Search results for: electron microscopy (SEM/TEM)

Authors: Rubens P. Costa, Marcelo A. Leigui de Oliveira

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The comprehension of features on the energy spectra, the chemical compositions, and the origins of Ultra-High Energy Cosmic Rays (UHECRs) - mainly atomic nuclei with energies above ~1.0 EeV (exa-electron volts) - are intrinsically linked to the problem of determining the magnitude of their deflections in cosmic magnetic fields on cosmological scales. In addition, as they propagate from the source to the observer, modifications are expected in their original energy spectra, anisotropy, and the chemical compositions due to interactions with low energy photons and matter. This means that any consistent interpretation of the nature and origin of UHECRs has to include the detailed knowledge of their propagation in a three-dimensional environment, taking into account the magnetic deflections and energy losses. The parameter space range for the magnetic fields in the universe is very large because the field strength and especially their orientation have big uncertainties. Particularly, the strength and morphology of the Extragalactic Magnetic Fields (EGMFs) remain largely unknown, because of the intrinsic difficulty of observing them. Monte Carlo simulations of charged particles traveling through a simulated magnetized universe is the straightforward way to study the influence of extragalactic magnetic fields on UHECRs propagation. However, this brings two major difficulties: an accurate numerical modeling of charged particles diffusion in magnetic fields, and an accurate numerical modeling of the magnetized Universe. Since magnetic fields do not cause energy losses, it is important to impose that the particle tracking method conserve the particle’s total energy and that the energy changes are results of the interactions with background photons only. Hence, special attention should be paid to computational effects. Additionally, because of the number of particles necessary to obtain a relevant statistical sample, the particle tracking method must be computationally efficient. In this work, we present an analysis of the propagation of ultra-high energy charged particles in the intergalactic medium. The EGMFs are considered to be coherent within cells of 1 Mpc (mega parsec) diameter, wherein they have uniform intensities of 1 nG (nano Gauss). Moreover, each cell has its field orientation randomly chosen, and a border region is defined such that at distances beyond 95% of the cell radius from the cell center smooth transitions have been applied in order to avoid discontinuities. The smooth transitions are simulated by weighting the magnetic field orientation by the particle's distance to the two nearby cells. The energy losses have been treated in the continuous approximation parameterizing the mean energy loss per unit path length by the energy loss length. We have shown, for a particle with the typical energy of interest the integration method performance in the relative error of Larmor radius, without energy losses and the relative error of energy. Additionally, we plotted the distance amplification from rectilinear propagation as a function of the traveled distance, particle's magnetic rigidity, without energy losses, and particle's energy, with energy losses, to study the influence of particle's species on these calculations. The results clearly show when it is necessary to use a full three-dimensional simulation.

Keywords: cosmic rays propagation, extragalactic magnetic fields, magnetic deflections, ultra-high energy

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Authors: Mohamed Sifan, Brabha Nagaratnam, Julian Thamboo, Keerthan Poologanathan

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Ultra high performance concrete (UHPC) is a type of cementitious material known for its exceptional strength, ductility, and durability. However, its production is often associated with high costs due to the significant amount of cementitious materials required and the use of fine powders to achieve the desired strength. The aim of this research is to explore the feasibility of developing cost-effective UHPC mixes using locally available materials. Specifically, the study aims to investigate the use of coarse limestone sand along with other sand types, namely, basalt sand, dolomite sand, and river sand for developing UHPC mixes and evaluating its performances. The study utilises the particle packing model to develop various UHPC mixes. The particle packing model involves optimising the combination of coarse limestone sand, basalt sand, dolomite sand, and river sand to achieve the desired properties of UHPC. The developed UHPC mixes are then evaluated based on their workability (measured through slump flow and mini slump value), compressive strength (at 7, 28, and 90 days), splitting tensile strength, and microstructural characteristics analysed through scanning electron microscope (SEM) analysis. The results of this study demonstrate that cost-effective UHPC mixes can be developed using locally available materials without the need for silica fume or fly ash. The UHPC mixes achieved impressive compressive strengths of up to 149 MPa at 28 days with a cement content of approximately 750 kg/m³. The mixes also exhibited varying levels of workability, with slump flow values ranging from 550 to 850 mm. Additionally, the inclusion of coarse limestone sand in the mixes effectively reduced the demand for superplasticizer and served as a filler material. By exploring the use of coarse limestone sand and other sand types, this study provides valuable insights into optimising the particle packing model for UHPC production. The findings highlight the potential to reduce costs associated with UHPC production without compromising its strength and durability. The study collected data on the workability, compressive strength, splitting tensile strength, and microstructural characteristics of the developed UHPC mixes. Workability was measured using slump flow and mini slump tests, while compressive strength and splitting tensile strength were assessed at different curing periods. Microstructural characteristics were analysed through SEM and energy dispersive X-ray spectroscopy (EDS) analysis. The collected data were then analysed and interpreted to evaluate the performance and properties of the UHPC mixes. The research successfully demonstrates the feasibility of developing cost-effective UHPC mixes using locally available materials. The inclusion of coarse limestone sand, in combination with other sand types, shows promising results in achieving high compressive strengths and satisfactory workability. The findings suggest that the use of the particle packing model can optimise the combination of materials and reduce the reliance on expensive additives such as silica fume and fly ash. This research provides valuable insights for researchers and construction practitioners aiming to develop cost-effective UHPC mixes using readily available materials and an optimised particle packing approach.

Keywords: cost-effective, limestone powder, particle packing model, ultra high performance concrete

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Authors: Giulia Fredi, Andrea Dorigato, Luca Fambri, Alessandro Pegoretti

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Thermal energy storage (TES) is the storage of heat for later use, thus filling the gap between energy request and supply. The most widely used materials for TES are the organic solid-liquid phase change materials (PCMs), such as paraffin. These materials store/release a high amount of latent heat thanks to their high specific melting enthalpy, operate in a narrow temperature range and have a tunable working temperature. However, they suffer from a low thermal conductivity and need to be confined to prevent leakage. These two issues can be tackled by confining PCMs with carbon nanotubes (CNTs). TES applications include the buildings industry, solar thermal energy collection and thermal management of electronics. In most cases, TES systems are an additional component to be added to the main structure, but if weight and volume savings are key issues, it would be advantageous to embed the TES functionality directly in the structure. Such multifunctional materials could be employed in the automotive industry, where the diffusion of lightweight structures could complicate the thermal management of the cockpit environment or of other temperature sensitive components. This work aims to produce epoxy/carbon structural laminates containing CNT-stabilized paraffin. CNTs were added to molten paraffin in a fraction of 10 wt%, as this was the minimum amount at which no leakage was detected above the melting temperature (45°C). The paraffin/CNT blend was cryogenically milled to obtain particles with an average size of 50 µm. They were added in various percentages (20, 30 and 40 wt%) to an epoxy/hardener formulation, which was used as a matrix to produce laminates through a wet layup technique, by stacking five plies of a plain carbon fiber fabric. The samples were characterized microstructurally, thermally and mechanically. Differential scanning calorimetry (DSC) tests showed that the paraffin kept its ability to melt and crystallize also in the laminates, and the melting enthalpy was almost proportional to the paraffin weight fraction. These thermal properties were retained after fifty heating/cooling cycles. Laser flash analysis showed that the thermal conductivity through the thickness increased with an increase of the PCM, due to the presence of CNTs. The ability of the developed laminates to contribute to the thermal management was also assessed by monitoring their cooling rates through a thermal camera. Three-point bending tests showed that the flexural modulus was only slightly impaired by the presence of the paraffin/CNT particles, while a more sensible decrease of the stress and strain at break and the interlaminar shear strength was detected. Optical and scanning electron microscope images revealed that these could be attributed to the preferential location of the PCM in the interlaminar region. These results demonstrated the feasibility of multifunctional structural TES composites and highlighted that the PCM size and distribution affect the mechanical properties. In this perspective, this group is working on the encapsulation of paraffin in a sol-gel derived organosilica shell. Submicron spheres have been produced, and the current activity focuses on the optimization of the synthesis parameters to increase the emulsion efficiency.

