammatory and immune-modulatory properties, known to regulate host responses to bacterial stimuli. This study aimed to investigate the effects of cinnamaldehyde on ligature-induced periodontitis in rats, and its impact on the modulation of human peripheral blood mononuclear cells (PBMC). Male Wistar rats were assigned into three groups:i) control: no ligature + vehicle; ii) ligature: ligature + vehicle; and iii) ligature + cinnamaldehyde (50 mg/kg); all treatments by daily oral gavage. After 14 days of induced periodontitis, the hemimandibles were collected for bone loss evaluation. The gingival levels of IL-1ß, MMP-9 and iNOS mRNA were evaluated. Nitric oxide (NO) was measured in both rat saliva and plasma. PBMC were stimulated with Aggregatibacter actinomycetemcomitans (Aa) in the presence or absence of cinnamaldehyde (5, 20 e 40 µM), and cytokine production was quantified in cell supernatant. Proliferating lymphocytes were taken for flow cytometer reading, while culture supernatants were used for IFN-γ and IL-10 assessment. The ligature group had both increased alveolar bone loss and gingival expression of IL-1ß, MMP-9 and iNOS compared to the control group. All parameters were attenuated by cinnamaldehyde treatment. Lower salivary but not plasma NO was detected in the cinnamaldehyde compared to the ligature group. Aa-stimulated PBMCs treated with cinnamaldehyde produced less IL-1ß; the compound also attenuated lymphocyte proliferation in a dose-dependent manner, as well as cell IL-10 production. Cinnamaldehyde treatment reduced periodontal bone loss, and downregulated key inflammatory mediators and human PBMC responses, pointing to novel potential therapeutic effects of this compound.

Innovative tailor made dextran based membranes with excellent non-inflammatory response: In vivo assessment.

In this work, dextran based membranes with potential to be used as implantable devices in Tissue Engineering and Regenerative Medicine (TERM) were prepared by a straightforward strategy. Briefly, two polymers approved by the Food and Drug Administration, viz. dextran and poly(ε-caprolactone) (PCL) were functionalized with methacrylate moieties, and subjected to photocrosslinking. Employing different weight ratios of each polymer in the formulations allowed to obtain transparent membranes with tunable physicochemical properties and low adverse host tissue response. Independently of the material, all formulations have shown to be thermally stable up to 300 °C whilst variations in the polymer ratio resulted in membranes with different glass transition temperatures (Tg) and flexibility. The swelling capacity ranged from 50% to 200%. On the other hand, in vitro hydrolytic degradation did not show to be material-dependent and all membranes maintained their structural integrity for more than 30 days, losing only 8-12% of their initial weight. Preliminary in vitro biological tests did not show any cytotoxic effect on seeded human dental pulp stem cells (hDPSCs), suggesting that, in general, all membranes are capable of supporting cell adhesion and viability. The in vivo biocompatibility of membranes implanted subcutaneously in rats' dorsum indicate that M100/0 (100%wt dextran) and M25/75 (25 %wt dextran) formulations can be classified as "slight-irritant" and "non-irritant", respectively. From the histological analysis performed on the main tissue organs it was not possible to detect any signs of fibrosis or necrosis thereby excluding the presence of toxic degradation by-products deposited or accumulated in these tissues. In combination, these results suggest that the newly developed formulations hold great potential as engineered devices for biomedical applications, where the biological response of cells and tissues are greatly dependent on the physical and chemical cues provided by the substrate.

Topical Application of Cinnamaldehyde Promotes Faster Healing of Skin Wounds Infected with Pseudomonas aeruginosa.

Wound healing can be delayed following colonization and infection with the common bacterium Pseudomonas aeruginosa. While multiple therapies are used for their treatment, these are ineffective, expensive, and labour-intensive. Thus, there is an enormous unmet need for the treatment of infected wounds. Cinnamaldehyde, the major component of cinnamon oil, is well known for its antimicrobial properties. Herein, we investigated the effects of sub-inhibitory concentrations of cinnamaldehyde in the virulence of P. aeruginosa. We also assessed its healing potential in P. aeruginosa-infected mouse skin wounds and the mechanisms involved in this response. Sub-inhibitory concentrations of cinnamaldehyde reduced P. aeruginosa metabolic rate and its ability to form biofilm and to cause haemolysis. Daily topical application of cinnamaldehyde on P. aeruginosa-infected skin wounds reduced tissue bacterial load and promoted faster healing. Lower interleukin-17 (IL-17), vascular endothelial growth factor (VEGF) and nitric oxide levels were detected in cinnamaldehyde-treated wound samples. Blockage of transient receptor potential ankyrin 1, the pharmacological target of cinnamaldehyde, abrogated its healing activity and partially reversed the inhibitory actions of this compound on VEGF and IL-17 generation. We suggest that topical application of sub-inhibitory concentrations of cinnamaldehyde may represent an interesting approach to improve the healing of P. aeruginosa-infected skin wounds.

