Enhanced HPLC Method for Boar Taint Quantification.

Boar taint is an unpleasant odour found in the carcasses of entire male pigs, resulting from androstenone and skatole accumulation during pubertal development, and impacting pork quality. This study proposes the validation of an adapted chromatographic method for quantifying skatole and androstenone in the pigs' liquid fat using fluorescence detection. A good chromatographic separation was achieved, with skatole (SKA) and androstenone (AND) elution at 4.4 and 9.9 min., respectively. An external calibration method was applied, with calibration curves correlation coefficient of 0.9999 for both analytes. Detection limit values were 1.53 and 16.02 ng/g for SKA and AND, respectively. SKA recovery was 99.72±2.34 % (2.34 % RSD) and 102.84±1.62 % (1.57 % RSD) for AND. Results showed good precision values (repeatability <2.46 % RSD for SKA, <6.85 % RSD for AND; intermediate precision <2.87 % RSD for SKA, <6.98 % RSD for AND). The method's robustness was tested and the values were within the reference ranges. The validation results proved that the adaptation of an existing method resulted in good assessments of robustness, reliability and accuracy.

Dendronized Ag/Au Nanomats: Antimicrobial Scaffold for Wound Healing Bandages.

Electrospun polymer nanofibers, due to high surface area-to-volume ratio, high porosity, good mechanical strength, and ease of functionalization, appear as promising multifunctional materials for biomedical applications. Thanks to their unidirectional structure, imitating the extracellular matrix (ECM), they can be used as scaffolds for cell adhesion and proliferation. In addition, the incorporation of active groups inside nanofiber can give properties for bactericides. The proposed nanomats incorporate nanoparticles templated within the electrospun nanofibers that prevent infections and stimulate tissue regeneration. The generated hybrid electrospun nanofibers are composed of a copolymer of L-lactide-block-ε-caprolactone (PL-b-CL), 70:30, blended with homopolymer polyvinylpyrrolidone (PVP) and gold (Au) nanoparticles. A low cytotoxicity and slightly increased immunoreactivity, stimulated by the nanomat, are observed. Moreover, the decoration of the hybrid nanomat with dendronized silver nanoparticles (Dend-Ag) improves their antibacterial activity against antibiotic-resistant Pseudomonas aeruginosa. The use of Dend-Ag for decorating offers several functional effects; namely, it enhances the antibacterial properties of the produced nanomats and induces a significant increase within macrophages' cytotoxicity. The unidirectional nanostructures of the generated hybrid nanomats demonstrate unique collective physio-chemical and biological properties suitable for a wide range of biomedical applications. Here, the antibacterial properties facilitate an optimal environment, contributing to accelerated wound healing.

Shell Formulation in Soft Gelatin Capsules: Design and Characterization.

Soft gelatin capsules (SGCs) are the most widely used pharmaceutical form after tablets. The active components, active pharmaceutical ingredients (APIs), or nutrients are dissolved, dispersed, or suspended in a liquid or semisolid fill, which is covered with a gelatin shell. Several factors can modify the properties of the gelatin shell and subsequently affect their operative handling during manufacturing process and the stability of the soft gelatin capsules. Three elements appear to be crucial: the shell formulation (type and content of the different components such as gelatins-source, extraction method-plasticizers, or additives); the manufacture and storage conditions (temperature, humidity, light) as well as the interactions between fill-shell formulas. Mechanical and thermal analysis arise as straightforward but highly useful tools to monitor the properties of the gelatin shell. This review provides an updated overview on the shell formulation and design. Additionally, it presents the uses of mechanical and thermal techniques to characterize and evaluate the impact of different parameters on the gelatin behavior over the production and stability of these pharmaceutical forms. This will help to detect changes that are yet not visible by visual inspection ensuring a suitable finished product over its shelf-life.

Potential anti-adhesion activity of novel carbosilane zwitterionic dendrimers against eukaryotic and prokaryotic pathogenic microorganisms.