Keywords: carbon fibers, carbon nanotubes, lightweight materials, multifunctional composites, thermal energy storage

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Authors: Ananya Barui, Provas Banerjee, Jyotirmoy Chatterjee

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Application of optical technology in medicine and biology has a long track-record. In this endeavor, OCT is able to attract both engineers and biologists to work together in the field of photonics for establishing a striking non-invasive imaging technology. In contrast to other in vivo imaging modalities like Raman imaging, confocal imaging, two-photon microscopy etc. which can perform in vivo imaging upto 100-200 micron depth due to limitation in numerical aperture or scattering, however, OCT can achieve high-resolution imaging upto few millimeters of tissue structures depending on their refractive index in different anatomical location. This tomographic system depends on interference of two light waves in an interferometer to produce a depth profile of specimen. In wound healing, frequent collection of biopsies for follow-up of repair process could be avoided by such imaging technique. Real time skin OCT (the optical biopsy) has efficacy in deeper and faster illumination of cutaneou tissue to acquire high resolution cross sectional images of their internal micro-structure. Swept Source-OCT (SS-OCT), a novel imaging technique, can generate high-speed depth profile (~ 2 mm) of wound at a sweeping rate of laser with micron level resolution and optimum coherent length of 5-6 mm. Normally multi-layered skin tissue depicts different optical properties along with variation in thickness, refractive index and composition (i.e. keratine layer, water, fat etc.) according to their anatomical location. For instance, stratum corneum, the upper-most and relatively dehydrated layer of epidermis reflects more light and produces more lucid and a sharp demarcation line with rest of the hydrated epidermal region. During wound healing or regeneration, optical properties of cutaneous tissue continuously altered with maturation of wound bed. More mature and less hydrated tissue component reflects more light and becomes visible as a brighter area in comparison to immature region which content higher amount water or fat that depicts as a darker area in OCT image. Non-healing wound possess prolonged inflammation and inhibits nascent proliferative stage. Accumulation of necrotic tissues also prevents the repair of non-healing wounds. Due to high resolution and potentiality to reflect the compositional aspects of tissues in terms of their optical properties, this tomographic method may facilitate in differentiating non-healing and acute wounds in addition to clinical observations. Non-invasive OCT offers better insight regarding specific biological status of tissue in health and pathological conditions, OCT images could be associated with histo-pathological ‘gold standard’. This correlated SS-OCT and microscopic evaluation of the wound edges can provide information regarding progressive healing and maturation of the epithelial components. In the context of searching analogy between two different imaging modalities, their relative performances in imaging of healing bed were estimated for probing an alternative approach. Present study validated utility of SS-OCT in revealing micro-anatomic structure in the healing bed with newer information. Exploring precise correspondence of OCT images features with histo-chemical findings related to epithelial integrity of the regenerated tissue could have great implication. It could establish the ‘optical biopsy’ as a potent non-invasive diagnostic tool for cutaneous pathology.

Keywords: histo-pathology, non invasive imaging, OCT, wound healing

Procedia PDF Downloads 254

Authors: Zuzana Tatarkova, Ivana Pilchova, Michal Cibulka, Martin Kolisek, Peter Racay, Peter Kaplan

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Introduction: Normal functioning of mitochondria is crucial for cardiac performance. Mitochondria undergo mitophagy and biogenesis, and mitochondrial proteins are subject to extensive post-translational modifications. The state of mitochondrial homeostasis reflects overall cellular fitness and longevity. Perturbed mitochondria produce less ATP, release greater amounts of reactive molecules, and are more prone to apoptosis. Therefore mitochondrial turnover is an integral aspect of quality control in which dysfunctional mitochondria are selectively eliminated through mitophagy. Currently, the progressive deterioration of physiological functions is seen as accumulation of modified/damaged proteins with limiting regenerative ability and disturbance of such affected protein-protein communication throughout aging in myocardial cells. Methodologies: For our study was used immunohistochemistry, biochemical methods: spectrophotometry, western blotting, immunodetection as well as more sophisticated 2D electrophoresis and mass spectrometry for evaluation protein-protein interactions and specific post-translational modification. Results and Discussion: Mitochondrial stress response to reactive species was evaluated as electron transport chain (ETC) complexes, redox-active molecules, and their possible communication. Protein-protein interactions revealed a strong linkage between age and ETC protein subunits. Redox state was strongly affected in senescent mitochondria with shift in favor of more pro-oxidizing condition within cardiomyocytes. Acute myocardial ischemia and ischemia-reperfusion (IR) injury affected ETC complexes I, II and IV with no change in complex III. Ischemia induced decrease in total antioxidant capacity, MnSOD, GSH and catalase activity with recovery in some extent during reperfusion. While MnSOD protein content was higher in IR group, activity returned to 95% of control. Nitric oxide is one of the biological molecules that can out compete MnSOD for superoxide and produce peroxynitrite. This process is faster than dismutation and led to the 10-fold higher production of nitrotyrosine after IR injury in adult with higher protection in senescent ones. 2D protein profiling revealed 140 mitochondrial proteins, 12 of them with significant changes after IR injury and 36 individual nitrotyrosine-modified proteins further identified by mass spectrometry. Linking these two groups, 5 proteins were altered after IR as well as nitrated, but only one showed massive nitration per lowering content of protein after IR injury in adult. Conclusions: Senescent cells have greater proportion of protein content, which might be modulated by several post-translational modifications. If these protein modifications are connected to functional consequences and protein-protein interactions are revealed, link may lead to the solution. Assume all together, dysfunctional proteostasis can play a causative role and restoration of protein homeostasis machinery is protective against aging and possibly age-related disorders. This work was supported by the project VEGA 1/0018/18 and by project 'Competence Center for Research and Development in the field of Diagnostics and Therapy of Oncological diseases', ITMS: 26220220153, co-financed from EU sources.

Keywords: aging heart, mitochondria, proteomics, redox state

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Authors: Sugandha Gupta, Arun Kumar Jha

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A large number of ferroelectric materials have been intensely investigated for applications in non-volatile ferroelectric random access memories (FeRAMs), piezoelectric transducers, actuators, pyroelectric sensors, high dielectric constant capacitors, etc. Bismuth layered ferroelectric materials such as Strontium Bismuth Tantalate (SBT) has attracted a lot of attention due to low leakage current, high remnant polarization and high fatigue endurance up to 1012 switching cycles. However, pure SBT suffers from various major limitations such as high dielectric loss, low remnant polarization values, high processing temperature, bismuth volatilization, etc. Significant efforts have been made to improve the dielectric and ferroelectric properties of this compound. Firstly, it has been reported that electrical properties vary with the Sr/ Bi content ratio in the SrBi2Ta2O9 compsition i.e. non-stoichiometric compositions with Sr-deficient / Bi excess content have higher remnant polarization values than stoichiometic SBT compositions. With the objective to improve structural, dielectric, ferroelectric and piezoelectric properties of SBT compound, rare earth holmium (Ho3+) was chosen as a donor cation for substitution onto the Bi2O2 layer. Moreover, hardly any report on holmium substitution in stoichiometric SrBi2Ta2O9 and non-stoichiometric Sr0.8Bi2.2Ta2O9 compositions were available in the literature. The holmium substituted SrBi2-xHoxTa2O9 (x= 0.00-2.0) and Sr0.8Bi2.2Ta2O9 (x=0.0 and 0.01) compositions were synthesized by the solid state reaction method. The synthesized specimens were characterized for their structural and electrical properties. X-ray diffractograms reveal single phase layered perovskite structure formation for holmium content in stoichiometric SBT samples up to x ≤ 0.1. The granular morphology of the samples was investigated using scanning electron microscope (Hitachi, S-3700 N). The dielectric measurements were carried out using a precision LCR meter (Agilent 4284A) operating at oscillation amplitude of 1V. The variation of dielectric constant with temperature shows that the Curie temperature (Tc) decreases on increasing the holmium content. The specimen with x=2.0 i.e. the bismuth free specimen, has very low dielectric constant and does not show any appreciable variation with temperature. The dielectric loss reduces significantly with holmium substitution. The polarization–electric field (P–E) hysteresis loops were recorded using a P–E loop tracer based on Sawyer–Tower circuit. It is observed that the ferroelectric property improve with Ho substitution. Holmium substituted specimen exhibits enhanced value of remnant polarization (Pr= 9.22 μC/cm²) as compared to holmium free specimen (Pr= 2.55 μC/cm²). Piezoelectric co-efficient (d33 values) was measured using a piezo meter system (Piezo Test PM300). It is observed that holmium substitution enhances piezoelectric coefficient. Further, the optimized holmium content (x=0.01) in stoichiometric SrBi2-xHoxTa2O9 composition has been substituted in non-stoichiometric Sr0.8Bi2.2Ta2O9 composition to obtain further enhanced structural and electrical characteristics. It is expected that a new class of ferroelectric materials i.e. Rare Earth Layered Structured Ferroelectrics (RLSF) derived from Bismuth Layered Structured Ferroelectrics (BLSF) will generate which can be used to replace static (SRAM) and dynamic (DRAM) random access memories with ferroelectric random access memories (FeRAMS).

Keywords: dielectrics, ferroelectrics, piezoelectrics, strontium bismuth tantalate

Procedia PDF Downloads 176

Authors: Adela Diepoltova, Klara Konecna, Ondrej Jandourek, Petr Nachtigal

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A loss of control over antibiotic-resistant pathogens has become a global issue due to severe and often untreatable infections. This state is reflected in complicated treatment, health costs, and higher mortality. All these factors emphasize the urgent need for the discovery and development of new anti-infectives. One of the most common pathogens mentioned in the phenomenon of antibiotic resistance are bacteria of the genus Staphylococcus. These bacterial agents have developed several mechanisms against the effect of antibiotics. One of them is biofilm formation. In staphylococci, biofilms are associated with infections such as endocarditis, osteomyelitis, catheter-related bloodstream infections, etc. To author's best knowledge, no validated and standardized methodology evaluating candidate compound activity against staphylococcal biofilms exists. However, a variety of protocols for in vitro drug activity testing has been suggested, yet there are often fundamental differences. Based on our experience, a key methodological step that leads to credible results is to form a robust biofilm with appropriate attributes such as firm adherence to the substrate, a complex arrangement in layers, and the presence of extracellular polysaccharide matrix. At first, for the purpose of drug antibiofilm activity evaluation, the focus was put on various conditions (supplementation of cultivation media by human plasma/fetal bovine serum, shaking mode, the density of initial inoculum) that should lead to reproducible and robust in vitro staphylococcal biofilm formation in microtiter plate model. Three model staphylococcal reference strains were included in the study: Staphylococcus aureus (ATCC 29213), methicillin-resistant Staphylococcus aureus (ATCC 43300), and Staphylococcus epidermidis (ATCC 35983). The total biofilm biomass was quantified using the Christensen method with crystal violet, and results obtained from at least three independent experiments were statistically processed. Attention was also paid to the viability of the biofilm-forming staphylococcal cells and the presence of extracellular polysaccharide matrix. The conditions that led to robust biofilm biomass formation with attributes for biofilms mentioned above were then applied by introducing an alternative method analogous to the commercially available test system, the Calgary Biofilm Device. In this test system, biofilms are formed on pegs that are incorporated into the lid of the microtiter plate. This system provides several advantages (in situ detection and quantification of biofilm microbial cells that have retained their viability after drug exposure). Based on our preliminary studies, it was found that the attention to the peg surface and substrate on which the bacterial biofilms are formed should also be paid to. Therefore, further steps leading to the optimization were introduced. The surface of pegs was coated by human plasma, fetal bovine serum, and L-polylysine. Subsequently, the willingness of bacteria to adhere and form biofilm was monitored. In conclusion, suitable conditions were revealed, leading to the formation of reproducible, robust staphylococcal biofilms in vitro for the microtiter model and the system analogous to the Calgary biofilm device, as well. The robustness and typical slime texture could be detected visually. Likewise, an analysis by confocal laser scanning microscopy revealed a complex three-dimensional arrangement of biofilm forming organisms surrounded by an extracellular polysaccharide matrix.

