Search results for: bone marrow cells

Authors: Marcel Verwey, Anna M. Joubert, Elsie M. Nolte, Wolfgang Dohle, Barry V. L. Potter, Anne E. Theron

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A tetrahydroisoquinolinone (THIQ) core can be used to mimic the A,B-ring of colchicine site-binding microtubule disruptors such as 2-methoxyestradiol in the design of anti-cancer agents. Steroidomimeric microtubule disruptors were synthesized by introducing C'2 and C'3 of the steroidal A-ring to C'6 and C'7 of the THIQ core and by introducing a decorated hydrogen bond acceptor motif projecting from the steroidal D-ring to N'2. For this in vitro study, four non-steroidal THIQ-based analogues were investigated and comparative studies were done between the non-sulphamoylated compound STX 3450 and the sulphamoylated compounds STX 2895, STX 3329 and STX 3451. The objective of this study was to investigate the modes of cell death induced by these four THIQ-based analogues in A549 lung carcinoma epithelial cells and metastatic breast adenocarcinoma MDA-MB-231 cells. Cytotoxicity studies to determine the half maximal growth inhibitory concentrations were done using spectrophotometric quantification via crystal violet staining and lactate dehydrogenase (LDH) assays. Microtubule integrity and morphologic changes of exposed cells were investigated using polarization-optical transmitted light differential interference contrast microscopy, transmission electron microscopy and confocal microscopy. Flow cytometric quantification was used to determine apoptosis induction and the effect that THIQ-based analogues have on cell cycle progression. Signal transduction pathways were elucidated by quantification of the mitochondrial membrane integrity, cytochrome c release and caspase 3, -6 and -8 activation. Induction of autophagic cell death by the THIQ-based analogues was investigated by morphological assessment of fluorescent monodansylcadaverine (MDC) staining of acidic vacuoles and by quantifying aggresome formation via flow cytometry. Results revealed that these non-steroidal microtubule disrupting analogues inhibited 50% of cell growth at nanomolar concentrations. Immunofluorescence microscopy indicated microtubule depolarization and the resultant mitotic arrest was further confirmed through cell cycle analysis. Apoptosis induction via the intrinsic pathway was observed due to depolarization of the mitochondrial membrane, induction of cytochrome c release as well as, caspase 3 activation. Potential involvement of programmed cell death type II was observed due to the presence of acidic vacuoles and aggresome formation. Necrotic cell death did not contribute significantly, indicated by stable LDH levels. This in vitro study revealed the induction of the intrinsic apoptotic pathway as well as possible involvement of autophagy after exposure to these THIQ-based analogues in both MDA-MB-231- and A549 cells. Further investigation of this series of anticancer drugs still needs to be conducted to elucidate the temporal, mechanistic and functional crosstalk mechanisms between the two observed programmed cell deaths pathways.

Keywords: apoptosis, autophagy, cancer, microtubule disruptor

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Authors: Maryam Firouzmandi, Moosa Miri

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Common reasons for early tooth loss are trauma, extraction due to caries or periodontal disease and congenital missing. The remaining space after tooth loss may cause functional and esthetic problems. Therefore restorative dentists should attempt to manage these spaces using conservative methods. The goal is to restore the lost esthetic and function, prevent phonetic, self-esteem and personality problems and tongue habits. Preserving alveolar bone is also of great importance during the growth stage. Purpose: When deciding about the management of the missing tooth, space implants are contradicted until the completion of dentoalveolar development. Even in adulthood, due to systemic or periodontal problems or biological and economic issues, the implant might not be indicated. In this article, the alternative conservative restorative methods of space maintenance are going to be discussed. Essix retainers are made chair-side as easy as forming a custom bleaching tray with some modifications. They are esthetically acceptable and not expensive. These temporaries provide support for the lips but could not be used during function. Mini-screw-supported temporaries are another option for maintaining the space, especially after orthodontic treatment when there is a time lag between the termination of orthodontic treatment and definitive restoration. Two techniques will be presented for this kind of restoration: Denture tooth pontic or a composite crown. The benefits are alveolar bone preservation, Physiologic pressure on the alveolar ridge to increase its density and even can be retained until the completion of the definitive treatment. Bonded fixed partial denture includes Maryland bridge, fiber-reinforced composite bridge, resin-bonded bridge, and ceramic bonded bridge. These types of bridges are recommended to be used after a pubertal growth spurt and a recent meta-analysis considered their clinical success similar to conventional FDPs and implant-supported crowns. However, they have several advantages that are going to be discussed by presenting some clinical examples. Practical instruction on how to construct an FRC bridge and a novel chair-side Maryland bridge will be given by means of clinical cases. Clinical relevance: minimally invasive options should always be considered and destruction of healthy enamel and dentin during the preparation phase should be avoided as much as possible.

Keywords: tooth missing, fiber-reinforced composite, Maryland, Essix retainers, screw-retained restoration

Procedia PDF Downloads 188

Authors: Flavien Marteau, Pedro Affonso Nóbrega, Pascal Biwole, Nicolas Autrusson, Iona De Bievre, Christian Beauger

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Effective water management is essential for the optimal performance of fuel cells. For this reason, many vehicle systems use a membrane humidifier, a passive device that humidifies the air before the cathode inlet. Although they offer good performance, humidifiers are voluminous, costly, and fragile, hence the desire to find an alternative. Direct water injection could be an option, although this method lacks maturity. It consists of injecting liquid water as a spray in the dry heated air coming out from the compressor. This work focuses on the evaluation of direct water injection and its performance compared to the membrane humidifier selected as a reference. Two architectures were experimentally tested to humidify an industrial 2 kW short stack made up of 20 cells of 150 cm² each. For the reference architecture, the inlet air is humidified with a commercial membrane humidifier. For the direct water injection architecture, a pneumatic nozzle was selected to generate a fine spray in the air flow with a Sauter mean diameter of about 20 μm. Initial performance was compared over the entire range of current based on polarisation curves. Then, the influence of various parameters impacting water management was studied, such as the temperature, the gas stoichiometry, and the water injection flow rate. The experimental results obtained confirm the possibility of humidifying the fuel cell using direct water injection. This study, however shows the limits of this humidification method, the mean cell voltage being significantly lower in some operating conditions with direct water injection than with the membrane humidifier. The voltage drop reaches 30 mV per cell (4 %) at 1 A/cm² (1,8 bara, 80 °C) and increases in more demanding humidification conditions. It is noteworthy that the heat of compression available is not enough to evaporate all the injected liquid water in the case of DWI, resulting in a mix of liquid and vapour water entering the fuel cell, whereas only vapour is present with the humidifier. Variation of the injection flow rate shows that part of the injected water is useless for humidification and seems to cross channels without reaching the membrane. The stack was successfully humidified thanks to direct water injection. Nevertheless, our work shows that its implementation requires substantial adaptations and may reduce the fuel cell stack performance when compared to conventional membrane humidifiers, but opportunities for optimisation have been identified.