Hydrogen peroxide-based products alter inflammatory and tissue damage-related proteins in the gingival crevicular fluid of healthy volunteers: a randomized trial.

Hydrogen peroxide (H2O2)-based products are effective in tooth whitening; however, their safety is controversial as they may harm patient tissues/cells. These effects are suggested to be concentration-dependent; nonetheless, to date, there are no reports on H2O2-mediated oxidative damage in the gingival tissue, and neither whether this can be detected in gingival crevicular fluid (GCF) samples. We hypothesize that H2O2 whitening products may cause collateral oxidative tissue damage following in office application. Therefore, H2O2 and nitric oxide (NO) levels were investigated in GCF samples obtained from patients undergoing dental bleaching with H2O2 at different concentrations, in a randomized, double-blind, split-mouth clinical trial. A proteomic analysis of these samples was also performed. H2O2-based whitening products promoted inflammation which was detected in GCF samples and lasted for longer following 35% H2O2 bleaching. This included time-dependent changes in NO levels and in the abundance of proteins associated with NO synthesis, oxidative stress, neutrophil regulation, nucleic acid damage, cell survival and/or tissue regeneration. Overall, H2O2-based products used in office promote inflammation irrespective of their concentration. As the inflammation caused by 35% H2O2 is longer, patients may benefit better from using lower concentrations of this bleaching product, as they may result in less tissue damage.

The influence of poly(ester amide) on the structural and functional features of 3D additive manufactured poly(ε-caprolactone) scaffolds.

The current research reports for the first time the use of blends of poly(ε-caprolactone) (PCL) and poly(ester amide) (PEA) for the fabrication of 3D additive manufactured scaffolds. Tailor made PEA was synthesized to afford fully miscible blends of PCL and PEA using different percentages (5, 10, 15 and 20% w/w). Stability, characteristic temperatures and material's compatibility were studied through thermal analyses (i.e., TGA, DSC). Even though DMTA and static compression tests demonstrated the possibility to improve the storage modulus, Young's modulus and maximum stress by increasing the amount of PEA, a decrease of hardness was found beyond a threshold concentration of PEA as the lowest values were achieved for PCL/PEA (20% w/w) scaffolds (from 0.39 ±â€¯0.03 GPa to 0.21 ±â€¯0.02 GPa in the analysed load range). The scaffolds presented a controlled morphology and a fully interconnected network of internal channels. The water contact angle measurements showed a clear increase of hydrophilicity resulting from the addition of PEA. This result was further corroborated with the improved adhesion and proliferation of human mesenchymal stem cells (hMSCs). The presence of PEA also influenced the cell morphology. Better cell spreading and a much higher and homogenous number of cells were observed for PCL/PEA scaffolds when compared to PCL ones.

Quasi-static and dynamic in vitro mechanical response of 3D printed scaffolds with tailored pore size and architectures.