Keywords: anti-biofilm drug activity screening, in vitro biofilm formation, microtiter plate model, the Calgary biofilm device, staphylococcal infections, substrate modification, surface coating

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Authors: Elvin P. Chizenga, Heidi Abrahamse

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Cancer cell resistance to therapy is the main cause of treatment failures and the poor prognosis of cancer convalescence. The progression of cervical cancer to other parts of the genitourinary system and the reported recurrence rates are overwhelming. Current treatments, including surgery, chemo and radiation have been inefficient in eradicating the tumor cells. These treatments are also associated with poor prognosis and reduced quality of life, including fertility loss. This has inspired the need for the development of new treatment modalities to eradicate cervical cancer successfully. Photodynamic Therapy (PDT) is a modern treatment modality that induces cell death by photochemical interactions of light and a photosensitizer, which in the presence of molecular oxygen, yields a set of chemical reactions that generate Reactive Oxygen Species (ROS) and other free radical species causing cell damage. Enhancing PDT using modified drug delivery can increase the concentration of the photosensitizer in the tumor cells, and this has the potential to maximize its therapeutic efficacy. In cervical cancer, all infected cells constitutively express genes of the E6 and E7 HPV viral oncoproteins, resulting in high concentrations of E6 and E7 in the cytoplasm. This provides an opportunity for active targeting of cervical cancer cells using immune-mediated drug delivery to maximize therapeutic efficacy. The use of nanoparticles in PDT has also proven effective in enhancing therapeutic efficacy. Gold nanoparticles (AuNps) in particular, are explored for their use in biomedicine due to their biocompatibility, low toxicity, and enhancement of drug uptake by tumor cells. In this present study, a biomolecule comprising of AuNPs, anti-E6 monoclonal antibodies, and Aluminium Phthalocyanine photosensitizer was synthesized for use in targeted PDT of cervical cancer. The AuNp-Anti-E6-Sulfonated Aluminium Phthalocyanine mix (AlPcSmix) photosensitizing biomolecule was synthesized by coupling AuNps and anti-E6 monoclonal antibodies to the AlPcSmix via Polyethylene Glycol (PEG) chemical links. The final product was characterized using Transmission Electron Microscope (TEM), Zeta Potential, Uv-Vis Spectrophotometry, Fourier Transform Infrared Spectroscopy (FTIR), and X-ray diffraction (XRD), to confirm its chemical structure and functionality. To observe its therapeutic role in treating cervical cancer, cervical cancer cells, HeLa cells were seeded in 3.4 cm² diameter culture dishes at a concentration of 5x10⁵ cells/ml, in vitro. The cells were treated with varying concentrations of the photosensitizing biomolecule and irradiated using a 673.2 nm wavelength of laser light. Post irradiation cellular responses were performed to observe changes in morphology, viability, proliferation, cytotoxicity, and cell death pathways induced. Dose-Dependent response of the cells to treatment was demonstrated as significant morphologic changes, increased cytotoxicity, and decreased cell viability and proliferation This study presented a synthetic biomolecule for targeted PDT of cervical cancer. The study suggested that PDT using this AuNp- Anti-E6- AlPcSmix photosensitizing biomolecule is a very effective treatment method for the eradication of cervical cancer cells, in vitro. Further studies in vivo need to be conducted to support the use of this biomolecule in treating cervical cancer in clinical settings.

Keywords: anti-E6 monoclonal antibody, cervical cancer, gold nanoparticles, photodynamic therapy

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Authors: Irina Alexandra Paun, Mona Mihailescu, Marian Zamfirescu, Catalin Romeo Luculescu, Adriana Maria Acasandrei, Cosmin Catalin Mustaciosu, Roxana Cristina Popescu, Maria Dinescu

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A major concern in bone tissue engineering is to develop complex 3D architectures that mimic the natural cells environment, facilitate the cells growth in a defined manner and allow the flow transport of nutrients and metabolic waste. In particular, porous structures of controlled pore size and positioning are indispensable for growing human-like bone structures. Another concern is to monitor both the structures and the seeded cells with high spatial resolution and without interfering with the cells natural environment. The present approach relies on laser-based technologies employed for fabricating porous biomimetic structures that support the growth of osteoblast-like cells and for their non-invasive 3D imaging. Specifically, the porous structures were built by two photon polymerization –direct writing (2PP_DW) of the commercially available photoresists IL-L780, using the Photonic Professional 3D lithography system. The structures consist of vertical tubes with micrometer-sized heights and diameters, in a honeycomb-like spatial arrangement. These were fabricated by irradiating the IP-L780 photoresist with focused laser pulses with wavelength centered at 780 nm, 120 fs pulse duration and 80 MHz repetition rate. The samples were precisely scanned in 3D by piezo stages. The coarse positioning was done by XY motorized stages. The scanning path was programmed through a writing language (GWL) script developed by Nanoscribe. Following laser irradiation, the unexposed regions of the photoresist were washed out by immersing the samples in the Propylene Glycol Monomethyl Ether Acetate (PGMEA). The porous structures were seeded with osteoblast like MG-63 cells and their osteogenic potential was tested in vitro. The cell-seeded structures were analyzed in 3D using the digital holographic microscopy technique (DHM). DHM is a marker free and high spatial resolution imaging tool, where the hologram acquisition is performed non-invasively i.e. without interfering with the cells natural environment. Following hologram recording, a digital algorithm provided a 3D image of the sample, as well as information about its refractive index, which is correlated with the intracellular content. The axial resolution of the images went down to the nanoscale, while the temporal scales ranged from milliseconds up to hours. The hologram did not involve sample scanning and the whole image was available in one frame recorded going over 200μm field of view. The digital holograms processing provided 3D quantitative information on the porous structures and allowed a quantitative analysis of the cellular response in respect to the porous architectures. The cellular shape and dimensions were found to be influenced by the underlying micro relief. Furthermore, the intracellular content gave evidence on the beneficial role of the porous structures in promoting osteoblast differentiation. In all, the proposed laser-based protocol emerges as a promising tool for the fabrication and non-invasive imaging of porous constructs for bone tissue engineering. Acknowledgments: This work was supported by a grant of the Romanian Authority for Scientific Research and Innovation, CNCS-UEFISCDI, project PN-II-RU-TE-2014-4-2534 (contract 97 from 01/10/2015) and by UEFISCDI PN-II-PT-PCCA no. 6/2012. A part of this work was performed in the CETAL laser facility, supported by the National Program PN 16 47 - LAPLAS IV.

Keywords: biomimetic, holography, laser, osteoblast, two photon polymerization

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Authors: Salah Jellali, Nusiba Suliman, Yassine Charabi, Jamal Al-Sabahi, Ahmed Al Raeesi, Malik Al-Wardy, Mejdi Jeguirim

