Speciation analysis of mercury in water samples by dispersive liquid-liquid microextraction coupled to capillary electrophoresis

In this study, a method of pretreatment and speciation analysis of mercury by dispersive liquid-liquid microextraction along with CE was developed. The method was based on the fact that mercury species including methylmercury (MeHg), ethylmercury (EtHg), phenylmercury (PhHg), and Hg(II) were complexed with 1-(2-pyridylazo)-2-naphthol to form hydrophobic chelates and l-cysteine could displace 1-(2-pyridylazo)-2-naphthol to form hydrophilic chelates with the four mercury species. Factors affecting complex formation and extraction efficiency, such as pH value, type, and volume of extractive solvent and disperser solvent, concentration of the chelating agent, ultrasonic time, and buffer solution were investigated. Under the optimal conditions, the enrichment factors were 102, 118, 547, and 46, and the LODs were 1.79, 1.62, 0.23, and 1.50 g/L for MeHg, EtHg, PhHg, and Hg(II), respectively. Method precisions (RSD, n = 5) were in the range of 0.29-0.54% for migration time, and 3.08-7.80% for peak area. Satisfactory recoveries ranging from 82.38 to 98.76% were obtained with seawater, lake, and tap water samples spiked at three concentration levels, respectively, with RSD (n = 5) of 1.98-7.18%. This method was demonstrated to be simple, convenient, rapid, cost-effective, and environmentally benign, and could be used as an ideal alternative to existing methods for analyzing trace residues of mercury species in water samples.In this study, a method of pretreatment and speciation analysis of mercury by dispersive liquid-liquid microextraction along with CE was developed. The method was based on the fact that mercury species including methylmercury (MeHg), ethylmercury (EtHg), phenylmercury (PhHg), and Hg(II) were complexed with 1-(2-pyridylazo)-2-naphthol to form hydrophobic chelates and l-cysteine could displace 1-(2-pyridylazo)-2-naphthol to form hydrophilic chelates with the four mercury species. Factors affecting complex formation and extraction efficiency, such as pH value, type, and volume of extractive solvent and disperser solvent, concentration of the chelating agent, ultrasonic time, and buffer solution were investigated. Under the optimal conditions, the enrichment factors were 102, 118, 547, and 46, and the LODs were 1.79, 1.62, 0.23, and 1.50 g/L for MeHg, EtHg, PhHg, and Hg(II), respectively. Method precisions (RSD, n = 5) were in the range of 0.29-0.54% for migration time, and 3.08-7.80% for peak area. Satisfactory recoveries ranging from 82.38 to 98.76% were obtained with seawater, lake, and tap water samples spiked at three concentration levels, respectively, with RSD (n = 5) of 1.98-7.18%. This method was demonstrated to be simple, convenient, rapid, cost-effective, and environmentally benign, and could be used as an ideal alternative to existing methods for analyzing trace residues of mercury species in water samples

Porous hydrogels from shark skin collagen crosslinked under dense carbon dioxide atmosphere

The possibility to fabricate marine collagen porous structures crosslinked with genipin under high pressure carbon dioxide is investigated. Collagen from shark skin is used to prepare prescaffolds by freeze-drying. The poor stability of the structures and low mechanical properties require crosslinking of the structures. Under dense CO2 atmosphere, crosslinking of collagen pre-scaffolds is allowed for 16 h. Additionally, the hydrogels are foamed and the scaffolds obtained present a highly porous structure. In vitro cell culture tests performed with a chondrocyte-like cell line show good cell adherence and proliferation, which is a strong indication of the potential of these scaffolds to be used in tissue cartilage tissue engineering.The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no. KBBE-2010-266033 (project SPECIAL) and from FEDER through POCTEP Project 0330_IBEROMARE_1_P. Portuguese Foundation for Science and Technology is also gratefully acknowledged for post-doc grants of J. Moreira-Silva and T.H. Silva

Silk-Fibroin/Methacrylated Gellan Gum Hydrogel as an novel scaffold for application in meniscus cell-based tissue engineering