Scaffold-based Tissue Engineering represents the most promising approach for the regeneration of load bearing skeletal tissues, in particular bone and cartilage. Scaffolds play major role in this process by providing a physical template for cells to adhere and proliferate whilst ensuring an adequate biomechanical support at the defect site. Whereas the quasi static mechanical properties of porous polymeric scaffolds are well documented, the response of these constructs under high strain compressive rates remain poorly understood. Therefore, this study investigates, for the first time, the influence of pore size and geometry on the mechanical behaviour of Polycaprolactone (PCL) scaffolds under quasi static and dynamic conditions. 3D printed scaffolds with varied pore sizes and geometries were obtained using different filament distances (FD) and lay-down patterns, respectively. In particular, by fixing the lay-down pattern at 0/90° and varying the FD between 480 and 980 µm it was possible to generate scaffolds with square pores with dimensions in the range of 150-650 µm and porosities of 59-79%. On the other hand, quadrangular, hexagonal, triangular and complex pore geometries with constant porosity (approx. 70%) were obtained at a fixed FD of 680 µm and imposing four different lay-down patterns of 0/90, 0/60/120, 0/45/90/135 and 0/30/60/90/120/150°, respectively. The mechanical response of printed scaffolds was assessed under two different compression loading regimes spanning five distinct strain rates, from 10-2 to 2000 s-1, using two different apparatus: a conventional screw-driven testing machine (Instron 4483) and a Split Hopkinson pressure bar (SHPB) equipped with a set of A201 Flexi-force™ (FF) force sensors and a pulse shaper. Our results show that the mechanical properties of PCL scaffolds are not strain rate sensitive between 1300 and 2000 s-1 and these strongly depend on the pore size (porosity) rather than pore geometry. Those findings are extremely relevant for the engineering of bone tissue scaffolds with enhanced mechanical stability by providing new data describing the mechanical response of these constructs at high strain rates as well as the at the transition between quasi static and dynamic regimes.

Transient Receptor Potential Canonical Channels 4 and 5 Mediate Escherichia coli-Derived Thioredoxin Effects in Lipopolysaccharide-Injected Mice.

Thioredoxin plays an essential role in bacterial antioxidant machinery and virulence; however, its regulatory actions in the host are less well understood. Reduced human Trx activates transient receptor potential canonical 5 (TRPC5) in inflammation, but there is no evidence of whether these receptors mediate bacterial thioredoxin effects in the host. Importantly, TRPC5 can form functional complexes with other subunits such as TRPC4. Herein, E. coli-derived thioredoxin induced mortality in lipopolysaccharide- (LPS-) injected mice, accompanied by reduction of leukocyte accumulation, regulation of cytokine release into the peritoneum, and impairment of peritoneal macrophage-mediated phagocytosis. Dual TRPC4/TRPC5 blockade by ML204 increased mortality and hypothermia in thioredoxin-treated LPS mice but preserved macrophage's ability to phagocytose. TRPC5 deletion did not alter body temperature but promoted additional accumulation of peritoneal leukocytes and inflammatory mediator release in thioredoxin-administered LPS mice. Thioredoxin diminished macrophage-mediated phagocytosis in wild-type but not TRPC5 knockout animals. TRPC5 ablation did not affect LPS-induced responses. However, ML204 caused mortality associated with exacerbated hypothermia and decreased peritoneal leukocyte numbers and cytokines in LPS-injected mice. These results suggest that bacterial thioredoxin effects under LPS stimuli are mediated by TRPC4 and TRPC5, shedding light on the additional mechanisms of bacterial virulence and on the pathophysiological roles of these receptors.

Treatment with either leflunomide or adalimumab reduces anaemia in patients with rheumatoid arthritis.

Rheumatoid arthritis is a chronic disease of the joints, which causes joint pain and disability. Anaemia is a frequent extra-articular manifestation in rheumatoid arthritis, affecting 30-70% of the patients; presenting a negative impact on patient´s quality of life. Some of the drugs used in rheumatoid arthritis treatment improve anaemia; but little is known on the beneficial effects of the anti-rheumatic leflunomide or the anti-TNFα adalimumab, in this parameter. We investigated the incidence of anaemia in rheumatoid arthritis patients treated or not with leflunomide or adalimumab. We also assessed whether anaemia correlates with disease activity. Anaemia was present in patients who had just been diagnosed with rheumatoid arthritis and had never taken disease modifying agents or biologicals (non-specific therapy group), but not in those taking either leflunomide or adalimumab. The erythrocyte sedimentation rate was increased in patients with non-specific therapy in comparison with those taking either leflunomide or adalimumab. Anaemia correlated with increased erythrocyte sedimentation rate. We suggest that leflunomide and adalimumab may be useful in treating anaemia in patients with rheumatoid arthritis.

Sensitivity Analysis of Different Shapes of a Plastic Optical Fiber-Based Immunosensor for Escherichia coli: Simulation and Experimental Results.