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Huge amounts of agricultural biomasses are worldwide produced. At the same time, large quantities of phosphorus are annually discharged into water bodies with possible serious effects onto the environment quality. The main objective of this work is to turn a local Omani biomass (date palm fronds wastes: DPFW) into an effective material for phosphorus recovery from aqueous and the reuse of this P-loaded material in agriculture as ecofriendly amendment. For this aim, the raw DPFW were firstly impregnated with 1 M salt separated solutions of CaCl₂, MgCl₂, FeCl₃, AlCl₃, and a mixture of MgCl₂/AlCl₃ for 24 h, and then pyrolyzed under N2 flow at 500 °C for 2 hours by using an adapted tubular furnace (Carbolite, UK). The synthetized biochars were deeply characterized through specific analyses concerning their morphology, structure, texture, and surface chemistry. These analyses included the use of a scanning electron microscope (SEM) coupled with an energy-dispersive X-Ray spectrometer (EDS), X-Ray diffraction (XRD), Fourier Transform Infrared (FTIR), sorption micrometrics, and X-ray Fluorescence (XRF) apparatus. Then, their efficiency in recovering phosphorus was investigated in batch mode for various contact times (1 min to 3 h), aqueous pH values (from 3 to 11), initial phosphorus concentrations (10-100 mg/L), presence of anions (nitrates, sulfates, and chlorides). In a second step, dynamic assays, by using laboratory columns (height of 30 cm and diameter of 3 cm), were performed in order to investigate the recovery of phosphorus by the modified biochar with a mixture of Mg/Al. The effect of the initial P concentration (25-100 mg/L), the bed depth height (3 to 8 g), and the flow rate (10-30 mL/min) was assessed. Experimental results showed that the biochars physico-chemical properties were very dependent on the type of the used modifying salt. The main affected parameters concerned the specific surface area, microporosity area, and the surface chemistry (pH of zero-point charge and available functional groups). These characteristics have significantly affected the phosphorus recovery efficiency from aqueous solutions. Indeed, the P removal efficiency in batch mode varies from about 5 mg/g for the Fe-modified biochar to more than 13 mg/g for the biochar functionalized with Mg/Al layered double hydroxides. Moreover, the P recovery seems to be a time dependent process and significantly affected by the pH of the aqueous media and the presence of foreign anions due to competition phenomenon. The laboratory column study of phosphorus recovery by the biochar functionalized with Mg/Al layered double hydroxides showed that this process is affected by the used phosphorus concentration, the flow rate, and especially the column bed depth height. Indeed, the phosphorus recovered amount increased from about 4.9 to more than 9.3 mg/g used biochar mass of 3 and 8 g, respectively. This work proved that salt-modified palm fronds-derived biochars could be considered as attractive and promising materials for phosphorus recovery from aqueous solutions even under dynamic conditions. The valorization of these P-loaded-modified biochars as eco-friendly amendment for agricultural soils is necessary will promote sustainability and circular economy concepts in the management of both liquid and solid wastes.

Keywords: date palm wastes, Mg/Al double-layered hydroxides functionalized biochars, phosphorus, recovery, sustainability, circular economy

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Authors: Ece A. Irmak, Amdulla O. Mekhrabov, M. Vedat Akdeniz

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There is a growing interest in the modeling and simulation of magnetic nanoalloys by various computational methods. Magnetic crystalline/amorphous nanoparticles (NP) are interesting materials from both the applied and fundamental points of view, as their properties differ from those of bulk materials and are essential for advanced applications such as high-performance permanent magnets, high-density magnetic recording media, drug carriers, sensors in biomedical technology, etc. As an important magnetic material, Fe-Ni based nanoalloys have promising applications in the chemical industry (catalysis, battery), aerospace and stealth industry (radar absorbing material, jet engine alloys), magnetic biomedical applications (drug delivery, magnetic resonance imaging, biosensor) and computer hardware industry (data storage). The physical and chemical properties of the nanoalloys depend not only on the particle or crystallite size but also on composition and atomic ordering. Therefore, computer modeling is an essential tool to predict structural, electronic, magnetic and optical behavior at atomistic levels and consequently reduce the time for designing and development of new materials with novel/enhanced properties. Although first-principles quantum mechanical methods provide the most accurate results, they require huge computational effort to solve the Schrodinger equation for only a few tens of atoms. On the other hand, molecular dynamics method with appropriate empirical or semi-empirical inter-atomic potentials can give accurate results for the static and dynamic properties of larger systems in a short span of time. In this study, structural evolutions, magnetic and electronic properties of Fe-Ni based nanoalloys have been studied by using molecular dynamics (MD) method in Large-scale Atomic/Molecular Massively Parallel Simulator (LAMMPS) and Density Functional Theory (DFT) in the Vienna Ab initio Simulation Package (VASP). The effects of particle size (in 2-10 nm particle size range) and temperature (300-1500 K) on stability and structural evolutions of amorphous and crystalline Fe-Ni bulk/nanoalloys have been investigated by combining molecular dynamic (MD) simulation method with Embedded Atom Model (EAM). EAM is applicable for the Fe-Ni based bimetallic systems because it considers both the pairwise interatomic interaction potentials and electron densities. Structural evolution of Fe-Ni bulk and nanoparticles (NPs) have been studied by calculation of radial distribution functions (RDF), interatomic distances, coordination number, core-to-surface concentration profiles as well as Voronoi analysis and surface energy dependences on temperature and particle size. Moreover, spin-polarized DFT calculations were performed by using a plane-wave basis set with generalized gradient approximation (GGA) exchange and correlation effects in the VASP-MedeA package to predict magnetic and electronic properties of the Fe-Ni based alloys in bulk and nanostructured phases. The result of theoretical modeling and simulations for the structural evolutions, magnetic and electronic properties of Fe-Ni based nanostructured alloys were compared with experimental and other theoretical results published in the literature.

Keywords: density functional theory, embedded atom model, Fe-Ni systems, molecular dynamics, nanoalloys

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Authors: E. Locatelli, I. Monaco, R. C. Martin, Y. Li, R. Pini, M. Chiariello, M. Comes Franchini

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Despite the many advantages of application of nanomaterials in the field of nanomedicine, increasing concerns have been expressed on their potential adverse effects on human health. There is urgency for novel green strategies toward novel materials with enhanced biocompatibility using safe reagents. Photothermal ablation therapy, which exploits localized heat increase of a few degrees to kill cancer cells, has appeared recently as a non-invasive and highly efficient therapy against various cancer types; anyway new agents able to generate hyperthermia when irradiated are needed and must have precise biocompatibility in order to avoid damage to healthy tissues and prevent toxicity. Recently, there has been increasing interest in magnesium as a biomaterial: it is the fourth most abundant cation in the human body, and it is essential for human metabolism. However magnesium nanoparticles (Mg NPs) have had limited diffusion due to the high reduction potential of magnesium cations, which makes NPs synthesis challenging. Herein, we report the synthesis of Mg NPs and their surface functionalization for the obtainment of a stable and biocompatible nanomaterial suitable for photothermal ablation therapy against cancer. We synthesized the Mg crystals by reducing MgCl2 with metallic lithium and exploiting naphthalene as an electron carrier: the lithium–naphthalene complex acts as the real reducing agent. Firstly, the nanocrystal particles were coated with the ligand 12-ethoxy ester dodecanehydroxamic acid, and then entrapped into water-dispersible polymeric micelles (PMs) made of the FDA-approved PLGA-b-PEG-COOH copolymer using the oil-in-water emulsion technique. Lately, we developed a more straightforward methodology by introducing chitosan, a highly biocompatible natural product, at the beginning of the process, simultaneously using lithium–naphthalene complex, thus having a one-pot procedure for the formation and surface modification of MgNPs. The obtained MgNPs were purified and fully characterized, showing diameters in the range of 50-300 nm. Notably, when coated with chitosan the particles remained stable as dry powder for more than 10 months. We proved the possibility of generating a temperature rise of a few to several degrees once MgNPs were illuminated using a 810 nm diode laser operating in continuous wave mode: the temperature rise resulted significant (0-15 °C) and concentration dependent. We then investigated potential cytotoxicity of the MgNPs: we used HN13 epithelial cells, derived from a head and neck squamous cell carcinoma and the hepa1-6 cell line, derived from hepatocellular carcinoma and very low toxicity was observed for both nanosystems. Finally, in vivo photothermal therapy was performed on xenograft hepa1-6 tumor bearing mice: the animals were treated with MgNPs coated with chitosan and showed no sign of suffering after the injection. After 12 hours the tumor was exposed to near-infrared laser light. The results clearly showed an extensive damage to tumor tissue after only 2 minutes of laser irradiation at 3Wcm-1, while no damage was reported when the tumor was treated with the laser and saline alone in control group. Despite the lower photothermal efficiency of Mg with respect to Au NPs, we consider MgNPs a promising, safe and green candidate for future clinical translations.

Keywords: chitosan, magnesium nanoparticles, nanomedicine, photothermal therapy

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Authors: Omaima A. Sharaf, Wafaa M. Abd El-Rahim, Hassan Moawad, Michael J. Sadowsky

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Textile industry is one of the important industries in the worldwide. It is known that the eco-friendly industrial and agricultural activities are significant for socio-economic stability of all countries. The absence of appropriate industrial waste water treatments is essential barrier for sustainable development in food and agricultural sectors especially in developing country like Egypt. Thus, the development of enzymatic bioremediation technology for textile dye removal will enhance the collaboration between scientists who develop the technology and industry where this technology will be implemented towards the safe disposal of the textile dye wastes. Highly efficient microorganisms are of most importance in developing and using highly effective biological treatment processes. Bacterial degradation of azo dyes is generally initiated by an enzymatic step that involves cleavage of azo linkages, usually with the aid of an azoreductase as electron donor. Thus, expanding the spectrum of microorganisms with high enzymatic activities as azoreductases and discovering novel azo-dye degrading enzymes, with enhanced stability and superior catalytic properties, are necessary for many environmental and industrial applications. Consequently, the use of molecular tools has become increasingly integrated into the understanding of enzyme properties and characterization. Researchers have utilized a gene cloning and expression methods as a tool to produce recombinant protein for decolorizing dyes more efficiently. Thus, presumptive evidence for the presence of genes encoding azoreductases in the genomes of selected local, and most potent azo-degrading strains were obtained by using specific oligonucleotides primers. These potent strains have been isolated from textile industrial wastewater in Egypt and identified using 16S rRNA sequence analysis as 'Lysinibacillus macrolidesB8, Brevibacillus parabrevisB11, Bacillus coagulansB7, and B. cereusB5'. PCR products of two full length genes designated as (AZO1;621bp and AZO2;534bp) were detected. BLASTx results indicated that AZO1 gene was corresponding to predicted azoreductase from of Bacillus sp. ABP14, complete genome, multispecies azoreductase [Bacillus], It was submitted to the gene bank by an accession no., BankIt2085371 AZO1 MG923210 (621bp; 207 amino acids). AZO1 was generated from the DNA of our identified strains Lysinibacillus macrolidesB8. On the other hand, AZO2 gene was corresponding to a predicted azoreductase from Bacillus cereus strain S2-8. Gene bank accession no. was BankIt2085839 AZO2 MG932081 (534bp;178 amino acids) and it was amplified from our Bacillus coagulansB7. Both genes were successfully cloned into pCR2.1TOPO (Invitrogen) and in pET28b+ vectors, then they transformed into E. coli DH5α and BL21(DE3) cells for heterologous expression studies. Our recombinant azoreductases (AZO1&AZO2) exhibited potential enzyme activity and efficiently decolorized an azo dye (Direct violet). They exhibited pH stability between 6 and 8 with optimum temperature up to 60°C and 37 °C after induction by 1mM and 1.5mM IPTG, for both AZO1 &AZO2, respectively. These results suggested that further optimization and purification of these recombinant proteins by using different heterologous expression systems will give great potential for the sustainable utilization of these recombinant enzymes in several industrial applications especially in wastewater treatments.