The development of biofilms on different surfaces continues to be a major public health problem. The antimicrobial resistance and the difficulty of finding drugs capable of combating these established biofilms generates the urgent need to find compounds that prevent cells from settling and establishing of these complex communities of microorganisms. Zwitterionic modification of nanomaterials allows the formation of a hydration layer, and this highly hydrophilic surface provides antifouling properties as well as a good biocompatibility by preventing non-specific interactions. Thus, they are appropriate candidates to prevent microbial adhesion to different surfaces and, in consequence, avoid biofilm formation. For this reason, we have incorporated zwitterionic moieties in multivalent systems, as are carbosilane dendrimers. Characterization of these systems was performed using nuclear magnetic resonance and mass spectrometry. It has been analysed if the new molecules have capacity to inhibit the biofilm formation in Candida albicans, Staphylococcus aureus and Pseudomonas aeruginosa. The results showed that they were more effective against S. aureus, observing a biofilm reduction of 81.5% treating with 32 mg/L of G2SiZWsf dendrimer and by 72.5% using 32 mg/L of the G3SiZWsf dendrimer. Finally, the absence of cytotoxicity was verified by haemolysis and cytotoxicity studies in human cells lines.

Carbosilane ruthenium metallodendrimer as alternative anti-cancer drug carrier in triple negative breast cancer mouse model: A preliminary study.

The carbosilane metallodendrimer G1-[[NCPh(o-N)Ru(η6- p-cymene)Cl]Cl]4 (CRD13), based on an arene Ru(II) complex coordinated to imino-pyridine surface groups, has been conjugated with anti-cancer drugs. Ruthenium in the positively-charged dendrimer structure allows this nanoparticle to be considered as an anticancer drug carrier, made more efficient because ruthenium has anticancer properties. The ability of CRD13 to form complexes with Doxorubicin (DOX), 5-Fluorouracil (5-Fu), and Methotrexate (MTX) has been evaluated using zeta potential measurement, transmission electron microscopy (TEM) and computer simulation. The results show that it forms stable nanocomplexes with all those drugs, enhancing their effectiveness against MDA-MB-231 cancer cells. In vivo tests indicate that the CRD13/DOX system caused a decrease of tumor weight in mice with triple negative breast cancer. However, the tumors were most visibly reduced when naked dendrimers were injected.

Mechanistic insight of lysozyme transport through the outer bacteria membrane with dendronized silver nanoparticles for peptidoglycan degradation.

Drug resistance has become a global problem, prompting the entire scientific world to seek alternative methods of dealing with resistant pathogens. Among the many alternatives to antibiotics, two appear to be the most promising: membrane permeabilizers and enzymes that destroy bacterial cell walls. Therefore, in this study, we provide insight into the mechanism of lysozyme transport strategies using two types of carbosilane dendronized silver nanoparticles (DendAgNPs), non-polyethylene glycol (PEG)-modified (DendAgNPs) and PEGylated (PEG-DendAgNPs), for outer membrane permeabilization and peptidoglycan degradation. Remarkably, studies have shown that DendAgNPs can build up on the surface of a bacterial cell, destroying the outer membrane, and thereby allowing lysozymes to penetrate inside the bacteria and destroy the cell wall. PEG-DendAgNPs, on the other hand, have a completely different mechanism of action. PEG chains containing a complex lysozyme resulted in bacterial aggregation and an increase in the local enzyme concentration near the bacterial membrane, thereby inhibiting bacterial growth. This is due to the accumulation of the enzyme in one place on the surface of the bacteria and penetration into it through slight damage of the membrane due to interactions of NPs with the membrane. The results of this study will help propel more effective antimicrobial protein nanocarriers.

A Framework for Using Epidemiology in Animal Welfare Science.

The potential advantages of using epidemiology in animal welfare research are substantial and are used with increased frequency. Collaboration between scientists of different fields, with different specific expertise is advantageous in the advancement of science. In this review, a framework to use epidemiology in animal welfare science is established. The different epidemiological study designs and analytical procedures are explored and put in an animal welfare scientific context. It is argued and demonstrated that epidemiology is used with advantage: in the identification of risk factors behind the development of maladaptation and abnormal behaviors; in the introduction of standardized procedures in research allowing comparisons between studies and facilitating the integration for evidence synthesis in systematic reviews and meta-analysis; by allowing animal welfare scientists to analyze complex settings such as farms or zoos. Mathematical modeling can also be used with advantage in risk assessment.