Keywords: cathode humidification, direct water injection, membrane humidifier, proton exchange membrane fuel cell

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Authors: Ya L. V., Benjamin Ong Wei Lin, Wanli Niu, Benjamin Seah Chin Tat

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This article introduces a methodology that improves reliability and cyclability of 2nd-life Li-ion battery system repurposed as an energy storage system (ESS). Most of the 2nd-life retired battery systems in the market have module/pack-level state-of-health (SOH) indicator, which is utilized for guiding appropriate depth-of-discharge (DOD) in the application of ESS. Due to the lack of cell-level SOH indication, the different degrading behaviors among various cells cannot be identified upon reaching retired status; in the end, considering end-of-life (EOL) loss and pack-level DOD, the repurposed ESS has to be oversized by > 1.5 times to complement the application requirement of reliability and cyclability. This proposed battery grading algorithm, using non-invasive methodology, is able to detect outlier cells based on historical voltage data and calculate cell-level historical maximum temperature data using semi-analytic methodology. In this way, the individual battery cell in the 2nd-life battery system can be graded in terms of SOH on basis of the historical voltage fluctuation and estimated historical maximum temperature variation. These grades will have corresponding DOD grades in the application of the repurposed ESS to enhance system reliability and cyclability. In all, this introduced battery grading algorithm is non-invasive, compatible with all kinds of retired Li-ion battery systems which lack of cell-level SOH indication, as well as potentially being embedded into battery management software for preventive maintenance and real-time cyclability optimization.

Keywords: battery grading algorithm, 2nd-life repurposing battery system, semi-analytic methodology, reliability and cyclability

Procedia PDF Downloads 187

Authors: A. Y. Tsidulko, T. M. Pankova, E. V. Grigorieva

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High-grade gliomas are the most frequent and most aggressive brain tumors which are characterized by active invasion of tumor cells into the surrounding brain tissue, where the extracellular matrix (ECM) plays a crucial role. Disruption of ECM can be involved in anticancer drugs effectiveness, side-effects and also in tumor relapses. The anti-inflammatory agent dexamethasone is a common drug used during high-grade glioma treatment for alleviating cerebral edema. Although dexamethasone is widely used in the clinic, its effects on normal brain tissue ECM remain poorly investigated. It is known that proteoglycans (PGs) are a major component of the extracellular matrix in the central nervous system. In our work, we studied the effects of dexamethasone on the ECM proteoglycans (syndecan-1, glypican-1, perlecan, versican, brevican, NG2, decorin, biglican, lumican) using RT-PCR in the experimental animal model. It was shown that proteoglycans in rat brain have age-specific expression patterns. In early post-natal rat brain (8 days old rat pups) overall PGs expression was quite high and mainly expressed PGs were biglycan, decorin, and syndecan-1. The overall transcriptional activity of PGs in adult rat brain is 1.5-fold decreased compared to post-natal brain. The expression pattern was changed as well with biglycan, decorin, syndecan-1, glypican-1 and brevican becoming almost equally expressed. PGs expression patterns create a specific tissue microenvironment that differs in developing and adult brain. Dexamethasone regimen close to the one used in the clinic during high-grade glioma treatment significantly affects proteoglycans expression. It was shown that overall PGs transcription activity is 1.5-2-folds increased after dexamethasone treatment. The most up-regulated PGs were biglycan, decorin, and lumican. The PGs expression pattern in adult brain changed after treatment becoming quite close to the expression pattern in developing brain. It is known that microenvironment in developing tissues promotes cells proliferation while in adult tissues proliferation is usually suppressed. The changes occurring in the adult brain after dexamethasone treatment may lead to re-activation of cell proliferation due to signals from changed microenvironment. Taken together obtained data show that dexamethasone treatment significantly affects the normal brain ECM, creating the appropriate microenvironment for tumor cells proliferation and thus can reduce the effectiveness of anticancer treatment and promote tumor relapses. This work has been supported by a Russian Science Foundation (RSF Grant 16-15-10243)

Keywords: dexamthasone, extracellular matrix, glioma, proteoglycan

Procedia PDF Downloads 184

Authors: Ife Elegbeleye, Nnditshedzeni Eric, Regina Maphanga, Femi Elegbeleye, Femi Agunbiade

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The global potential of other renewable energy sources such as wind, hydroelectric, bio-mass, and geothermal is estimated to be approximately 13 %, with hydroelectricity constituting a larger percentage. Sunlight provides by far the largest of all carbon-neutral energy sources. More energy from the sunlight strikes the Earth in one hour (4.3 × 1020 J) than all the energy consumed on the planet in a year (4.1 × 1020 J), hence, solar energy remains the most abundant clean, renewable energy resources for mankind. Photovoltaic (PV) devices such as silicon solar cells, dye sensitized solar cells are utilized for harnessing solar energy. Polyene-diphenylaniline organic molecules are important sets of molecules that has stirred many research interest as photosensitizers in TiO₂ semiconductor-based dye sensitized solar cells (DSSCs). The advantages of organic dye molecule over metal-based complexes are higher extinction coefficient, moderate cost, good environmental compatibility, and electrochemical properties. The polyene-diphenylaniline organic dyes with basic configuration of donor-π-acceptor are affordable, easy to synthesize and possess chemical structures that can easily be modified to optimize their photocatalytic and spectral properties. The enormous interest in polyene-diphenylaniline dyes as photosensitizers is due to their fascinating spectral properties which include visible light to near infra-red-light absorption. In this work, density functional theory approach via GPAW software, Avogadro and ASE were employed to study the effect of methoxy functionalized group on the spectral properties of polyene-diphenylaniline dyes and their photons absorbing characteristics in the visible region to near infrared region of the solar spectrum. Our results showed that the two-phenyl based complexes D5 and D7 exhibits maximum absorption peaks at 750 nm and 850 nm, while D9 and D11 with methoxy group shows maximum absorption peak at 800 nm and 900 nm respectively. The highest absorption wavelength is notable for D9 and D11 containing additional polyene and methoxy groups. Also, D9 and D11 chromophores with the methoxy group shows lower energy gap of 0.98 and 0.85 respectively than the corresponding D5 and D7 dyes complexes with energy gap of 1.32 and 1.08. The analysis of their electron injection kinetics ∆Ginject into the band gap of TiO₂ shows that D9 and D11 with the methoxy group has higher electron injection kinetics of -2.070 and -2.030 than the corresponding polyene-diphenylaniline complexes without the addition of polyene group with ∆Ginject values of -2.820 and -2.130 respectively. Our findings suggest that the addition of functionalized group as an extension of the organic complexes results in higher light harvesting efficiencies and bathochromic shift of the absorption spectra to higher wavelength which suggest higher current densities and open circuit voltage in DSSCs. The study suggests that the photocatalytic properties of organic chromophores/complexes with donor-π-acceptor configuration can be enhanced by the addition of functionalized groups.