Conventional pathogen detection methods require trained personnel, specialized laboratories and can take days to provide a result. Thus, portable biosensors with rapid detection response are vital for the current needs for in-loco quality assays. In this work the authors analyze the characteristics of an immunosensor based on the evanescent field in plastic optical fibers with macro curvature by comparing experimental with simulated results. The work studies different shapes of evanescent-wave based fiber optic sensors, adopting a computational modeling to evaluate the probes with the best sensitivity. The simulation showed that for a U-Shaped sensor, the best results can be achieved with a sensor of 980 µm diameter by 5.0 mm in curvature for refractive index sensing, whereas the meander-shaped sensor with 250 µm in diameter with radius of curvature of 1.5 mm, showed better sensitivity for either bacteria and refractive index (RI) sensing. Then, an immunosensor was developed, firstly to measure refractive index and after that, functionalized to detect Escherichia coli. Based on the results with the simulation, we conducted studies with a real sensor for RI measurements and for Escherichia coli detection aiming to establish the best diameter and curvature radius in order to obtain an optimized sensor. On comparing the experimental results with predictions made from the modelling, good agreements were obtained. The simulations performed allowed the evaluation of new geometric configurations of biosensors that can be easily constructed and that promise improved sensitivity.

Transient Receptor Potential Ankyrin 1 Channel Expression on Peripheral Blood Leukocytes from Rheumatoid Arthritic Patients and Correlation with Pain and Disability.

Patients with rheumatoid arthritis (RA) suffer from pain and joint disability. The transient receptor potential ankyrin 1 (TRPA1) channel expressed on sensory neurones and non-neuronal cells mediates pain transduction and inflammation and it has been implicated in RA. However, there is little information on the contribution of TRPA1 for human disease. Here, we investigated the expression of TRPA1 on peripheral blood leukocytes and the circulating levels of its endogenous activators 4-hydroxynonenal (4-HNE) and hydrogen peroxide (H2O2) in RA patients treated or not with the anti-rheumatic leflunomide (LFN) or the anti-TNFα adalimumab (ADA). We also assessed whether TRPA1 expression correlates with joint pain and disability, in addition to the immune changes in RA. TRPA1 expression on peripheral blood leukocytes correlated with pain severity and disability. TRPA1 levels on these cells were associated with the numbers of polymorphonuclear and the activation of CD14+ cells. No correlations were found between the lymphocyte population and TRPA1 expression, pain or disability. Patients recently diagnosed with RA expressed increased levels of TRPA1 on their leukocytes whilst treatment with either LFN or ADA down-regulated this receptor probably by reducing the numbers of polymorphonuclears and the activation of CD14+ cells. We suggest that the activation levels of CD14+ cells, the numbers of PMNs in the peripheral blood and the expression of TRPA1 on peripheral blood leukocytes correlate with RA progression, affecting joint pain sensitivity and loss of function.

Cinnamaldehyde modulates LPS-induced systemic inflammatory response syndrome through TRPA1-dependent and independent mechanisms.

Cinnamaldehyde is a natural essential oil suggested to possess anti-bacterial and anti-inflammatory properties; and to activate transient receptor potential ankyrin 1 (TRPA1) channels expressed on neuronal and non-neuronal cells. Here, we investigated the immunomodulatory effects of cinnamaldehyde in an in vivo model of systemic inflammatory response syndrome (SIRS) induced by lipopolysaccharide. Swiss mice received a single oral treatment with cinnamaldehyde 1 h before LPS injection. To investigate whether cinnamaldehyde effects are dependent on TRPA1 activation, animals were treated subcutaneously with the selective TRPA1 antagonist HC-030031 5 min prior to cinnamaldehyde administration. Vehicle-treated mice were used as controls. Cinnamaldehyde ameliorated SIRS severity in LPS-injected animals. Diminished numbers of circulating mononuclear cells and increased numbers of peritoneal mononuclear and polymorphonuclear cell numbers were also observed. Cinnamaldehyde augmented the number of peritoneal Ly6C(high) and Ly6C(low) monocyte/macrophage cells in LPS-injected mice. Reduced levels of nitric oxide, plasma TNFα and plasma and peritoneal IL-10 were also detected. Additionally, IL-1ß levels were increased in the same animals. TRPA1 antagonism by HC-030031 reversed the changes in the number of circulating and peritoneal leukocytes in cinnamaldehyde-treated animals, whilst increasing the levels of peritoneal IL-10 and reducing peritoneal IL-1ß. Overall, cinnamaldehyde modulates SIRS through TRPA1-dependent and independent mechanisms.

Fructans, ascorbate peroxidase, and hydrogen peroxide in ryegrass exposed to ozone under contrasting meteorological conditions.