Immobilization of Alcalase on Silica Supports Modified with Carbosilane and PAMAM Dendrimers.

Enzyme immobilization is a powerful strategy for enzyme stabilization and recyclability. Materials covered with multipoint molecules are very attractive for this goal, since the number of active moieties to attach the enzyme increases with respect to monofunctional linkers. This work evaluates different dendrimers supported on silica to immobilize a protease enzyme, Alcalase. Five different dendrimers were employed: two carbosilane (CBS) dendrimers of different generations (SiO2-G0Si-NH2 and SiO2-G1Si-NH2), a CBS dendrimer with a polyphenoxo core (SiO2-G1O3-NH2), and two commercial polyamidoamine (PAMAM) dendrimers of different generations (SiO2-G0PAMAM-NH2 and SiO2-G1PAMAM-NH2). The results were compared with a silica support modified with a monofunctional molecule (2-aminoethanethiol). The effect of the dendrimer generation, the immobilization conditions (immobilization time, Alcalase/SiO2 ratio, and presence of Ca2+ ions), and the digestion conditions (temperature, time, amount of support, and stirring speed) on Alcalase activity has been evaluated. Enzyme immobilization and its activity were highly affected by the kind of dendrimer and its generation, observing the most favorable behavior with SiO2-G0PAMAM-NH2. The enzyme immobilized on this support was used in two consecutive digestions and, unlike CBS supports, it did not retain peptides released in the digestion.

Local People Standings on Existing Farm Animal Welfare Legislation in the BRIC Countries and the USA. Comparison with Western European Legislation.

This study explored the demand for improved farm animal welfare (FAW) legislation in the BRIC countries and the USA. Results are discussed in comparison to Europe. Interviewees ranked their willingness to support or oppose introduction of more FAW-friendly laws in their country. A multinomial logistic regression was fit to the data (p < 0.001), with the parameters "country × gender" (p < 0.001) and "country × age" (p < 0.001) found significant. Americans, Russian women, and older Brazilian men are very supportive. The age effect is also felt in India, where older people are more supportive. Chinese, American men, and younger Indians are less supportive. Russian males are the group that oppose the most, followed by younger Brazilians and Indians. The law and its application vary a lot between countries. Nevertheless, the societal willingness to improve FAW legislation is high in all countries. The willingness is higher in Europe. The different cultural backgrounds, the socio-economic factors, and the social, economic, and environmental sustainability are enough reasons to create barriers to policy harmonization in the global trade of farm animal products.

The effect of surface modification of dendronized gold nanoparticles on activation and release of pyroptosis-inducing pro-inflammatory cytokines in presence of bacterial lipopolysaccharide in monocytes.

Biomedical applications of gold nanoparticles (AuNPs) may be limited by their toxicological effects. Although surface-modified AuNPs can induce apoptosis, less is known about whether they can induce other types of cell death. Pyroptosis, an inflammatory type of programmed cell death, can be induced in immune cells, especially macrophages, by bacterial endotoxins. Therefore, in this study, dendronized AuNPs were combined with bacterial lipopolysaccharides (LPSs) as the main stimulators of pro-inflammatory responses to test the induction of pyroptosis in THP-1 myeloid cell line. These AuNPs induced caspase-1 activity (3-4 times more compared to control) and enhanced the release of interleukin (IL)-18 and IL-1ß without inducing gasdermin D cleavage and related pore formation. The production of pro-inflammatory cytokines occurred mainly visible during LPS treatment, although their secretion was observed only after administration of dendronized AuNPs (release of IL-1ß to supernatant up to 80 pg/mL). These findings suggest that dendronized AuNPs can induce pyroptosis-like inflammatory mechanisms and that these mechanisms are enhanced in the presence of bacterial LPS. The intensity of this effect was dependent on AuNP surface modification. These results shed new light on the cytotoxicity of metal NPs, including immune responses, indicating that surface modifications play crucial roles in their nanotoxicological effects.