Keywords: azoreductases, decolorization, enzyme activity, gene cloning and expression

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Authors: Tianfang Zhang, Luxi He, Zhengbin He, Songlin Yi

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Moso bamboo is a common non-wood forest resource in Asia that is widely used in construction, furniture, and other fields. Influenced by the heterogeneous structure and various hygroscopic groups of bamboo, the deformation occurs as moisture absorption and desorption when the environment temperature and humidity conditions change. Thermal modification is a well-established commercial technology for improving the dimensional stability of bamboo. However, the higher energy consumption and carbon emissions limit its further development. Previous studies have indicated that inorganic salt-assisted thermal modification could lead to significant reductions in moisture absorption and energy consumption. Represented by metal chlorides, it could show Lewis acid properties when dissolved in water, generating metal ion ligand complexes. In addition, ultrasonic treatment, as an efficient and environmentally friendly physical treatment method, improved the accessibility of pretreatment chemical impregnation agents and intensified mass and heat transfer during reactions. To save energy and reduce deformation, this study elucidates the influence of zinc chloride-ultrasonic treatment on the physicochemical properties of heat-treated bamboo, and the details of the bamboo deformation mechanism with Lewis acid are explained. Three sets of parameters (inorganic salt concentration, ultrasonic frequency and heat treatment temperature) were designed, and an optimized process was proposed to clarify this scientific question, that is: 5% (w/w) zinc chloride solution, 40 kHz ultrasonic waves and heat treatment at 160 °C. The samples were characterized by different means to analyze changes in their macroscopic features, pore structure, chemical structure and chemical composition. The results suggested that the maximum weight loss rate was reduced by at least 19.75%. The maximum thermal degradation peak of hemicellulose was significantly shifted forward. The hygroscopicity was reduced by 10.15%, the relative crystallinity was increased by 4.4%, the surface contact angle was increased by 25.2%, and the color change was increased by 23.60 in the optimal condition. From the electron microscope observation, the treated surface became rougher, and cracks appeared in some weaker areas, accelerating starch loss and removing granular attachments around the pits. By ion diffusion, zinc ions diffused into hemicellulose and a partial amorphous region of cellulose. Parts of the cell wall structure were subjected to swelling and degradation, leading to the broken state of parenchyma cells. From the Raman spectrum, compared to conventional thermal modifications, hemicellulose thermal degradation and lignin migration is promoted by Lewis acid under dilute acid-thermal condition. As shown in this work, the combined Lewis acid and ultrasonic pretreatment as an environmentally friendly, safe, and efficient physic-chemical combined pretreatment method improved the dimensional stability of Moso bamboo and lowered the thermal degradation conditions. This method has great potential for development in the field of bamboo heat treatment, and it might provide some guidance for making dark bamboo flooring.

Keywords: Moso bamboo, Lewis acid, ultrasound, heat treatment

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Authors: Martyna Ekiert, Natalia Grzechnik, Joanna Karbowniczek, Urszula Stachewicz, Andrzej Mlyniec

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Tendon grafting is a common procedure performed to treat tendon rupture. Before the surgical procedure, tissues intended for grafts (i.e., Achilles tendon) are stored in ultra-low temperatures for a long time and also may be subjected to unfavorable conditions, such as repetitive freezing (F) and thawing (T). Such storage protocols may highly influence the graft mechanical properties, decrease its functionality and thus increase the risk of complications during the transplant procedure. The literature reports on the influence of multiple F/T cycles on internal structure and mechanical properties of tendons stay inconclusive, confirming and denying the negative influence of multiple F/T at the same time. An inconsistent research methodology and lack of clear limit of F/T cycles, which disqualifies tissue for surgical graft purposes, encouraged us to investigate the issue of multiple F/T cycles by the mean of biomechanical tensile tests supported with Scanning Electron Microscope (SEM) imaging. The study was conducted on male bovine Achilles tendon-derived from the local abattoir. Fresh tendons were cleaned of excessive membranes and then sectioned to obtained fascicle bundles. Collected samples were randomly assigned to 6 groups subjected to 1, 2, 4, 6, 8 and 12 cycles of freezing-thawing (F/T), respectively. Each F/T cycle included deep freezing at -80°C temperature, followed by thawing at room temperature. After final thawing, thin slices of the side part of samples subjected to 1, 4, 8 and 12 F/T cycles were collected for SEM imaging. Then, the width and thickness of all samples were measured to calculate the cross-sectional area. Biomechanical tests were performed using the universal testing machine (model Instron 8872, INSTRON®, Norwood, Massachusetts, USA) using a load cell with a maximum capacity of 250 kN and standard atmospheric conditions. Both ends of each fascicle bundle were manually clamped in grasping clamps using abrasive paper and wet cellulose wadding swabs to prevent tissue slipping while clamping and testing. Samples were subjected to the testing procedure including pre-loading, pre-cycling, loading, holding and unloading steps to obtain stress-strain curves for representing tendon stretching and relaxation. The stiffness of AT fascicles bundle samples was evaluated in terms of modulus of elasticity (Young’s modulus), calculated from the slope of the linear region of stress-strain curves. SEM imaging was preceded by chemical sample preparation including 24hr fixation in 3% glutaraldehyde buffered with 0.1 M phosphate buffer, washing with 0.1 M phosphate buffer solution and dehydration in a graded ethanol solution. SEM images (Merlin Gemini II microscope, ZEISS®) were taken using 30 000x mag, which allowed measuring a diameter of collagen fibrils. The results confirm a decrease in fascicle bundles Young’s modulus as well as a decrease in the diameter of collagen fibrils. These results confirm the negative influence of multiple F/T cycles on the mechanical properties of tendon tissue.

Keywords: biomechanics, collagen, fascicle bundles, soft tissue

Procedia PDF Downloads 101

Authors: Leila Noori, Rosario Barone, Francesca Rappa, Antonella Marino Gammazza, Alessandra Maria Vitale, Giuseppe Donato Mangano, Giusy Sentiero, Filippo Macaluso, Kathryn H. Myburgh, Francesco Cappello, Federica Scalia

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The neuromuscular system controls, directs, and allows movement of the body through the action of neural circuits, which include motor neurons, sensory neurons, and skeletal muscle fibers. Protein homeostasis of the involved cytotypes appears crucial to maintain the correct and prolonged functions of the neuromuscular system, and both neuronal cells and skeletal muscle fibers express significant quantities of protein chaperones, the molecular machinery responsible to maintain the protein turnover. Genetic mutations or defective post-translational modifications of molecular chaperones (i.e., genetic or acquired chaperonopathies) may lead to neuromuscular disorders called as neurochaperonopathies. The limited knowledge of the effects of the defective chaperones on skeletal muscle fibers and neurons impedes the progression of therapeutic approaches. A distinct genetic variation of CCT5 gene encoding for the subunit 5 of the chaperonin CCT (Chaperonin Containing TCP1; also known as TRiC, TCP1 Ring Complex) was recently described associated with severe distal motor neuropathy by our team. In this study, we investigated the histopathological abnormalities of the skeletal muscle biopsy of the pediatric patient affected by the mutation Leu224Val in the CCT5 subunit. We provide molecular and structural features of the diseased skeletal muscle tissue that we believe may be useful to identify undiagnosed cases of this rare genetic disorder. We investigated the histological abnormalities of the affected tissue via hematoxylin and eosin staining. Then we used immunofluorescence and qPCR techniques to explore the expression and distribution of CCT5 in diseased and healthy skeletal muscle tissue. Immunofluorescence and immunohistochemistry assays were performed to study the sarcomeric and structural proteins of skeletal muscle, including actin, myosin, tubulin, troponin-T, telethonin, and titin. We performed Western blot to examine the protein expression of CCT5 and some heat shock proteins, Hsp90, Hsp60, Hsp27, and α-B crystallin, along with the main client proteins of the CCT5, actin, and tubulin. Our findings revealed muscular atrophy, abnormal morphology, and different sizes of muscle fibers in affected tissue. The swollen nuclei and wide interfiber spaces were seen. Expression of CCT5 had been decreased and showed a different distribution pattern in the affected tissue. Altered expression, distribution, and bandage pattern were detected by confocal microscopy for the interested muscular proteins in tissue from the patient compared to the healthy control. Protein levels of the studied Hsps normally located at the Z-disk were reduced. Western blot results showed increased levels of the actin and tubulin proteins in the diseased skeletal muscle biopsy compared to healthy tissue. Chaperones must be expressed at high levels in skeletal muscle to counteract various stressors such as mechanical, oxidative, and thermal crises; therefore, it seems relevant that defects of molecular chaperones may result in damaged skeletal muscle fibers. So far, several chaperones or cochaperones involved in neuromuscular disorders have been defined. Our study shows that alteration of the CCT5 subunit is associated with the damaged structure of skeletal muscle fibers and alterations of chaperone system components and paves the way to explore possible alternative substrates of chaperonin CCT. However, further studies are underway to investigate the CCT mechanisms of action to design applicable therapeutic strategies.