Introduction: Knee meniscus injury is highly prevalent and there is a demand for new cost-effective treatment solutions. Tissue engineering (TE) and regenerative medicine strategies using acellular scaffolds are being used in clinical application for total or partial meniscus replacement [1]. Although this strategy has been considered as a safe and promising approach, progressive volume reduction of the implant and early failure have been described. Advances in the field of meniscus TE are required and greatly depend on increased knowledge of meniscus biology, improvement of biomaterials and cell-based therapies [2]. Advanced scaffolds for meniscus TE should possess adequate mechanics, biodegradability and biocompatibility, and also be able to mimic and preserve the asymmetric vascular network of this complex tissue, i.e. enable controlling the segmental vascularization during the regeneration process. Silk fibroin scaffolds derived from Bombyx mori cocoon have been recognized as a versatile biomaterial for application in meniscus TE [3]. The purpose of this study is to: 1) contribute to the knowledge of meniscus aiming future clinical applications (namely, the aspects dealing with the characterization of cellular phenotypes and density, biomechanics and extracellular matrix composition) and 2) to develop an alternative and viable silk fibroin scaffold possessing adequate properties for either use in acellular or cellular approaches for partial and/or total meniscus replacement, and combine it with the methacrylated gellan gum hydrogel (iGG-MA) hydrogel, which is able to prevent the ingrowth of endothelial cells and blood vessels into the hydrogels [4,5]. Patients & Methods: Morphologically intact menisci were collected from 44 human donors (12 male, 32 female). All menisci (30 lateral and 14 medial) were divided into anterior, middle and posterior segments prior to mechanical, biological or histological characterization. Human meniscus cells (HMC´s) were isolated using an enzymatic standard protocol. HMC´s phenotype was characterized by flow cytometry analysis. Haematoxylin and eosin (H&E), safranin-O and collagen I staining were performed for segmental characterization of the extracellular matrix. For the evaluation of the viscoelastic properties, dynamic mechanical analysis (DMA) was performed using fresh tissue samples. The three segments of menisci were cut in cylindrical shapes with 4 mm diameter and analyzed at 37ºC in PBS (pH 7.4). The microstructure of freeze-dried meniscus was investigated by micro-computed tomography (micro-CT) analysis. Silk-based scaffolds (10 and 12 wt%) were produced by means of combining salt leaching and freeze-drying methods [3], in order to match human tissue biological and biomechanical features. HMC’s were seeded onto the different silk scaffolds at a cell density of 5x104 cells/disc. Then, the cell-laden scaffolds were cultured in static conditions, for times of culturing up to 21 days. After specific times of culturing (from 1 day up to 21 days), HMC´s adhesion, viability and proliferation were investigated by scanning electron microscopy (SEM), calcein-AM assay and DNA quantification tests, respectively. In addition, the mechanical properties of the cell-loaded scaffolds were evaluated by DMA. The HMC’s-laden hydrogel/silk scaffolds were produced by encapsulating the HMC’s into a 2 wt% iGG-MA hydrogel, followed by impregnation onto the 12 wt% silk scaffold. A chorioallantoic membrane (CAM) assay was used to investigate in vivo the control of segmental vascularization of the acellular and cell-laden hydrogel/silk scaffolds by the effect of iGG-MA hydrogel, until day 14 of embryonic development. Results & Discussion: The biological characterization of this meniscus tissue, although not yet completely accomplished, has evolved significantly in the last few years. In this work, DMA analysis has shown that medial meniscus has significantly higher stiffness (E' and Tan d) than lateral meniscus. There is also significant regional variation form anterior to posterior menisci segments regarding biomechanical features. Age, gender and bone mass index (BMI) also influences meniscus stiffness. The FACS analysis revealed that cells isolated from the human meniscus are a mixed population of cells, i.e. fibrochondrocyte-like and MSCs (cells are positive for CD105, CD73 and CD90, and lack CD34 and CD45). HMC’s maintained their phenotype for 21 days when cultured in tissue culture polystyrene plate (2D). The micro-CT analysis revealed that the human freeze-dried meniscus possessed a mean porosity of 58.0±20.3% and interconectivity of 41.9%±53.7. The mean pore size and trabeculae thickness was 220.7±111.5 µm and 159.7±78.6 µm, respectively. The knowledge arising from the present study allowed us to develop a novel polymeric scaffold made of silk fibroin, which was subsequently characterized without cells and after cell-loading. SEM analysis revealed that the HMC´s adhered to the surface of the scaffolds. The viability assay and DNA quantification showed that HMC´s were viable and proliferated well when cultured onto both silk-10 and silk-12 scaffolds, until 21 days. DMA analysis has shown that the moduli of the acellular scaffolds immersed in culture medium for 14 days were 27.6 ± 7.9 kPa and 61.1 ± 0.4 at 10 Hz, for silk-10 e silk-12, respectively. By its turn, the moduli determined at 10 Hz of the cell-laden scaffolds cultured after 14 days of culturing were 48.2± 19.8 and 140.1 ± 15.6 kPa, for silk-10 and silk-12, respectively. The in vivo study allowed investigating the number of macroscopic blood vessels converging to the implants. The evaluation of possible inflammation and endothelial cells ingrowths was performed by histological (H&E staining) and immunohistochemical methods (SNA-lectin staining). Results have shown that iGG-MA hydrogel prevented the endothelial cells adhesion and blood vessels infiltration into the HMC’s hydrogel/silk scaffolds, after 4 days of implantation, even in the presence of VEGF