Ozone (O3) is the most abundant tropospheric oxidant as well as an important component of photochemical pollution. Once inside the plant, ozone can produce reactive oxygen species that change the antioxidative pool and the carbohydrate metabolism. The current study aimed to analyze whether the contents and the composition of the fructan, the ascorbate peroxidase activity, and the H2O2 accumulation were changed in Lolium multiflorum ssp. italicum cv. Lema plants as response to short-term exposure to ozone and/or to different meteorological conditions, in two contrasting seasons (winter and summer). Results showed that higher solar radiation tends to decrease fructose content and, along with temperature, increases the ascorbate peroxidase (APX) activity. Such activity and levels of fructans practically did not vary during the time the experiment was being done, but APX daylight variation was modified by the ozone. Thus, the higher levels of this pollutant decreased the APX activity and increased fructose content, as well as changed the size of the fructan chains. Hydrogen peroxide (H2O2) accumulation was higher in plants that were fumigated with ozone when compared to the control, and it decreased throughout the day. As a conclusion, fructan contents increased when the APX activity decreased. It suggested that fructans could also help the defense system when there is a reduction on the APX activity in the plant.

Resistance training suppresses intra-abdominal fatty acid synthesis in ovariectomized rats.

Ovarian hormone loss is associated with a shift in fat distribution to intra-abdomin al adipose tissue (intra-AAT) depots and with lipid metabolism disorders, which predisposes individuals to developing insulin resistance. Resistance training (RT) prevents increases in intra-AAT after ovarian hormone loss. However, the molecular mechanisms underlying these changes remain unclear. We investigated the effects of ovariectomy and RT on gene expression related to lipogenesis and fat oxidation in the intra-AAT of ovariectomized rats. Sprague-Dawley rats (n=6/group) were divided into the groups: sham-sedentary, ovariectomized-sedentary, sham-RT and ovariectomized-RT. RT groups performed a 10-week climbing program on a ladder with progressive overload. Intra-AAT was subjected to morphometric and mRNA analysis. Ovariectomized-sedentary group had larger adipocytes and higher expression of peroxisome proliferator-activated receptor-γ (PPAR-γ), sterol regulatory element-binding protein-1c (SREBP-1c), stearoyl-CoA desaturase-1 (SCD-1), acetyl-CoA carboxylase (ACC), hormone-sensitive lipase (HSL) and lower expression of the oxidative carnitinepalmitoyltransferase-I (CPT-1). RT counteracted OVX-induced increases in PPAR-γ and SCD-1 and decreased SREBP-1c. ACC and HSL were downregulated in ovariectomized-RT compared with the ovariectomized-sedentary group. Ovariectomized-RT group had the highest CPT-1 gene expression. Adipocyte size decreased in ovariectomized-RT group. Results suggest that RT reduces intra-AAT adipocyte size in ovariectomized rats by suppressing intra-AAT fatty acid synthesis and enhancing fatty acid ß-oxidation.

Assessment of the ozone tolerance of two soybean cultivars (Glycine max cv. Sambaíba and Tracajá) cultivated in Amazonian areas.

Brazilian soybean cultivars (Glycine max Sambaíba and Tracajá) routinely grown in Amazonian areas were exposed to filtered air (FA) and filtered air enriched with ozone (40 and 80 ppb, 6 h/day for 5 days) to assess their level of tolerance to this pollutant by measuring changes in key biochemical, physiological, and morphological indicators of injury and in enzymatic and non-enzymatic antioxidants. Sambaíba plants were more sensitive to ozone than Tracajá plants, as revealed by comparing indicator injury responses and antioxidant stimulations. Sambaíba exhibited higher visible leaf injury, higher stomatal conductance, and a severe decrease in the carbon assimilation rate. Higher ozone level (80 ppb) caused an increase in cell death in both cultivars. Levels of malondialdehyde and hydrogen peroxide also increased in Tracajá exposed under 80 ppb. Sambaíba plants exhibited decreases in ascorbate and glutathione levels and in enzymatic activities associated with these antioxidants. The higher tolerance of the Tracajá soybean appeared to be indicated by reduced physiological injuries and lower stomatal conductance, which might decrease the influx of ozone and enhance oxidation-reduction reactions involving catalase, ascorbate peroxidase, ascorbate, and glutathione, most likely stimulated by higher hydrogen peroxide.