Keywords: molecular chaperones, neurochaperonopathy, neuromuscular system, protein homeostasis

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Authors: Wonil Nam, Xiang Ren, Inyoung Kim, Masoud Agah, Wei Zhou

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Despite its ultrasensitive detection capability, surface-enhanced Raman spectroscopy (SERS) faces challenges as a quantitative biochemical analysis tool due to the significant dependence of local field intensity in hotspots on nanoscale geometric variations of plasmonic nanostructures. Therefore, despite enormous progress in plasmonic nanoengineering of high-performance SERS devices, it is still challenging to quantitatively correlate the measured SERS signals with the actual molecule concentrations at hotspots. A significant effort has been devoted to developing SERS calibration methods by introducing internal standards. It has been achieved by placing Raman tags at plasmonic hotspots. Raman tags undergo similar SERS enhancement at the same hotspots, and ratiometric SERS signals for analytes of interest can be generated with reduced dependence on geometrical variations. However, using Raman tags still faces challenges for real-world applications, including spatial competition between the analyte and tags in hotspots, spectral interference, laser-induced degradation/desorption due to plasmon-enhanced photochemical/photothermal effects. We show that electronic Raman scattering (ERS) signals from metallic nanostructures at hotspots can serve as the internal calibration standard to enable quantitative SERS analysis and improve biostatistical analysis. We perform SERS with Au-SiO₂ multilayered metal-insulator-metal nano laminated plasmonic nanostructures. Since the ERS signal is proportional to the volume density of electron-hole occupation in hotspots, the ERS signals exponentially increase when the wavenumber is approaching the zero value. By a long-pass filter, generally used in backscattered SERS configurations, to chop the ERS background continuum, we can observe an ERS pseudo-peak, IERS. Both ERS and SERS processes experience the |E|⁴ local enhancements during the excitation and inelastic scattering transitions. We calibrated IMRS of 10 μM Rhodamine 6G in solution by IERS. The results show that ERS calibration generates a new analytical value, ISERS/IERS, insensitive to variations from different hotspots and thus can quantitatively reflect the molecular concentration information. Given the calibration capability of ERS signals, we performed label-free SERS analysis of living biological systems using four different breast normal and cancer cell lines cultured on nano-laminated SERS devices. 2D Raman mapping over 100 μm × 100 μm, containing several cells, was conducted. The SERS spectra were subsequently analyzed by multivariate analysis using partial least square discriminant analysis. Remarkably, after ERS calibration, MCF-10A and MCF-7 cells are further separated while the two triple-negative breast cancer cells (MDA-MB-231 and HCC-1806) are more overlapped, in good agreement with the well-known cancer categorization regarding the degree of malignancy. To assess the strength of ERS calibration, we further carried out a drug efficacy study using MDA-MB-231 and different concentrations of anti-cancer drug paclitaxel (PTX). After ERS calibration, we can more clearly segregate the control/low-dosage groups (0 and 1.5 nM), the middle-dosage group (5 nM), and the group treated with half-maximal inhibitory concentration (IC50, 15 nM). Therefore, we envision that ERS calibrated SERS can find crucial opportunities in label-free molecular profiling of complicated biological systems.

Keywords: cancer cell drug efficacy, plasmonics, surface-enhanced Raman spectroscopy (SERS), SERS calibration

Procedia PDF Downloads 110

Authors: K. Karapetyan, F. Tkhruni, A. Aghajanyan, T. S. Balabekyan, L. Arstamyan

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Introduction: Globalization of the food supply has created the conditions favorable for the emergence and spread of food-borne and especially dangerous pathogens (EDP) in developing countries. The fresh-cut fruit and vegetable industry is searching for alternatives to replace chemical treatments with biopreservative approaches that ensure the safety of the processed foods product. Antimicrobial compounds of lactic acid bacteria (LAB) possess bactericidal or bacteriostatic activity against intestinal pathogens, spoilage organisms and food-borne pathogens such as Listeria monocytogenes, Staphylococcus aureus and Salmonella. Endemic strains of LAB were isolated. The strains, showing broad spectrum of antimicrobial activity against food spoiling microorganisms, were selected. The genotyping by 16S rRNA sequencing, GS-PCR, RAPD PCR methods showed that they were presented by Lactobacillus rhamnosus109, L.plantarum 65, L.plantarum 66 and Enterococcus faecium 64 species. LAB are deposited in "Microbial Depository Center" (MDC) SPC "Armbiotechnology". Methods: LAB strains were isolated from different dairy products from rural households from the highland regions of Armenia. Serially diluted samples were spread on MRS (Merck, Germany) and hydrolyzed milk agar (1,2 % w/v). Single colonies from each LAB were individually inoculated in liquid MRS medium and incubated at 37oC for 24 hours. Culture broth with biomass was centrifuged at 10,000 g during 20 min for obtaining of cell free culture broth (CFC). The antimicrobial substances from CFC broth were purified by the combination of adsorption-desorption and ion-exchange chromatography methods. Separation of bacteriocins was performed using a HPLC method on "Avex ODS" C18 column. Mass analysis of peptides recorded on the device API 4000 in the electron ionization mode. The spot-on-lawn method on the test culture plated in the solid medium was applied. The antimicrobial activity is expressed in arbitrary units (AU/ml). Results. Purification of CFC broth of LAB allowed to obtain partially purified antimicrobial preparations which contains bacteriocins with broad spectrum of antimicrobial activity. Investigation of their main biochemical properties shown, that inhibitory activity of preparations is partially reduced after treatment with proteinase K, trypsin, pepsin, suggesting a proteinaceous nature of bacteriocin-like substances containing in CFC broth. Preparations preserved their activity after heat treatment (50-121 oC, 20 min) and were stable in the pH range 3–8. The results of SDS PAAG electrophoresis show that L.plantarum 66 and Ent.faecium 64 strains have one bacteriocin (BCN) with maximal antimicrobial activity with approximate molecular weight 2.0-3.0 kDa. From L.rhamnosus 109 two BCNs were obtained. Mass spectral analysis indicates that these bacteriocins have peptide bonds and molecular weight of BCN 1 and BCN 2 are approximately 1.5 kDa and 700 Da. Discussion: Thus, our experimental data shown, that isolated endemic strains of LAB are able to produce bacteriocins with high and different inhibitory activity against broad spectrum of microorganisms of different taxonomic group, such as Salmonella sp., Esherichia coli, Bacillus sp., L.monocytogenes, Proteus mirabilis, Staph. aureus, Ps. aeruginosa. Obtained results proved the perspectives for use of endemic strains in the preservation of foodstuffs. Acknowledgments: This work was realized with financial support of the Project Global Initiatives for Preliferation Prevention (GIPP) T2- 298, ISTC A-1866.

Keywords: antimicrobial activity, bacteriocins, endemic strains, food safety

Procedia PDF Downloads 541

Authors: L. Malic, X. Zhang, D. Brassard, L. Clime, J. Daoud, C. Luebbert, V. Barrere, A. Boutin, S. Bidawid, N. Corneau, J. Farber, T. Veres

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The incidence of infections caused by foodborne pathogens such as Listeria monocytogenes (L. monocytogenes) poses a great potential threat to public health and safety. These issues are further exacerbated by legal repercussions due to “zero tolerance” food safety standards adopted in developed countries. Unfortunately, a large number of related disease outbreaks are caused by pathogens present in extremely low counts currently undetectable by available techniques. The development of highly sensitive and rapid detection of foodborne pathogens is therefore crucial, and requires robust and efficient pre-analytical sample preparation. Immunomagnetic separation is a popular approach to sample preparation. Microfluidic chips combined with external magnets have emerged as viable high throughput methods. However, external magnets alone are not suitable for the capture of nanoparticles, as very strong magnetic fields are required. Devices that incorporate externally applied magnetic field and microstructures of a soft magnetic material have thus been used for local field amplification. Unfortunately, very complex and costly fabrication processes used for integration of soft magnetic materials in the reported proof-of-concept devices would prohibit their use as disposable tools for food and water safety or diagnostic applications. We present a sample preparation magnetic microfluidic device implemented in low-cost thermoplastic polymers using fabrication techniques suitable for mass-production. The developed magnetic capture chip (M-chip) was employed for rapid capture and release of L. monocytogenes conjugated to immunomagnetic nanoparticles (IMNs) in buffer and beef filtrate. The M-chip relies on a dense array of Nickel-coated high-aspect ratio pillars for capture with controlled magnetic field distribution and a microfluidic channel network for sample delivery, waste, wash and recovery. The developed Nickel-coating process and passivation allows generation of switchable local perturbations within the uniform magnetic field generated with a pair of permanent magnets placed at the opposite edges of the chip. This leads to strong and reversible trapping force, wherein high local magnetic field gradients allow efficient capture of IMNs conjugated to L. monocytogenes flowing through the microfluidic chamber. The experimental optimization of the M-chip was performed using commercially available magnetic microparticles and fabricated silica-coated iron-oxide nanoparticles. The fabricated nanoparticles were optimized to achieve the desired magnetic moment and surface functionalization was tailored to allow efficient capture antibody immobilization. The integration, validation and further optimization of the capture and release protocol is demonstrated using both, dead and live L. monocytogenes through fluorescence microscopy and plate- culture method. The capture efficiency of the chip was found to vary as function of listeria to nanoparticle concentration ratio. The maximum capture efficiency of 30% was obtained and the 24-hour plate-culture method allowed the detection of initial sample concentration of only 16 cfu/ml. The device was also very efficient in concentrating the sample from a 10 ml initial volume. Specifically, 280% concentration efficiency was achieved in 17 minutes only, demonstrating the suitability of the system for food safety applications. In addition, flexible design and low-cost fabrication process will allow rapid sample preparation for applications beyond food and water safety, including point-of-care diagnosis.