Structural and Luminescence Properties of Silica-Based Hybrids Containing New Silylated-Diketonato Europium(III) Complex

A new betadiketonate ligand displaying a trimethoxysilyl group as grafting function and a diketone moiety as complexing site (TTA-Si = 4,4,4-trifluoro-2-(3-trimethoxysilyl)propyl)-1-3-butanedione (C4H3S)COCH[(CH2)3Si(OCH3)3]COCF3) and its highly luminescent europium(III) complex [Eu(TTA-Si)3] have been synthesized and fully characterized. Luminescent silica-based hybrids have been prepared as well with this new complex grafted on the surface of dense silica nanoparticles (28 (+/-3 nm) or on mesoporous silica particles. The covalent bonding of Eu(TTA-Si)3 inside the core of uniform silica nanoparticles (40 (+/- 5 nm) was also achieved. Luminescence properties are discussed in relation to the europium chemical environment involved in each of the three hybrids. The general methodology proposed allowed high grafting ratios and overcame chelate release and tendency to agglomeration, and it could be applied to any silica matrix (in the core or at the surface, nanosized or not, dense or mesoporous) and therefore numerous applications such as luminescent markers and luminophors could be foreseen

Load Shifting and Enhancing Energy Savings with Dynamic Ventilation Strategies in Multi-Family Residential Buildings

There is an increasing focus on the time at which energy is used in buildings both to reduce utility costs and carbon emissions in response to time-dependent grid signals. One method to shift electrical load out of peak pricing hours is to use batteries, but they have high first costs and also incur an energy penalty due to round trip efficiency and other losses. Another method is to use thermal storage to offset heating and cooling. Similarly, mechanical ventilation systems can also be controlled to shift energy use to periods of the day with lower energy, cost, and environmental impacts by varying the ventilation rate while still meeting ventilation code requirements. Mechanical ventilation systems in large multi-family residential buildings are mostly central air systems with either manually balanced dampers or constant airflow regulator (CAR) dampers that aim to provide a constant ventilation airflow rate to each apartment. ASHRAE 62.2 allows for dynamic ventilation rate systems in these buildings as long as the average relative exposure rate and the peak relative exposure rate during occupied periods are no more than 1 and 5, respectively, for any time interval that cannot exceed an hour. In this study, we used EnergyPlus simulations to examine energy end-use profiles for a large multi-family building under design in San Jose, California. We considered a balanced ventilation system using a central dedicated outdoor air supply (DOAS) system. We tested different load-shifting scenarios with multiple parameters to explore how the ventilation airflow rate can be varied to shift load, while also assessing energy and utility cost impacts. The parameters we assessed in each scenario were: the presence of a centralized ERV system or not; ventilation design sizing; and length of load shifting time period. All dynamic ventilation cases, with and without ERV systems, resulted in energy and operational cost savings relative to the constant ventilation cases when compared to providing the same amount of load shifting using batteries, and all tested strategies met ASHRAE 62.2 requirements. The results show that after accounting for the battery penalty typically associated with load shifting, all dynamic ventilation cases reviewed result in improved energy savings when compared to the constant ventilation strategy