Keywords: renewable energy resource, solar energy, dye sensitized solar cells, polyene-diphenylaniline organic chromophores

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Authors: Moustafa Osman Mohammed

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The photocurrent generations are influencing ultra-high efficiency solar cells based on self-assembled quantum dot (QD) nanostructures. Nanocrystal quantum dots (QD) provide a great enhancement toward solar cell efficiencies through the use of quantum confinement to tune absorbance across the solar spectrum enabled multi-exciton generation. Based on theoretical predictions, QDs have potential to improve systems efficiency in approximate regular electrons excitation intensity greater than 50%. In solar cell devices, an intermediate band formed by the electron levels in quantum dot systems. The spatial architecture is exploring how can solar cell integrate and produce not only high open circuit voltage (> 1.7 eV) but also large short-circuit currents due to the efficient absorption of sub-bandgap photons. In the proposed QD system, the structure allows barrier material to absorb wavelengths below 700 nm while multi-photon processes in the used quantum dots to absorb wavelengths up to 2 µm. The assembly of the electronic model is flexible to demonstrate the atoms and molecules structure and material properties to tune control energy bandgap of the barrier quantum dot to their respective optimum values. In terms of energy virtual conversion, the efficiency and cost of the electronic structure are unified outperform a pair of multi-junction solar cell that obtained in the rigorous test to quantify the errors. The milestone toward achieving the claimed high-efficiency solar cell device is controlling the edge causes of energy bandgap between the barrier material and quantum dot systems according to the media design limits. Despite this remarkable potential for high photocurrent generation, the achievable open-circuit voltage (Voc) is fundamentally limited due to non-radiative recombination processes in QD solar cells. The orientation of voltage recovery system is compared theoretically with experimental Voc variation in mediation upper–limit obtained one diode modeling form at the cells with different bandgap (Eg) as classified in the proposed spatial architecture. The opportunity for improvement Voc is valued approximately greater than 1V by using smaller QDs through QD solar cell recovery systems as confined to other micro and nano operations states.

Keywords: nanotechnology, photovoltaic solar cell, quantum systems, renewable energy, environmental modeling

Procedia PDF Downloads 138

Authors: Albert Mufundirwa, Satoru Yoshioka, K. Ogi, Takeharu Sugiyama, George F. Harrington, Bretislav Smid, Benjamin Cunning, Kazunari Sasaki, Akari Hayashi, Stephen M. Lyth

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Polymer electrolyte membrane fuel cells (PEMFCs) are electrochemical energy conversion devices used for portable, residential and vehicular applications due to their low emissions, high efficiency, and quick start-up characteristics. However, PEMFCs generally use expensive, Pt-based electrocatalysts as electrode catalysts. Due to the high cost and limited availability of platinum, research and development to either drastically reduce platinum loading, or replace platinum with alternative catalysts is of paramount importance. A combination of high surface area supports and nano-structured active sites is essential for effective operation of catalysts. We synthesize carbon foam supports by thermal decomposition of sodium ethoxide, using a template-free, gram scale, cheap, and scalable pyrolysis method. This carbon foam has a high surface area, highly porous, three-dimensional framework which is ideal for electrochemical applications. These carbon foams can have surface area larger than 2500 m²/g, and electron microscopy reveals that they have micron-scale cells, separated by few-layer graphene-like carbon walls. We applied this carbon foam as a platinum catalyst support, resulting in the improved electrochemical surface area and mass activity for the oxygen reduction reaction (ORR), compared to carbon black. Similarly, silver-decorated carbon foams showed higher activity and efficiency for electrochemical carbon dioxide conversion than silver-decorated carbon black. A promising alternative to Pt-catalysts for the ORR is iron-impregnated nitrogen-doped carbon catalysts (Fe-N-C). Doping carbon with nitrogen alters the chemical structure and modulates the electronic properties, allowing a degree of control over the catalytic properties. We have adapted our synthesis method to produce nitrogen-doped carbon foams with large surface area, using triethanolamine as a nitrogen feedstock, in a novel bottom-up protocol. These foams are then infiltrated with iron acetate (FeAc) and pyrolysed to form Fe-N-C foams. The resulting Fe-N-C foam catalysts have high initial activity (half-wave potential of 0.68 VRHE), comparable to that of commercially available Pt-free catalysts (e.g., NPC-2000, Pajarito Powder) in acid solution. In alkaline solution, the Fe-N-C carbon foam catalysts have a half-wave potential of 0.89 VRHE, which is higher than that of NPC-2000 by almost 10 mVRHE, and far out-performing platinum. However, the durability is still a problem at present. The lessons learned from X-ray absorption spectroscopy (XAS), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), and electrochemical measurements will be used to carefully design Fe-N-C catalysts for higher performance PEMFCs.

Keywords: carbon-foam, polymer electrolyte membrane fuel cells, platinum, Pt-free, Fe-N-C, ORR

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Authors: Jihad Daba, Jean-Pierre Dubois

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Multi path fading noise degrades the performance of cellular communication, most notably in femto- and pico-cells in 3G and 4G systems. When the wireless channel consists of a small number of scattering paths, the statistics of fading noise is not analytically tractable and poses a serious challenge to developing closed canonical forms that can be analysed and used in the design of efficient and optimal receivers. In this context, noise is multiplicative and is referred to as stochastically local fading. In many analytical investigation of multiplicative noise, the exponential or Gamma statistics are invoked. More recent advances by the author of this paper have utilized a Poisson modulated and weighted generalized Laguerre polynomials with controlling parameters and uncorrelated noise assumptions. In this paper, we investigate the statistics of multi-diversity stochastically local area fading channel when the channel consists of randomly distributed Rayleigh and Rician scattering centers with a coherent specular Nakagami-distributed line of sight component and an underlying doubly stochastic Poisson process driven by a lognormal intensity. These combined statistics form a unifying triply stochastic filtered marked Poisson point process model.