Keywords: array of pillars, bacteria isolation, immunomagnetic sample preparation, polymer microfluidic device

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Authors: Tom S. Owens, Tom A. Calverley, Benjamin S. Stacey, Christopher J. Marley, George Rose, Lewis Fall, Gareth L. Jones, Priscilla Williams, John P. R. Williams, Martin Steggall, Damian M. Bailey

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Introduction and aims: Sports-related concussion (SRC) represents a significant and growing public health concern in rugby union, yet remains one of the least understood injuries facing the health community today. Alongside increasing SRC incidence rates, there is concern that prior recurrent concussion may contribute to long-term neurologic sequelae in later-life. This may be due to an accelerated decline in cerebral perfusion, a major risk factor for neurocognitive decline and neurodegeneration, though the underlying mechanisms remain to be established. The present study hypothesised that recurrent concussion in current professional rugby union players would result in elevated systemic oxidative-nitrosative stress, reflected by a free radical-mediated reduction in nitric oxide (NO) bioavailability and impaired cerebrovascular and cognitive function. Methodology: A longitudinal study design was adopted across the 2017-2018 rugby union season. Ethical approval was obtained from the University of South Wales Ethics Committee. Data collection is ongoing, and therefore the current report documents result from the pre-season and first half of the in-season data collection. Participants were initially divided into two subgroups; 23 professional rugby union players (aged 26 ± 5 years) and 22 non-concussed controls (27 ± 8 years). Pre-season measurements were performed for cerebrovascular function (Doppler ultrasound of middle cerebral artery velocity (MCAv) in response to hypocapnia/normocapnia/hypercapnia), cephalic venous concentrations of the ascorbate radical (A•-, electron paramagnetic resonance spectroscopy), NO (ozone-based chemiluminescence) and cognition (neuropsychometric tests). Notational analysis was performed to assess contact in the rugby group throughout each competitive game. Results: 1001 tackles and 62 injuries, including three concussions were observed across the first half of the season. However, no associations were apparent between number of tackles and any injury type (P > 0.05). The rugby group expressed greater oxidative stress as indicated by increased A•- (P < 0.05 vs. control) and a subsequent decrease in NO bioavailability (P < 0.05 vs. control). The rugby group performed worse in the Ray Auditory Verbal Learning Test B (RAVLT-B, learning, and memory) and the Grooved Pegboard test using both the dominant and non-dominant hands (visuomotor coordination, P < 0.05 vs. control). There were no between-group differences in cerebral perfusion at baseline (MCAv: 54 ± 13 vs. 59 ± 12, P > 0.05). Likewise, no between-group differences in CVRCO2Hypo (2.58 ± 1.01 vs. 2.58 ± 0.75, P > 0.05) or CVRCO2Hyper (2.69 ± 1.07 vs. 3.35 ± 1.28, P > 0.05) were observed. Conclusion: The present study identified that the rugby union players are characterized by impaired cognitive function subsequent to elevated systemic-oxidative-nitrosative stress. However, this appears to be independent of any functional impairment in cerebrovascular function. Given the potential long-term trajectory towards accelerated cognitive decline in populations exposed to SRC, prophylaxis to increase NO bioavailability warrants consideration.

Keywords: cognition, concussion, mild traumatic brain injury, rugby

Procedia PDF Downloads 138

Authors: Glauco M. M. M. Lustosa, João Paulo C. Costa, Sonia M. Zanetti, Mario Cilense, Leinig Antônio Perazolli, Maria Aparecida Zaghete

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The contamination of the environment has been one of the biggest problems of our time, mostly due to developments of many industries. SnO2 is an n-type semiconductor with band gap about 3.5 eV and has its electrical conductivity dependent of type and amount of modifiers agents added into matrix ceramic during synthesis process, allowing applications as sensing of gaseous pollutants on ambient. The chemical synthesis by polymeric precursor method consists in a complexation reaction between tin ion and citric acid at 90 °C/2 hours and subsequently addition of ethyleneglycol for polymerization at 130 °C/2 hours. It also prepared polymeric resin of zinc, cobalt and niobium ions. Stoichiometric amounts of the solutions were mixed to obtain the systems (Zn, Nb)-SnO2 and (Co, Nb) SnO2 . The metal immobilization reduces its segregation during the calcination resulting in a crystalline oxide with high chemical homogeneity. The resin was pre-calcined at 300 °C/1 hour, milled in Atritor Mill at 500 rpm/1 hour, and then calcined at 600 °C/2 hours. X-Ray Diffraction (XDR) indicated formation of SnO2 -rutile phase (JCPDS card nº 41-1445). The characterization by Scanning Electron Microscope of High Resolution showed spherical ceramic powder nanostructured with 10-20 nm of diameter. 20 mg of SnO2 -based powder was kept in 20 ml of isopropyl alcohol and then taken to an electrophoretic deposition (EPD) system. The EPD method allows control the thickness films through the voltage or current applied in the electrophoretic cell and by the time used for deposition of ceramics particles. This procedure obtains films in a short time with low costs, bringing prospects for a new generation of smaller size devices with easy integration technology. In this research, films were obtained in an alumina substrate with interdigital electrodes after applying 2 kV during 5 and 10 minutes in cells containing alcoholic suspension of (Zn, Nb)-SnO2 and (Co, Nb) SnO2 of powders, forming a sensing layer. The substrate has designed integrated micro hotplates that provide an instantaneous and precise temperature control capability when a voltage is applied. The films were sintered at 900 and 1000 °C in a microwave oven of 770 W, adapted by the research group itself with a temperature controller. This sintering is a fast process with homogeneous heating rate which promotes controlled growth of grain size and also the diffusion of modifiers agents, inducing the creation of intrinsic defects which will change the electrical characteristics of SnO2 -based powders. This study has successfully demonstrated a microfabricated system with an integrated micro-hotplate for detection of CO and NO2 gas at different concentrations and temperature, with self-heating SnO2 - based nanoparticles films, being suitable for both industrial process monitoring and detection of low concentrations in buildings/residences in order to safeguard human health. The results indicate the possibility for development of gas sensors devices with low power consumption for integration in portable electronic equipment with fast analysis. Acknowledgments The authors thanks to the LMA-IQ for providing the FEG-SEM images, and the financial support of this project by the Brazilian research funding agencies CNPq, FAPESP 2014/11314-9 and CEPID/CDMF- FAPESP 2013/07296-2.

Keywords: chemical synthesis, electrophoretic deposition, self-heating, gas sensor

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Authors: Achraf Al Faraj, Asma Sultana Shaik, Baraa Al Sayed

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Specific and effective targeting of drug delivery systems (DDS) to cancerous sites remains a major challenge for a better diagnostic and therapy. Recently, SWCNTs with their unique physicochemical properties and the ability to cross the cell membrane show promising in the biomedical field. The purpose of this study was first to develop a biocompatible iron oxide tagged SWCNTs as diagnostic nanoprobes to allow their noninvasive detection using MRI and their preferential targeting in a breast cancer murine model by placing an optimized flexible magnet over the tumor site. Magnetic targeting was associated to specific antibody-conjugated SWCNTs active targeting. The therapeutic efficacy of doxorubicin-conjugated SWCNTs was assessed, and the superiority of diffusion-weighted (DW-) MRI as sensitive imaging biomarker was investigated. Short Polyvinylpyrrolidone (PVP) stabilized water soluble SWCNTs were first developed, tagged with iron oxide nanoparticles and conjugated with Endoglin/CD105 monoclonal antibodies. They were then conjugated with doxorubicin drugs. SWCNTs conjugates were extensively characterized using TEM, UV-Vis spectrophotometer, dynamic light scattering (DLS) zeta potential analysis and electron spin resonance (ESR) spectroscopy. Their MR relaxivities (i.e. r1 and r2*) were measured at 4.7T and their iron content and metal impurities quantified using ICP-MS. SWCNTs biocompatibility and drug efficacy were then evaluated both in vitro and in vivo using a set of immunological assays. Luciferase enhanced bioluminescence 4T1 mouse mammary tumor cells (4T1-Luc2) were injected into the right inguinal mammary fat pad of Balb/c mice. Tumor bearing mice received either free doxorubicin (DOX) drug or SWCNTs with or without either DOX or iron oxide nanoparticles. A multi-pole 10x10mm high-energy flexible magnet was maintained over the tumor site during 2 hours post-injections and their properties and polarity were optimized to allow enhanced magnetic targeting of SWCNTs toward the primary tumor site. Tumor volume was quantified during the follow-up investigation study using a fast spin echo MRI sequence. In order to detect the homing of SWCNTs to the main tumor site, susceptibility-weighted multi-gradient echo (MGE) sequence was used to generate T2* maps. Apparent diffusion coefficient (ADC) measurements were also performed as a sensitive imaging biomarker providing early and better assessment of disease treatment. At several times post-SWCNT injection, histological analysis were performed on tumor extracts and iron-loaded SWCNT were quantified using ICP-MS in tumor sites, liver, spleen, kidneys, and lung. The optimized multi-poles magnet revealed an enhanced targeting of magnetic SWCNTs to the primary tumor site, which was found to be much higher than the active targeting achieved using antibody-conjugated SWCNTs. Iron-loading allowed their sensitive noninvasive tracking after intravenous administration using MRI. The active targeting of doxorubicin through magnetic antibody-conjugated SWCNTs nanoprobes was found to considerably decrease the primary tumor site and may have inhibited the development of metastasis in the tumor-bearing mice lung. ADC measurements in DW-MRI were found to significantly increase in a time-dependent manner after the injection of DOX-conjugated SWCNTs complexes.