Bacteria capture with magnetic nanoparticles modified with cationic carbosilane dendritic systems.

Bacteria elimination from water sources is key to obtain drinkable water. Hence, the design of systems with ability to interact with bacteria and remove them from water is an attractive proposal. A diversity of polycationic macromolecules has shown bactericide properties, due to interactions with bacteria membranes. In this work, we have grafted cationic carbosilane (CBS) dendrons and dendrimers on the surface of iron oxide magnetic nanoparticles (MNP), leading to NP (ca. 10 nm) that interact with bacteria by covering bacteria membrane. Application of an external magnetic field removes MNP from solution sweeping bacteria attached to them. The interaction of the MNP with Gram-positive S. aureus bacteria is more sensible to the size of dendritic system covering the MNP, whereas interaction with Gram-negative E. coli bacteria is more sensible to the density of cationic groups. Over 500 ppm of NPM, MNP covered with dendrons captured over 90% of both type of bacteria, whereas MNP covered with dendrimers were only able to capture S. aureus bacteria (over 90%) but not E. coli bacteria. Modified MNP were characterized by transmission electron microscopy (TEM), thermogravimetric analysis (TGA), Fourier-transform infrared spectroscopy (FTIR), Z potential and dynamic light scattering (DLS). Interaction with bacteria was analyzed by UV, TEM and scanning electron microscopy (SEM). Moreover, the possibility to recycle cationic dendronized MNP was explored.

Heterofunctional carbosilane polyphenolic dendrons: new antioxidants platforms.

Reactive oxygen species (ROS) play a critical role in different human pathophysiological processes. ROS, together with nitrogen reactive species, generated as by-products of cellular metabolism or external factors, affects intracellular redox homeostasis. Redox-active groups found in proteins and other compounds such as polyphenols are involved in maintaining intracellular redox homeostasis. In this work, a new family of heterofunctional first-generation carbosilane dendrons functionalised with different polyphenols at the focal point and dimethylammonium groups at the periphery has been obtained through two synthetic strategies: reductive amination and straightforward amidation reaction. Their antioxidant activity has been evaluated through two spectrophotometric methods: ferric reducing antioxidant power (FRAP) assay and 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay to establish a correlation between the number of hydroxyl groups and the antioxidant activity.

The Antibacterial Effect of PEGylated Carbosilane Dendrimers on P. aeruginosa Alone and in Combination with Phage-Derived Endolysin.

The search for new microbicide compounds is of an urgent need, especially against difficult-to-eradicate biofilm-forming bacteria. One attractive option is the application of cationic multivalent dendrimers as antibacterials and also as carriers of active molecules. These compounds require an adequate hydrophilic/hydrophobic structural balance to maximize the effect. Herein, we evaluated the antimicrobial activity of cationic carbosilane (CBS) dendrimers unmodified or modified with polyethylene glycol (PEG) units, against planktonic and biofilm-forming P. aeruginosa culture. Our study revealed that the presence of PEG destabilized the hydrophilic/hydrophobic balance but reduced the antibacterial activity measured by microbiological cultivation methods, laser interferometry and fluorescence microscopy. On the other hand, the activity can be improved by the combination of the CBS dendrimers with endolysin, a bacteriophage-encoded peptidoglycan hydrolase. This enzyme applied in the absence of the cationic CBS dendrimers is ineffective against Gram-negative bacteria because of the protective outer membrane shield. However, the endolysin-CBS dendrimer mixture enables the penetration through the membrane and then deterioration of the peptidoglycan layer, providing a synergic antimicrobial effect.

Organometallic dendrimers based on Ruthenium(II) N-heterocyclic carbenes and their implication as delivery systems of anticancer small interfering RNA.