Keywords: cellular communication, femto and pico-cells, stochastically local area fading channel, triply stochastic filtered marked Poisson point process

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Authors: Savitha Janakiraman, Shivakumar M. C

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Anidulafungin, an advanced class of antifungal agent used for the treatment of chronic fungal infections, is derived from Echinocandin B nucleus, an intermediate metabolite of Echinocandin B produced by Aspergillus nidulans. The enzyme acylase derived from the fermentation broth of Actinoplanes utahensis (NRRL 12052) plays a key role in the bioconversion of echinocandin B to echinocandin B nucleus. The membrane-bound nature of acylase and low levels of expression contributes to the rate-limiting process of enzymatic deacylation, hence low yields of ECB nucleus and anidulafungin. In the present study, this is addressed through novel genetic engineering approaches of overexpression and heterologous expression studies, immobilization of whole cells of Actinoplanes utahensis (NRRL 12052) and Co-cultivation studies. Overexpression of the acylase gene in Actinoplanes utahensis (NRRL 12052) was done by increasing the gene copy number to increase the echinocandin B nucleus production. Echinocandin B acylase gene, under the control of a PermE* promoter, was cloned in pSET152 vector and introduced into Actinoplanes utahensis (NRRL12052) by a ɸC31-directed site-specific recombination method. The resultant recombinant strain (C2-18) showed a 3-fold increase in acylase expression, which was confirmed by HPLC analysis. Pichia pastoris is one of the most effective and versatile host systems for the production of heterologous proteins. The ECB acylase gene was cloned into pPIC9K vector with AOX1 promoter and was transformed into Pichia pastoris (GS115). The acylase expression was confirmed by protein expression and bioconversion studies. The heterologous expression of acylase in Pichia pastoris, is a milestone in the development of antifungals. Actively growing cells of Actinoplanes utahensis (NRRL 12052) were immobilized and tested for bioconversion ability which showed >90% conversion in each cycle. The stability of immobilized cell beads retained the deacylation ability up to 60 days and reusability was confirmed up to 4 cycles. The significant findings from the study have revealed that immobilization of whole cells of Actinoplanes utahensis (NRRL 12052) could be an alternative option for bioconversion of echinocandin B to echinocandin B nucleus, which has not been reported to date. The concept of co-cultivation of Aspergillus nidulans and Actinoplanes utahensis strains for the production of the echinocandin B nucleus was also carried out in order to produce echinocandin B nucleus. The process completely reduced the ECB purification step and, therefore, could be recommended as an ingenious method to improve the yield of the ECB nucleus.

Keywords: acylase, anidulafungin, antifungals, Aspergillus nidulans

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Authors: Eyyup Murat Karakurt, Yan Huang, Mehmet Kaya, Huseyin Demirtas

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In this study, the effect of the porosity effect of Ti-20Zr alloy produced by powder metallurgy as a biomaterial was investigated experimentally. The Ti based alloys (Ti-20%Zr (at.) were produced under 300 MPa, for 6 h at 1200 °C. Afterward, the microstructure of the Ti-based alloys was analyzed by optical analysis, scanning electron microscopy, energy dispersive spectrometry. Moreover, compression tests were applied to determine the mechanical behaviour of samples. As a result, highly porous Ti-20Zr alloys exhibited an elastic modulus close to human bone. The results later were compared theoretically and experimentally.

Keywords: porosity effect, Ti based alloys, elastic modulus, compression test

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Authors: Rahul Madathiparambil Visalakshan, Alex Cavallaro, John Hayball, Krasimir Vasilev

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The role of biomaterial surface nanotopograhy on fibrinogen adsorption and unfolding, and the subsequent immune response were studied. Inconsistent topography and varying chemical functionalities along with a lack of reproducibility pose a challenge in determining the specific effects of nanotopography or chemistry on proteins and cells. It is important to have a well-defined nanotopography with a homogeneous chemistry to study the real effect of nanotopography on biological systems. Therefore, we developed a technique that can produce well-defined and highly reproducible topography to identify the role of specific roughness, size, height and density with the presence of homogeneous chemical functionality. Using plasma polymerisation of oxazoline monomers and immobilized gold nanoparticles we created surfaces with an equal number density of nanoparticles of different sizes. This surface was used to study the role of surface nanotopography and the interplay of surface chemistry on proteins and immune cells. The effect of nanotopography on fibrinogen adsorption was investigated using Quartz Cristal Microbalance with Dissipation and micro BCA. The mass of fibrinogen adsorbed on the surface increased with increasing size of nano-topography. Protein structural changes up on adsorption to the nano rough surface was studied using circular dichroism spectroscopy. Fibrinogen unfolding varied depending on the specific nanotopography of the surfaces. It was revealed that the in vitro immune response to the nanotopography surfaces changed due to this protein unfolding.

Keywords: biomaterial inflammation, protein and cell responses, protein unfolding, surface nanotopography

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Authors: Mohammed N. Al Kindi, Mazin Al Khabouri, Khalsa Al Lamki, Tommasso Pappuci, Giovani Romeo, Nadia Al Wardy

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Hereditary hearing loss is a heterogeneous group of complex disorders with an overall incidence of one in every five hundred newborns presented as syndromic and non-syndromic forms. Cadherin-related 23 (CDH23) is one of the listed deafness causative genes. CDH23 is found to be expressed in the stereocilia of hair cells and the retina photoreceptor cells. Defective CDH23 has been associated mostly with prelingual severe-to-profound sensorineural hearing loss (SNHL) in either syndromic (USH1D) or non-syndromic SNHL (DFNB12). An Omani family diagnosed clinically with severe-profound sensorineural hearing loss was genetically analysed by whole exome sequencing technique. A novel homozygous missense variant, c.A7451C (p.D2484A), in exon 53 of CDH23 was detected. One hundred and thirty control samples were analysed where all were negative for the detected variant. The variant was analysed in silico for pathogenicity verification using several mutation prediction software. The variant proved to be a pathogenic mutation and is reported for the first time in Oman and worldwide. It is concluded that in silico mutation prediction analysis might be used as a useful molecular diagnostics tool benefiting both genetic counseling and mutation verification. The aspartic acid 2484 alanine missense substitution might be the main disease-causing mutation that damages CDH23 function and could be used as a genetic hearing loss marker for this particular Omani family.