Keywords: single-walled carbon nanotubes, nanomedicine, magnetic resonance imaging, cancer diagnosis and therapy

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Authors: Michael Sigruener, Dirk Muscat, Nicole Struebbe

Abstract:

Fiber reinforcement is a state of the art to enhance mechanical properties in plastics. For concrete and civil engineering, steel reinforcements are commonly used. Steel reinforcements show disadvantages in their chemical resistance and weight, whereas polymer fibers' major problems are in fiber-matrix adhesion and mechanical properties. In spite of these facts, longevity and easy handling, as well as chemical resistance motivate researches to develop a polymeric material for fiber reinforced concrete. Adhesion and interfacial mechanism in fiber-polymer-composites are already studied thoroughly. For polymer fibers used as concrete reinforcement, the bonding behavior still requires a deeper investigation. Therefore, several differing polymers (e.g., polypropylene (PP), polyamide 6 (PA6) and polyetheretherketone (PEEK)) were spun into fibers via single screw extrusion and monoaxial stretching. Fibers then were embedded in a concrete matrix, and Single-Fiber-Pull-Out-Tests (SFPT) were conducted to investigate bonding characteristics and microstructural interface of the composite. Differences in maximum pull-out-force, displacement and slope of the linear part of force vs displacement-function, which depicts the adhesion strength and the ductility of the interfacial bond were studied. In SFPT fiber, debonding is an inhomogeneous process, where the combination of interfacial bonding and friction mechanisms add up to a resulting value. Therefore, correlations between polymeric properties and pull-out-mechanisms have to be emphasized. To investigate these correlations, all fibers were introduced to a series of analysis such as differential scanning calorimetry (DSC), contact angle measurement, surface roughness and hardness analysis, tensile testing and scanning electron microscope (SEM). Of each polymer, smooth and abraded fibers were tested, first to simulate the abrasion and damage caused by a concrete mixing process and secondly to estimate the influence of mechanical anchoring of rough surfaces. In general, abraded fibers showed a significant increase in maximum pull-out-force due to better mechanical anchoring. Friction processes therefore play a major role to increase the maximum pull-out-force. The polymer hardness affects the tribological behavior and polymers with high hardness lead to lower surface roughness verified by SEM and surface roughness measurements. This concludes into a decreased maximum pull-out-force for hard polymers. High surface energy polymers show better interfacial bonding strength in general, which coincides with the conducted SFPT investigation. Polymers such as PEEK or PA6 show higher bonding strength in smooth and roughened fibers, revealed through high pull-out-force and concrete particles bonded on the fiber surface pictured via SEM analysis. The surface energy divides into dispersive and polar part, at which the slope is correlating with the polar part. Only polar polymers increase their SFPT-function slope due to better wetting abilities when showing a higher bonding area through rough surfaces. Hence, the maximum force and the bonding strength of an embedded fiber is a function of polarity, hardness, and consequently surface roughness. Other properties such as crystallinity or tensile strength do not affect bonding behavior. Through the conducted analysis, it is now feasible to understand and resolve different effects in pull-out-behavior step-by-step based on the polymer properties itself. This investigation developed a roadmap on how to engineer high adhering polymeric materials for fiber reinforcement of concrete.

Keywords: fiber-matrix interface, polymeric fibers, fiber reinforced concrete, single fiber pull-out test

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Authors: Reham K. Sebaih, Afaf I. Shehata , Awatif A. Hindi, Tarek Gheith, Amal A. Hazzani Anas Al-Orjan

Abstract:

Staphylococci spp are ubiquitous gram-positive bacteria is often associated with infections, especially nosocomial infections, and antibiotic resistanceStudy pathogenic bacteria and its use as a tool in the technology of Nano biology and molecular genetics research of the latest research trends of modern characterization and definition of different multiresistant of bacteria including Staphylococci. The Staphylococci are widespread all over the world and particularly in Saudi Arabia The present work study was conducted to evaluate the effect of five different types of nanoparticles (biosynthesized zinc oxide, Spherical and rod of each silver and gold nanoparticles) and their antibacterial impact on the Staphylococcus species. Ninety-six isolates of Staphylococcus species. Staphylococcus aureus, Staphylococcus epidermidis, MRSA were collected from different sources during the period between March 2011G to June 2011G. All isolates were isolated from inpatients and outpatients departments at Royal Commission Hospital in Yanbu Industrial, Saudi Arabia. High percentage isolation from males(55%) than females (45%). Staphylococcus epidermidis from males was (47%), (28%), and(25%). For Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus (MRSA. Isolates from females were Staphylococcus aureus with higher percent of (47%), (30%), and (23%) for MRSA, Staphylococcus epidermidis. Staphylococcus aureus from wound swab were the highest percent (51.42%) followed by vaginal swab (25.71%). Staphylococcus epidermidis were founded with higher percentage in blood (37.14%) and wound swab (34.21%) respectively related to other. The highest percentage of methicillin-resistant Staphylococcus aureus (MRSA)(80.77%) were isolated from wound swab, while those from nostrils were (19.23%). Staphylococcus species were isolates in highest percentage from hospital Emergency department with Staphylococcus aureus (59.37%), Methicillin-resistant Staphylococcus aureus (MRSA) (28.13%)and Staphylococcus epidermidis (12.5%) respectively. Evaluate the antibacterial property of Zinc oxide, Silver, and Gold nanoparticles as an alternative to conventional antibacterial agents Staphylococci isolates from hospital sources we screened them. Gold and Silver rods Nanoparticles to be sensitive to all isolates of Staphylococcus species. Zinc oxide Nanoparticles gave sensitivity impact range(52%) and (48%). The Gold and Silver spherical nanoparticles did not showed any effect on Staphylococci species. Zinc Oxide Nanoparticles gave bactericidal impact (25%) and bacteriostatic impact (75%) for of Staphylococci species. Detecting the association of nanoparticles with Staphylococci isolates imaging by scanning electron microscope (SEM) of some bacteriostatic isolates for Zinc Oxide nanoparticles on Staphylococcus aureus, Staphylococcus epidermidis and Methicillin resistant Staphylococcus aureus(MRSA), showed some Overlapping Bacterial cells with lower their number and appearing some appendages with deformities in external shape. Molecular analysis was applied by Multiplex polymerase chain reaction (PCR) used for the identification of genes within Staphylococcal pathogens. A multiplex polymerase chain reaction (PCR) method has been developed using six primer pairs to detect different genes using 50bp and 100bp DNA ladder marker. The range of Molecular gene typing ranging between 93 bp to 326 bp for Staphylococcus aureus and Methicillin resistant Staphylococcus aureus by TSST-1,mecA,femA and eta, while the bands border were from 546 bp to 682 bp for Staphylococcus epidermidis using icaAB and atlE. Sixteen isolation of Staphylococcus aureus and Methicillin resistant Staphylococcus aureus were positive for the femA gene at 132bp,this allowed the using of this gene as an internal positive control, fifteen isolates of Staphylococcus aureus and Methicillin resistant Staphylococcus aureus were positive for mecA gene at163bp.This gene was responsible for antibiotic resistant Methicillin, Two isolates of Staphylococcus aureus and Methicillin resistant Staphylococcus aureus were positive for the TSST-1 gene at326bp which is responsible for toxic shock syndrome in some Staphylococcus species, None were positive for eta gene at 102bpto that was responsible for Exfoliative toxins. Six isolates of Staphylococcus epidermidis were positive for atlE gene at 682 bp which is responsible for the initial adherence, three isolates of Staphylococcus epidermidis were positive for icaAB gene at 546bp that are responsible for mediates the formation of the biofilm. In conclusion, this study demonstrates the ability of the detection of the genes to discriminate between infecting Staphylococcus strains and considered biological tests, they may potentiate the clinical criteria used for the diagnosis of septicemia or catheter-related infections.

Keywords: multiplex polymerase chain reaction, toxic shock syndrome, Staphylococcus aureus, nosocomial infections

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