Performance and Operation of the CMS Electromagnetic Calorimeter

The operation and general performance of the CMS electromagnetic calorimeter using cosmic-ray muons are described. These muons were recorded after the closure of the CMS detector in late 2008. The calorimeter is made of lead tungstate crystals and the overall status of the 75848 channels corresponding to the barrel and endcap detectors is reported. The stability of crucial operational parameters, such as high voltage, temperature and electronic noise, is summarised and the performance of the light monitoring system is presented

Multi-scale, whole-system models of liver metabolic adaptation to fat and sugar in non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is a serious public health issue associated with high fat, high sugar diets. However, the molecular mechanisms mediating NAFLD pathogenesis are only partially understood. Here we adopt an iterative multi-scale, systems biology approach coupled to in vitro experimentation to investigate the roles of sugar and fat metabolism in NAFLD pathogenesis. The use of fructose as a sweetening agent is controversial; to explore this, we developed a predictive model of human monosaccharide transport, signalling and metabolism. The resulting quantitative model comprising a kinetic model describing monosaccharide transport and insulin signalling integrated with a hepatocyte-specific genome-scale metabolic network (GSMN). Differential kinetics for the utilisation of glucose and fructose were predicted, but the resultant triacylglycerol production was predicted to be similar for monosaccharides; these predictions were verified by in vitro data. The role of physiological adaptation to lipid overload was explored through the comprehensive reconstruction of the peroxisome proliferator activated receptor alpha (PPARα) regulome integrated with a hepatocyte-specific GSMN. The resulting qualitative model reproduced metabolic responses to increased fatty acid levels and mimicked lipid loading in vitro. The model predicted that activation of PPARα by lipids produces a biphasic response, which initially exacerbates steatosis. Our data support the evidence that it is the quantity of sugar rather than the type that is critical in driving the steatotic response. Furthermore, we predict PPARα-mediated adaptations to hepatic lipid overload, shedding light on potential challenges for the use of PPARα agonists to treat NAFLD

Towards the design of 3D multiscale instructive tissue engineering constructs: Current approaches and trends

The design of 3D constructs with adequate properties to instruct and guide cells both in vitro and in vivo is one of the major focuses of tissue engineering. Successful tissue regeneration depends on the favorable crosstalk between the supporting structure, the cells and the host tissue so that a balanced matrix production and degradation is achieved. Herein, the major occurring events and players in normal and regenerative tissue are overviewed. These have been inspiring the selection or synthesis of instructive cues to include into the 3D constructs. We further highlight the importance of a multiscale perception of the range of features that can be included on the biomimetic structures. Lastly, we focus on the current and developing tissue-engineering approaches for the preparation of such 3D constructs: top-down, bottom-up and integrative. Bottom-up and integrative approaches present a higher potential for the design of tissue engineering devices with multiscale features and higher biochemichal control than top-down strategies, and are the main focus of this review.The research leading to these results has received funding from the European Research Council grant agreement ERC-2012-ADG-20120216-321266 for the project ComplexiTE. Portuguese Foundation for Science and Technology is gratefully acknowledged for the fellowship of Sara M. Oliveira (SFRH/BD/70107/2010)