Keywords: Cdh23, d2484a, in silico, Oman

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Authors: Lista Andriyati, Nurpudji A Taslim

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The human immunodeficiency virus (HIV) patients have potential nutritional and metabolic problems. HIV is a virus that attacks cells T helper and impairs the function of immune cells. Infected individuals gradually become immunodeficient, results in increased susceptibility to a wide range of infections such as tuberculosis (TB). Malnutrition has destructive effects on the immune system and host defense mechanisms. Effective and proper nutritional therapies are important to improve medical outcomes and quality of life, which is associated with functional improvement. A case of 38-years old man admitted to hospital with loss of consciousness and was diagnosed HIV infection and relapse lung TB with severe malnutrition, fever, oral candidiasis, anemia (6.3 g/dL), severe hypoalbuminemia (1.9 g/dL), severe hypokalemia (2.2 mmol/L), immune depletion (1085 /µL) and elevated liver enzyme (ALT 1198/AST 375 U/L). Nutritional intervention by giving 2300 kcal of energy, protein 2 g/IBW/day, carbohydrate 350 g, fat 104 g through enteral and parenteral nutrition. Supplementations administered are zinc, vitamin A, vitamin B1, vitamin B6, vitamin B12, vitamin C, vitamin D, and snakehead fish extract high content of protein albumin (Pujimin®). After 46 days, there are clinical and metabolic improvement in Hb (6.3 to 11.2 g/dL), potassium (2.2 to 3.4 mmol/L), albumin (1.9 to 2.3 g/dL), ALT 1198 to 47/AST 375 to 68 U/L) and improved awareness. In conclusion, nutritional therapy in HIV infection with adequate macronutrients and micronutrients fulfillment and immunonutrition is very important to avoid cachexia and to improve nutritional status and immune disfunction.

Keywords: HIV, hypoalbuminemia, malnutrition, tuberculosis

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Authors: Zeytu G. Asfaw, Serkalem K. Abrha, Demisew G. Degefu

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Background: HIV/AIDS remains a major public health problem in Ethiopia and heavily affecting people of productive and reproductive age. We aimed to compare the performance of Parametric Survival Analysis and Bayesian Survival Analysis using simulations and in a real dataset application focused on determining predictors of HIV patient survival. Methods: A Parametric Survival Models - Exponential, Weibull, Log-normal, Log-logistic, Gompertz and Generalized gamma distributions were considered. Simulation study was carried out with two different algorithms that were informative and noninformative priors. A retrospective cohort study was implemented for HIV infected patients under Highly Active Antiretroviral Therapy in Alamata General Hospital, North Ethiopia. Results: A total of 320 HIV patients were included in the study where 52.19% females and 47.81% males. According to Kaplan-Meier survival estimates for the two sex groups, females has shown better survival time in comparison with their male counterparts. The median survival time of HIV patients was 79 months. During the follow-up period 89 (27.81%) deaths and 231 (72.19%) censored individuals registered. The average baseline cluster of differentiation 4 (CD4) cells count for HIV/AIDS patients were 126.01 but after a three-year antiretroviral therapy follow-up the average cluster of differentiation 4 (CD4) cells counts were 305.74, which was quite encouraging. Age, functional status, tuberculosis screen, past opportunistic infection, baseline cluster of differentiation 4 (CD4) cells, World Health Organization clinical stage, sex, marital status, employment status, occupation type, baseline weight were found statistically significant factors for longer survival of HIV patients. The standard error of all covariate in Bayesian log-normal survival model is less than the classical one. Hence, Bayesian survival analysis showed better performance than classical parametric survival analysis, when subjective data analysis was performed by considering expert opinions and historical knowledge about the parameters. Conclusions: Thus, HIV/AIDS patient mortality rate could be reduced through timely antiretroviral therapy with special care on the potential factors. Moreover, Bayesian log-normal survival model was preferable than the classical log-normal survival model for determining predictors of HIV patients survival.

Keywords: antiretroviral therapy (ART), Bayesian analysis, HIV, log-normal, parametric survival models

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Authors: Nouf Alharbi, James O'shea

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For decades, renewable energy sources have received considerable global interest due to the increase in fossil fuel consumption. The abundant energy produced by sunlight makes dye-sensitised solar cells (DSSCs) a promising alternative compared to conventional silicon and thin film solar cells due to their transparency and tunable colours, which make them suitable for applications such as windows and glass facades. The transfer of an excited electron onto the surface is an important procedure in the DSSC system, so different groups of dye molecules were studied on the rutile TiO2 (110) surface. Currently, the study of organic dyes has become an interest of researchers due to ruthenium being a rare and expensive metal, and metal-free organic dyes have many features, such as high molar extinction coefficients, low manufacturing costs, and ease of structural modification and synthesis. There are, of course, some groups that have developed organic dyes and exhibited lower light-harvesting efficiency ranging between 4% and 8%. Since most dye molecules are complicated or fragile to be deposited by thermal evaporation or sublimation in the ultra-high vacuum (UHV), all dyes (i.e, D5, SC4, and R6) in this study were deposited in situ using the electrospray deposition technique combined with X-ray photoelectron spectroscopy (XPS) as an alternative method to obtain high-quality monolayers of titanium dioxide. These organic molecules adsorbed onto rutile TiO2 (110) are explored by XPS, which can be used to obtain element-specific information on the chemical structure and study bonding and interaction sites on the surface.

Keywords: dyes, deposition, electrospray, molecules, organic, rutile, sensitised, XPS

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Authors: Aliaa M. El-Borai, Ehab A. Beltagy, Eman E. Gadallah, Samy A. ElAssar

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Bioremediation technology is now used for treatment instead of traditional metal removal methods. A strain was isolated from Marsa Alam, Red sea, Egypt showed high resistance to high lead concentration and was identified by the 16S rRNA gene sequencing technique as Halomonas sp. ES015. Medium optimization was carried out using Plackett-Burman design, and the most significant factors were yeast extract, casamino acid and inoculums size. The optimized media obtained by the statistical design raised the removal efficiency from 84% to 99% from initial concentration 250 ppm of lead. Moreover, Box-Behnken experimental design was applied to study the relationship between yeast extract concentration, casamino acid concentration and inoculums size. The optimized medium increased removal efficiency to 97% from initial concentration 500 ppm of lead. Immobilized Halomonas sp. ES015 cells on sponge cubes, using optimized medium in loop bioremediation column, showed relatively constant lead removal efficiency when reused six successive cycles over the range of time interval. Also metal removal efficiency was not affected by flow rate changes. Finally, the results of this research refer to the possibility of lead bioremediation by free or immobilized cells of Halomonas sp. ES015. Also, bioremediation can be done in batch cultures and semicontinuous cultures using column technology.