The first systematic analysis of 3D rapid prototyped poly(ε-caprolactone) scaffolds manufactured through BioCell printing: the effect of pore size and geometry on compressive mechanical behaviour and in vitro hMSC viability.

Novel additive manufacturing processes are increasingly recognized as ideal techniques to produce 3D biodegradable structures with optimal pore size and spatial distribution, providing an adequate mechanical support for tissue regeneration while shaping in-growing tissues. With regard to the mechanical and biological performances of 3D scaffolds, pore size and geometry play a crucial role. In this study, a novel integrated automated system for the production and in vitro culture of 3D constructs, known as BioCell Printing, was used only to manufacture poly(ε-caprolactone) scaffolds for tissue engineering; the influence of pore size and shape on their mechanical and biological performances was investigated. Imposing a single lay-down pattern of 0°/90° and varying the filament distance, it was possible to produce scaffolds with square interconnected pores with channel sizes falling in the range of 245-433 µm, porosity 49-57% and a constant road width. Three different lay-down patterns were also adopted (0°/90°, 0°/60/120° and 0°/45°/90°/135°), thus resulting in scaffolds with quadrangular, triangular and complex internal geometries, respectively. Mechanical compression tests revealed a decrease of scaffold stiffness with the increasing porosity and number of deposition angles (from 0°/90° to 0°/45°/90°/135°). Results from biological analysis, carried out using human mesenchymal stem cells, suggest a strong influence of pore size and geometry on cell viability. On the other hand, after 21 days of in vitro static culture, it was not possible to detect any significant variation in terms of cell morphology promoted by scaffold topology. As a first systematic analysis, the obtained results clearly demonstrate the potential of the BioCell Printing process to produce 3D scaffolds with reproducible well organized architectures and tailored mechanical properties.

Improved osteoblast cell affinity on plasma-modified 3-D extruded PCL scaffolds.

Cellular adhesion and proliferation inside three-dimensional synthetic scaffolds represent a major challenge in tissue engineering. Besides the surface chemistry of the polymers, it is well recognized that scaffold internal architecture, namely pore size/shape and interconnectivity, has a strong effect on the biological response of cells. This study reports for the first time how polycaprolactone (PCL) scaffolds with controlled micro-architecture can be effectively produced via bioextrusion and used to enhance the penetration of plasma deposited species. Low-pressure nitrogen-based coatings were employed to augment cell adhesion and proliferation without altering the mechanical properties of the structures. X-ray photoelectron spectroscopy carried out on different sections of the scaffolds indicates a uniform distribution of nitrogen-containing groups throughout the entire porous structure. In vitro biological assays confirm that plasma deposition sensitively promotes the activity of Saos-2 osteoblast cells, leading to a homogeneous colonization of the PCL scaffolds.

Seasonal characterization of antioxidant responses in plants of Ipomoea nil cv. Scarlet O'Hara / Caracterização sazonal de respostas antioxidativas em plantas de Ipomoea nil cv. Scarlet O'Hara

Reactive oxygen species can be produced in leaf cells during normal aerobic metabolism or in a variety of exogenous factors, which may cause oxidative damage to plants, unless they have an efficient antioxidant defense system, consisting of enzymatic and non-enzymatic substances. This work raised the hypothesis that plants of Ipomoea nil cv. Scarlet O'Hara, a native species and ornamental vine of the tropics, might tolerate oxidative stress factors imposed by natural fluctuations in weather conditions through changes in the antioxidant profile.The objective of this study was to determine the variations in three leaf antioxidants in plants growing inside a greenhouse without air pollutants and exposed to varying meteorological conditions throughout the four seasons of the year and to observe if such variations are related to the oscillations in meteorological factors. Four experimental campaigns were carried out, one in each season of 2006. Each campaign lasted 28 days and started with 45 plants. Ascorbic acid (AA) concentrations and superoxide dismutase (SOD) and peroxidase (POD) activities were determined in leaves of five plants in nine sampling days of each campaign. The antioxidant responses oscillated throughout the year. The highest values were found during the spring. This seasonal antioxidant profile was associated to variations in temperature, relative humidity and global radiation. Plants of this cultivar may then tolerate oxidative stress naturally imposed by meteorological conditions.