With the purpose of obtaining a new dendritic system against cancer, this paper is focused on the synthesis of spherical carbosilane metallodendrimers of different generations holding Ru(II) N-heterocyclic carbene (NHC) on the periphery from the imidazolium precursors. Both imidazolium salt dendrimers and their metallodendrimers counterparts showed promising anticancer activity, similar to cisplatin, mainly at high generations. In addition, both families of second and third generations were able to form dendriplexes with anticancer small interfering RNA (siRNA), protecting the cargo against RNAse and being able to internalize it in HEPG2 (human liver cancer) tumour cells. The characterization and effectiveness of the dendriplexes were evaluated by various analytical techniques such as zeta potential, electrophoresis and circular dichroism, the stability of the system and the protective nature of the dendrimer estimated using RNAse and the internalization of dendriplexes by confocal microscopy. The major advantage observed with the ruthenium metallodendrimers with respect to the imidazolium salts precursors was in cellular uptake, where the internalization of Mcl-1-FITC siRNA (myeloid cell leukaemia-1 fluorescein labelled siRNA) proceeded more efficiently. Therefore, we propose here that both imidazolium and Ru metallodendrimers are interesting candidates in cancer due to their double action, as anticancer per se and as carrier for anticancer siRNA, providing in this way a combined action.

Insight on the Structure-to-Activity of Carbosilane Metallodendrimers in the Fight against Staphylococcus aureus Biofilms.

Biofilm formation is a critical health concern, involved in most human bacterial infections. Combatting this mechanism, which increases resistance to traditional antibiotics and host immune defences, requires novel therapeutic approaches. The remarkable biocide activity and the monodispersity of carbosilane metallodendrimers make them excellent platforms to evaluate the impact of different structural parameters on the biological activity. In this work, we explore the influence of iminopyridine ring substituents on the antibacterial activity against planktonic and biofilm Staphylococcus aureus. New families of first-generation Ru(II) and Cu(II) metallodendrimers were synthesised and analysed, in comparison to the non-substituted counterparts. The results showed that the presence of methyl or methoxy groups in meta position to the imine bond decreased the overall positive charge on the metal ion and, subsequently, the activity against planktonic bacteria. However, it seemed a relevant parameter to consider for the prevention of biofilm formation, if they contribute to increasing the overall lipophilicity. An optimum balance of the charge and lipophilicity of the metallodrug, accomplished through structural design, will provide effective biocide agents against bacteria biofilms.

Promising PEGylated cationic dendrimers for delivery of miRNAs as a possible therapy against HIV-1 infection.

BACKGROUND: The appearance of resistance against new treatments and the fact that HIV-1 can infect various cell types and develop reservoirs and sanctuaries makes it necessary to develop new therapeutic approaches to overcome those failures. RESULTS: Studies of cytotoxicity, genotoxicity, complexes formation, stability, resistance, release and particle size distribution confirmed that G2-SN15-PEG, G3-SN31-PEG, G2-SN15-PEG-FITC and G3-SN31-PEG-FITC dendrimers can form complexes with miRNAs being biocompatible, stable and conferring protection to these nucleic acids. Confocal microscopy and flow cytometry showed effective delivery of these four dendrimers into the target cells, confirming their applicability as delivery systems. Dendriplexes formed with the dendrimers and miRNAs significantly inhibited HIV-1 infection in PBMCs. CONCLUSIONS: These dendrimers are efficient delivery systems for miRNAs and they specifically and significantly improved the anti-R5-HIV-1 activity of these RNA molecules.

Comparison of the effects of dendrimer, micelle and silver nanoparticles on phospholipase A2 structure.

The interaction of nanoparticles (NP) with proteins (the so-called 'protein corona') is a huge challenge in attempting to apply them in personalized nanomedicine. We have analyzed the interaction between A) two 'soft' NPs (a cationic phosphorus dendrimer of generation 3; a cationic phosphorus amphiphilic dendron of generation 2), and B) one 'hard' nanoparticle (silver NP covered with cationic carbosilane dendritic moieties); and membrane-bound protein phospholipase A2 from bovine pancreas. The hard and soft NPs have differences in the nature of their interactions with phospholipase A2. This enzyme surrounds hard AgNP, whereas dendrimer and amphiphilic dendron form aggregates/micelles with phospholipase A2. There is a difference in action of phospholipase A2 bound to the core of dendrimer, and of micelles formed from non-covalent interactions between the amphiphilic dendron. These data are important in understanding the nature of interaction between different kinds of nanoparticles and proteins.