Keywords: bioremediation, lead, Box–Behnken, Halomonas sp. ES015, loop bioremediation, Plackett-Burman

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Authors: Geeta Gahlawat, Sanjeev Kumar Soni

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An imprudent use of environmentally hazardous petrochemical-based plastics and limited availability of fossil fuels have provoked research interests towards production of biodegradable plastics - polyhydroxyalkanoate (PHAs). However, the industrial application of PHAs based products is primarily restricted by their high cost of recovery and extraction protocols. Moreover, solvents used for the extraction and purification are toxic and volatile which causes adverse environmental hazards. Development of efficient downstream recovery strategies along with utilization of non-toxic solvents will accelerate their commercialization. In this study, various extraction strategies were designed for sustainable and cost-effective recovery of PHAs from Cupriavidus necator using non-toxic environment friendly solvents viz. 1,2-propylene carbonate, ethyl acetate, isoamyl alcohol, butyl acetate. The effect of incubation time i.e. 10, 30 and 50 min and temperature i.e. 60, 80, 100, 120°C was tested to identify the most suitable solvent. PHAs extraction using a recyclable solvent, 1,2 propylene carbonate, showed the highest recovery yield (90%) and purity (93%) at 120°C and 30 min incubation. Ethyl acetate showed the better capacity to recover PHAs from cells than butyl acetate. Extraction with ethyl acetate exhibited high recovery yield and purity of 96% and 92%, respectively at 100°C. Effect of non-toxic surfactant such as linear alkylbenzene sulfonic acid (LAS) was also studied at 40, 60 and 80°C, and detergent pH range of 3.0, 5.0, 7.0 and 9.0 for the extraction of PHAs from the cells. LAS gave highest yield of 86% and purity of 88% at temperature 80°C and 5.0 pH.

Keywords: polyhydroxyalkanoates, Cupriavidus necator, extraction, recovery yield

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Authors: Mousa Saleh Daoud

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Rickets is a condition that affects the development of bones in children. It causes soft bones, which can become bowed or curved, this bending and curvature is evident in the age of Walking. The most common cause of rickets is dietary deficiency of vitamin D or Lack of exposure to sunlight or both together. The link between vitamin D and rickets has been known for many years and is well understood by doctors and scientists. If a child does not get enough of the vitamin D, the bones cannot form hard outer shells. This is why they become soft and weak. This study was conducted on children who reviewed by our medical clinic between the years 2011-2013. The study included 400 children, aged between one and six years. 11 children had clear clinical manifestations of rickets of varying degrees and all of them due to lack of vitamin D except for one case of rickets resistant to vitamin D. 389 cases ranged between natural and deficiency in vitamin D without clinical manifestations of Rickets.

Keywords: rickts, bone metabolic diseases, vitamin D, child

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Authors: Seok Woo Chang, Kee Yeon Kum, Kwang Shik Bae, WooCheol Lee

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Aim: The objective of this study was to compare the biocompatibilities and mineralization potential of ProRoot MTA and newly developed calcium phosphate based cement, Capseal. Materials and Methods: The biocompatibilities and mineralization-related gene expressions (Bone sialoprotein (BSP) and osteocalcin (OCN)) of ProRoot MTA and Capseal were also compared by a methylthiazol tetrazolium (MTT) assay and reverse transcription-polymerization chain reaction (RT-PCR) analysis on 1, 3, and 7 days, respectively. Empty rings were used as control group. The results were statistically analyzed by Kruskal-Wallis test with a Bonferroni correction. P-value of < 0.05 was considered significant. Results: The biocompatibilities of ProRoot MTA and Capseal were equally favorable. ProRoot MTA and Capseal affected the messenger RNA expression of osteocalcin and osteonectin. Conclusions: Based on the results, both ProRoot MTA and Capseal could be a useful biomaterial in clinical endodontics.

Keywords: biocompatibility, calcium silicate cement, MTT, RT-PCR

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Authors: L. Achab, F. Iachachene

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In microvessels, red blood cells (RBCs) exhibit a tendency to migrate towards the vessel center, establishing a core-annular flow pattern. The core region, marked by a high concentration of RBCs, is governed by significantly non-Newtonian viscosity. Conversely, the annular layer, composed of cell-free plasma, is characterized by Newtonian low viscosity. This property enables the plasma layer to act as a lubricant for the vessel walls, efficiently reducing resistance to the movement of blood cells. In this study, we investigate the factors influencing blood flow in microvessels and the thickness of the annular plasma layer using a non-miscible fluids approach in a 2D axisymmetric geometry. The governing equations of an incompressible unsteady flow are solved numerically through the Volume of Fluid (VOF) method to track the interface between the two immiscible fluids. To model blood viscosity in the core region, we adopt the Quemada constitutive law which is accurately captures the shear-thinning blood rheology over a wide range of shear rates. Our results are then compared to an established theoretical approach under identical flow conditions, particularly concerning the radial velocity profile and the thickness of the annular plasma layer. The simulation findings for low Reynolds numbers, demonstrate a notable agreement with the theoretical solution, emphasizing the pivotal role of blood’s rheological properties in the core region in determining the thickness of the annular plasma layer.

Keywords: core-annular flows, microvessels, Quemada model, plasma layer thickness, volume of fluid method

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Authors: H. Agbaria, A. Salman, M. Huleihel, G. Beck, D. H. Rich, S. Mordechai, J. Kapelushnik

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Viral and bacterial infections are responsible for variety of diseases. These infections have similar symptoms like fever, sneezing, inflammation, vomiting, diarrhea and fatigue. Thus, physicians may encounter difficulties in distinguishing between viral and bacterial infections based on these symptoms. Bacterial infections differ from viral infections in many other important respects regarding the response to various medications and the structure of the organisms. In many cases, it is difficult to know the origin of the infection. The physician orders a blood, urine test, or 'culture test' of tissue to diagnose the infection type when it is necessary. Using these methods, the time that elapses between the receipt of patient material and the presentation of the test results to the clinician is typically too long ( > 24 hours). This time is crucial in many cases for saving the life of the patient and for planning the right medical treatment. Thus, rapid identification of bacterial and viral infections in the lab is of great importance for effective treatment especially in cases of emergency. Blood was collected from 50 patients with confirmed viral infection and 50 with confirmed bacterial infection. White blood cells (WBCs) and plasma were isolated and deposited on a zinc selenide slide, dried and measured under a Fourier transform infrared (FTIR) microscope to obtain their infrared absorption spectra. The acquired spectra of WBCs and plasma were analyzed in order to differentiate between the two types of infections. In this study, the potential of FTIR microscopy in tandem with multivariate analysis was evaluated for the identification of the agent that causes the human infection. The method was used to identify the infectious agent type as either bacterial or viral, based on an analysis of the blood components [i.e., white blood cells (WBC) and plasma] using their infrared vibrational spectra. The time required for the analysis and evaluation after obtaining the blood sample was less than one hour. In the analysis, minute spectral differences in several bands of the FTIR spectra of WBCs were observed between groups of samples with viral and bacterial infections. By employing the techniques of feature extraction with linear discriminant analysis (LDA), a sensitivity of ~92 % and a specificity of ~86 % for an infection type diagnosis was achieved. The present preliminary study suggests that FTIR spectroscopy of WBCs is a potentially feasible and efficient tool for the diagnosis of the infection type.