As espécies reativas de oxigênio podem ser produzidas em células de folhas durante o metabolismo aeróbico normal ou o sob uma diversidade de fatores exógenos que, por sua vez, podem causar danos oxidativos às plantas, a menos que estas tenham um eficiente sistema de defesa antioxidativo, formado por substâncias enzimáticas e não enzimáticas. Neste trabalho, levantou-se a hipótese de que as plantas de Ipomoea nil cv. Scarlet O'Hara, uma espécie trepadeira ornamental e nativa dos trópicos, podem tolerar fatores de estresse oxidativo imposto por oscilações naturais nas condições meteorológicas por meio de mudanças no perfil antioxidativo. Assim, este trabalho objetivou determinar as variações em três espécies antioxidantes foliares em plantas crescidas em casa de vegetação sob ar filtrado e expostas a condições meteorológicas variáveis ao longo das quatro estações do ano de 2006, bem como verificar se tais variações estão relacionadas às oscilações de fatores meteorológicos. Para tanto, realizaram-se quatro campanhas experimentais. Cada campanha durou 28 dias e começou com 45 plantas. Concentrações de ácido ascórbico (AA) e as atividades de superóxido dismutase (SOD) e peroxidase (POD) foram determinadas em folhas de cinco plantas distintas e retiradas da casa de vegetação em nove dias de amostragem de cada campanha. As respostas antioxidativas oscilaram durante todo o ano, sendo os maiores valores encontrados durante a primavera. Este perfil sazonal de antioxidantes foi associado às variações de temperatura, umidade relativa e radiação global. As plantas desta cultivar podem, então, tolerar o estresse oxidativo naturalmente imposto pelas condições meteorológicas.

Seasonal characterization of antioxidant responses in plants of Ipomoea nil cv. Scarlet O'Hara.

Reactive oxygen species can be produced in leaf cells during normal aerobic metabolism or in a variety of exogenous factors, which may cause oxidative damage to plants, unless they have an efficient antioxidant defense system, consisting of enzymatic and non-enzymatic substances. This work raised the hypothesis that plants of Ipomoea nil cv. Scarlet O'Hara, a native species and ornamental vine of the tropics, might tolerate oxidative stress factors imposed by natural fluctuations in weather conditions through changes in the antioxidant profile.The objective of this study was to determine the variations in three leaf antioxidants in plants growing inside a greenhouse without air pollutants and exposed to varying meteorological conditions throughout the four seasons of the year and to observe if such variations are related to the oscillations in meteorological factors. Four experimental campaigns were carried out, one in each season of 2006. Each campaign lasted 28 days and started with 45 plants. Ascorbic acid (AA) concentrations and superoxide dismutase (SOD) and peroxidase (POD) activities were determined in leaves of five plants in nine sampling days of each campaign. The antioxidant responses oscillated throughout the year. The highest values were found during the spring. This seasonal antioxidant profile was associated to variations in temperature, relative humidity and global radiation. Plants of this cultivar may then tolerate oxidative stress naturally imposed by meteorological conditions.

MIRU-VNTR typing adds discriminatory value to groups of Mycobacterium bovis and Mycobacterium caprae strains defined by spoligotyping.

The value of Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeats (MIRU-VNTR) as a genotyping technique for Mycobacterium bovis and Mycobacterium caprae, has been confirmed in different countries and epidemiological scenarios. However, a standardized panel of loci has not yet been adopted for these species, since allelic diversity of each locus differs among countries. To determine the most discriminatory loci, a panel of 181 M. bovis and M. caprae strains representing 12 spoligotypes was created. The panel included strains from the three predominant spoligotypes previously isolated in Portugal; strains from spoligotyping group SB0140, prevalent in the British Isles but also detected in Portugal; strains from spoligotypes common to cattle and wildlife species and strains from the M. caprae spoligotyping group SB0157. MIRU-VNTR analysis of these strains, targeting 8 selected loci, produced 87 different profiles (h=0.99), being VNTR3232, QUB11a, ETR-B and ETR-A the most discriminatory loci (h=0.96). A single M. bovis spoligotyping group could be differentiated - up to 44 MIRU-VNTR profiles. These results emphasize the high genotype diversity of Portuguese isolates compared with other countries. MIRU-VNTR typing was superior to spoligotyping for identifying multi-genotype infected herds and the combination of the two genotyping methods by a hierarchical approach confirmed the genetic relatedness of M. bovis isolates between cattle and wildlife.