Elaborated study of Cu(II) carbosilane metallodendrimers bearing substituted iminopyridine moieties as antitumor agents.

Iminopyridine-decorated carbosilane metallodendrimers have recently emerged as a promising strategy in the treatment of cancer diseases. Their unique features such as the nanometric size, the multivalent nature and the structural perfection offer an extraordinary platform to explore structure-to-property relationships. Herein, we showcase the outstanding impact on the antitumor activity of a parameter not explored before: the iminopyridine substituents in meta position. New Cu(II) carbosilane metallodendrimers, bearing methyl or methoxy substituents in the pyridine ring, were synthesized and thoroughly characterized. Electron Paramagnetic Resonance (EPR) was exploited to unveil the properties of the metallodendrimers. This study confirmed the presence of different coordination modes of the Cu(II) ion (Cu-N2O2, Cu-N4 and Cu-O4), whose ratios were determined by the structural features of the dendritic molecules. These metallodendrimers exhibited IC50 values in the low micromolar range (<6 µM) in tumor cell lines such as HeLa and MCF-7. The subsequent in vitro assays on both healthy (PBMC) and tumor (U937) myeloid cells revealed two key facts which improved the cytotoxicity and selectivity of the metallodrug: First, maximizing the Cu-N2O2 coordination mode; second, adequately selecting the pair ring-substituent/metal-counterion. The most promising candidates, G1(-CH3)Cl (8) and G1(-OCH3)NO3(17), exhibited a substantial increase in the antitumor activity in U937 tumor cells, compared to the non-substituted counterparts, probably through two different ROS-production pathways.

Immune cell arrival kinetics to peritoneum and role during murine-experimental trichomoniasis.

Trichomonas vaginalis causes trichomoniasis, an inflammatory process related to an increased rate of HIV transmission. In order to study T. vaginalis infection response in a microorganism-free environment, an infection model was established providing a host­parasite interaction system useful to study the interplay between immune cells and the parasite. Infected mice peritoneal cells were immunophenotyped at different times after infection using flow cytometry. Neutrophils and macrophages showed the most relevant increase from third to 12th day post-infection. A high number of B lymphocytes were present on 15th day post-infection, and an increase in memory T cells was observed on sixth day post-infection. The levels of NO increased at day 10 post-infection; no significant influence was observed on T. vaginalis clearance. Increased viability of T. vaginalis was observed when the NETs inhibitors, metformin and Cl− amidine, were administrated, highlighting the importance of this mechanism to control parasite infection (43 and 86%, respectively). This report presents a comprehensive cell count of the immune cells participating against trichomoniasis in an in vivo interaction system. These data highlight the relevance of innate mechanisms such as specific population changes of innate immune cells and their impact on the T. vaginalis viability.

Immobilization of thermolysin enzyme on dendronized silica supports. Evaluation of its feasibility on multiple protein hydrolysis cycles.

This work evaluates different dendrimer-silica supports for the immobilization of enzymes by multipoint covalent binding. Thermolysin was immobilized on two dendrimers (PAMAM and carbosilane) with two different generations (zero (G0) and first (G1)). Results were compared with a control, a silica support functionalized with a monofunctional molecule. Dendrimers increased the number of available sites to bind the enzyme. Despite the enzyme was immobilized on all supports, G0 dendrimers immobilized a 30% more enzyme than G1. Thermolysin immobilized on G0 dendrimer supports showed the highest activity and could be employed in three consecutive hydrolysis cycles. Optimal immobilization time was 1 h while optimal protein loading was 25 mg enzyme/100 mg support. Enzyme activity was promoted when using 5 mg of immobilized enzyme at 750 rpm, 60 °C, and 2 h of hydrolysis. Under these conditions, the activity of thermolysin increased up to the 78% of the free enzyme activity. Kinetics of the hydrolysis reaction using the immobilized thermolysin was also studied and compared with the obtained using the free thermolysin. The addition of ZnCl2 and NaCl during the immobilization procedure increased thermolysin activity in the second (22% more) and in the third (14% more) hydrolysis clycles.