Keywords: viral infection, bacterial infection, linear discriminant analysis, plasma, white blood cells, infrared spectroscopy

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Authors: Suthida Panwong, Jeeraporn Pekkoh, Yingmanee Tragoolpua, Aussara Panya

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Dengue virus (DENV) infection is a major public health problem in many countries, especially in tropical and subtropical countries. DENV infection causes dengue fever that can progress to serious conditions of dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), relevant to a high risk of mortality. However, there are no effective treatments available against the manifestation and fatalities. Currently, natural extracts have been widely used for the treatment of infectious diseases because to harmless, do not cumulate in health, or have lower side effects. Chlorella spp. is a microalgae with anti-viral activity, but there is not much report to support its ability to inhibit DENV infection. Thus, this study aimed to investigate the inhibitory effect of ethanolic extract from Chlorella sp. AARL G049, which was explored in Thailand on inhibition of DENV-2 infection. The inhibitory effect on viral infection was assessed using a foci-forming assay (FFA), which revealed that a concentration of 125 µg/mL could inhibit viral infection in Vero cells by 75.45±8.06% when treated at the same time as DENV-2 infection. Moreover, the extract at an equal concentration effectively reduced viral protein synthesis by 90.51±5.48% when assessed in human cell lines using enzyme-linked immunosorbent assay (ELISA). Concordantly, the number of infected cells after treatment was reduced as measured by immunofluorescent assay (IFA). Therefore, the finding of this study supports the potential use of Chlorella sp. extract to suppress DENV infection.

Keywords: viral infection, flavivirus, treatment, natural extract

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Authors: Zahra Khoshnood, Reza Khoshnood

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The aim of present study was to monitor the presence of Trichodina sp. in Rainbow trout, Oncorhynchus mykiss collected from various fish farms in the western provinces of Iran during January, 2013- January, 2014. Out of 675 sampled fish 335, (49.16%) were infested with Trichodina. The highest prevalence was observed in the spring and winter followed by autumn and summer. In general, the intensity of infection was low except in cases where outbreaks of Trichodiniasis endangered the survival of fish in some ponds. In light infestation Trichodina is usually present on gills, fins and skin of apparently healthy fish. Clinical signs of Trichodiniasis only appear on fish with heavy infections and cases of moderate ones that are usually exposed to one or more stress factors including, rough handling during transportation from ponds, overcrowdness, malnutrition, high of free ammonia and low of oxygen concentration. Clinical signs of Trichodiniasis in sampled fish were sluggish movement, loss of appetite, black coloration, necrosis and ulcer on different parts of the body, detached scales and excessive accumulation of mucous in gill pouches. The most obvious histopathological changes in diseased fish were sloughing of the epidermal layer, aggregation of leucocytes and melanine-carrying cells (between the dermis and hypodermis) and proliferative changes including hyperplasia and hypertrophy of the epithelial lining cells of gill filaments which resulted in fusion of secondary lamellae. Control of Trichodiniasis, has been achieved by formalin bath treatment at a concentration of 250 ppm for one hour.

Keywords: gill, histopathology, rainbow trout, Trichodina

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Authors: Balaji Devanathan, Gokul G, Raveena Divya, Abhishek Yadav, Sudhir K.Gupta

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Background: Age estimation is a common problem in administrative settings, medico legal cases, and among athletes competing in different sports. Age estimation is a problem in medico legal problems that arise in hospitals when there has been a criminal abortion, when consenting to surgery or a general physical examination, when there has been infanticide, impotence, sterility, etc. Medical imaging progress has benefited forensic anthropology in various ways, most notably in the area of determining bone age. An efficient method for researching the epiphyseal union and other differences in the body's bones and joints is multi-slice computed tomography. There isn't a significant database on Indians available. So to obtain an Indian based database author has performed this original study. Methodologies: The appearance and fusion of ossification centre of sternoclavicular joint is evaluated, and grades were assigned accordingly. Using MSCT scans, we examined the relationship between the age of the deceased and alterations in the sternoclavicular joint during the appearance and union in 500 instances, 327 men and 173 females, in the age range of 0 to 25 years. Results: According to our research in both the male and female groups, the ossification centre for the medial end of the clavicle first appeared between the ages of 18.5 and 17.1 respectively. The age range of the partial union was 20.4 and 20.2 years old. The earliest age of complete fusion was 23 years for males and 22 years for females. For fusion of their sternebrae into one, age range is 11–24 years for females and 17–24 years. The fusion of the third and fourth sternebrae was completed by 11 years. The fusions of the first and second and second and third sternebrae occur by the age of 17 years. Furthermore, correlation and reliability were carried out which yielded significant results. Conclusion: With numerous exceptions, the projected values are consistent with a large number of the previously developed age charts. These variations may be caused by the ethnic or regional heterogeneity in the ossification pattern among the population under study. The pattern of bone maturation did not significantly differ between the sexes, according to the study. The study's age range was 0 to 25 years, and for obvious reasons, the majority of the occurrences occurred in the last five years, or between 20 and 25 years of age. This resulted in a comparatively smaller study population for the 12–18 age group, where age estimate is crucial because of current legal requirements. It will require specialized PMCT research in this age range to produce population standard charts for age estimate. The medial end of the clavicle is one of several ossification foci that are being thoroughly investigated since they are challenging to assess with a traditional X-ray examination. Combining the two has been shown to be a valid result when it comes to raising the age beyond eighteen.

Keywords: age estimation, sternoclavicular joint, medial clavicle, computed tomography

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Authors: Nasrin Takzaree, Gholamreza Hassanzadeh, Abbas Hadjiakhoondi, Mohammadreza Rouini

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Introduction: Today herbal medicine are extensively used for various therapeutic reasons. Whereas Achillea millefolium L. comprises different chemical compounds it is used in classic and modern medicine for different purposes. Concerning the family planning as a principle matter, the idea of using specific herbal medicine is of great importance. Purpose: To investigate the effects of Achillea millefolium L. extract on fertility power and spermatogenesis process in male mature Wistar rats and the anti-fertility effects of this extract in male genital system. Material and methods: In this study 32 male mature Wistar rats were randomly divided in to 4 experimental groups. 1st experimental group included 8 rats receiving Achillea millefolium extract at the dose of 200 mg/kg intraperitoneally. Second and third groups received the extract the same at the doses of 400 and 800 mg/kg respectively. 4th group was considered as control group in which the parenteral distilled water was administered. after 20 days, rats were sacrificed and the spermatogenesis process was histologically examined. Results: In experimental groups receiving high doses of extract comparing with control group, thickness in seminiferous tubules basal membrane, decrease in germinal epithelium cells, congestion in testicular tissue, disarrangement in germinal epithelium cells as well as decrease in cellular condense were observed (p<0.001). Conclusion: Findings suggest that alcoholic extract of Achillea millefolium at high concentrations lead to the structural alterations and changes in spermatogenesis in testicular tissue.

Keywords: spermatogenesis, alcoholic extract of Achillea millefolium L., testis, Wistar rat

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Authors: Kolja Them, Johannes Salamon, Harald Ittrich, Michael Kaul, Tobias Knopp

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Superparamagnetic iron oxide nanoparticles (SPIONs) are nanoscale magnets which can be biologically functionalized for biomedical applications. Stem cell therapies to repair damaged tissue, magnetic fluid hyperthermia for cancer therapy and targeted drug delivery based on SPIONs are prominent examples where the visualization of a preferably low concentrated SPION distribution is essential. In 2005 a new method for tomographic SPION imaging has been introduced. The method named magnetic particle imaging (MPI) takes advantage of the nanoparticles magnetization change caused by an oscillating, external magnetic field and allows to directly image the time-dependent nanoparticle distribution. The SPION magnetization can be changed by the electron spin dynamics as well as by a mechanical rotation of the nanoparticle. In this work different calibration methods in MPI are investigated for image reconstruction of magnetically labeled stem cells. It is shown that a calibration using rotationally immobilized SPIONs provides a higher quality of stem cell images with fewer artifacts than a calibration using mobile SPIONs. The enhancement of the image quality and the reduction of artifacts enables the localization and identification of a smaller number of magnetically labeled stem cells. This is important for future medical applications where low concentrations of functionalized SPIONs interacting with biological matter have to be localized.

Keywords: biomedical imaging, iron oxide nanoparticles, magnetic particle imaging, stem cell imaging

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Authors: Stanislaw Kosmynka

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Although the genesis of the activity of fighting salafist radicalism in Spain dates back to the 80’s, the development of extremism of this kind manifested itself only in the next decade. Its first permanently functioning structures in this country in the second half of 90’s of 20th century came from Algieria and Syria. At the same time it should be emphasized that this distinction is in many dimensions conventional, the more so because they consisted also of immigrants from other coutries of Islam, particularly from Morocco. The paper seeks to understand the radical salafist challenge for Spain in the context of some terrorist networks consisted of immigrants from Morocco. On the eve of the new millennium Moroccan jihadists played an increasingly important role. Although the activity of these groups had for many years mainly logistical and propaganda character, the bomb attack carried out on 11 March 2004 in Madrid constituted an expression of open forms of terrorism, directed against the authorities and society of Spain and reflected the narration of representatives of the trend of the global jihad. The people involved in carrying out that act of violence were to a large extent Moroccan immigrants; also in the following years among the cells of radicals in Spain Moroccans stood out many times. That is why the forms and directions of activity of these extremists in Spain, also after 11th March 2004 and in the actual context of the impact of Islamic State, are worth presenting. The paper is focused on threats to the security of Spain and the region and remains connected with the issues of mutual relations of the society of a host country with immigrant communities which to a large degree come from this part of Maghreb.

Keywords: jihadi terrorism, Morocco, radical salafism, security, Spain, terrorist cells, threat

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Authors: Tarek Aly

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The shoulder is the most mobile joint whose stability requires the interaction of both dynamic and static stabilizers. Its wide range of movement predisposes to a high susceptibility to dislocation, accounting for nearly 50% of all dislocations. This trauma typically results in ligament injury (e.g., labral tear, capsular strain) or bony fracture (e.g., loss of glenoid or humeral head bone), which frequently causes recurrent instability. Patients with significant glenoid defects may require Latarjet procedure, which involves transferring the coracoid to the antero-inferior glenoid rim. In spite of outstanding results, 15 to 30% of cases suffer complications. In this article, we discuss the diagnosis of recurrent shoulder instability, the surgical technique and various complications of Latarjet procedure.

Keywords: recurrent, anterior gleno-humeral instability, latarjet, unstable shoulder

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Authors: Alena Cejkova, Martina Paldrychova, Jana Michailidu, Olga Matatkova, Jan Masak

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Increasing the resistance of pathogenic microorganisms to many antibiotics is a serious threat to the treatment of infectious diseases and cleaning medical instruments. It should be added that the resistance of microbial populations growing in biofilms is often up to 1000 times higher compared to planktonic cells. Biofilm formation in a number of microorganisms is largely influenced by the quorum sensing regulatory mechanism. Finding external factors such as natural substances or physical processes that can interfere effectively with quorum sensing signal molecules should reduce the ability of the cell population to form biofilm and increase the effectiveness of antibiotics. The present work is devoted to the effect of chitosan as a representative of natural substances with anti-biofilm activity and non- thermal plasma (NTP) alone or in combination with polymyxin B on biofilm formation of Pseudomonas aeruginosa. Particular attention was paid to the influence of these agents on the level of quorum sensing signal molecules (acyl-homoserine lactones) during planktonic and biofilm cultivations. Opportunistic pathogenic strains of Pseudomonas aeruginosa (DBM 3081, DBM 3777, ATCC 10145, ATCC 15442) were used as model microorganisms. Cultivations of planktonic and biofilm populations in 96-well microtiter plates on horizontal shaker were used for determination of antibiotic and anti-biofilm activity of chitosan and polymyxin B. Biofilm-growing cells on titanium alloy, which is used for preparation of joint replacement, were exposed to non-thermal plasma generated by cometary corona with a metallic grid for 15 and 30 minutes. Cultivation followed in fresh LB medium with or without chitosan or polymyxin B for next 24 h. Biofilms were quantified by crystal violet assay. Metabolic activity of the cells in biofilm was measured using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) colorimetric test based on the reduction of MTT into formazan by the dehydrogenase system of living cells. Activity of N-acyl homoserine lactones (AHLs) compounds involved in the regulation of biofilm formation was determined using Agrobacterium tumefaciens strain harboring a traG::lacZ/traR reporter gene responsive to AHLs. The experiments showed that both chitosan and non-thermal plasma reduce the AHLs level and thus the biofilm formation and stability. The effectiveness of both agents was somewhat strain dependent. During the eradication of P. aeruginosa DBM 3081 biofilm on titanium alloy induced by chitosan (45 mg / l) there was an 80% decrease in AHLs. Applying chitosan or NTP on the P. aeruginosa DBM 3777 biofilm did not cause a significant decrease in AHLs, however, in combination with both (chitosan 55 mg / l and NTP 30 min), resulted in a 70% decrease in AHLs. Combined application of NTP and polymyxin B allowed reduce antibiotic concentration to achieve the same level of AHLs inhibition in P. aeruginosa ATCC 15442. The results shown that non-thermal plasma and chitosan have considerable potential for the eradication of highly resistant P. aeruginosa biofilms, for example on medical instruments or joint implants.

Keywords: anti-biofilm activity, chitosan, non-thermal plasma, opportunistic pathogens

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