Search results for: adipose tissue-derived stem cell injections

Authors: E. Mosiniewicz-Szablewska, P. Suchocki, P. C. Morais

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Despite the significant progress in cancer treatment, there is a need to search for new therapeutic methods in order to minimize side effects. Chemotherapy, the main current method of treating cancer, is non-selective and has a number of limitations. Toxicity to healthy cells is undoubtedly the biggest problem limiting the use of many anticancer drugs. The problem of how to kill cancer without harming a patient can be solved by using organic selenium (IV) compounds. Organic selenium (IV) compounds are a new class of materials showing a strong anticancer activity. They are first organic compounds containing selenium at the +4 oxidation level and therefore they eliminate the multidrug-resistance for all tumor cell lines tested so far. These materials are capable of selectively killing cancer cells without damaging the healthy ones. They are obtained by the incorporation of selenous acid (H2SeO3) into molecules of fatty acids of sunflower oil and therefore, they are inexpensive to manufacture. Attaching these compounds to magnetic carriers enables their precise delivery directly to the tumor area and the simultaneous application of the magnetic hyperthermia, thus creating a huge opportunity to effectively get rid of the tumor without any side effects. Polylactic-co-glicolic acid (PLGA) nanocapsules loaded with maghemite (-Fe2O3) nanoparticles and organic selenium (IV) compounds are successfully prepared by nanoprecipitation method. In vitro antitumor activity of the nanocapsules were evidenced using murine melanoma (B16-F10), oral squamos carcinoma (OSCC) and murine (4T1) and human (MCF-7) breast lines. Further exposure of these cells to an alternating magnetic field increased the antitumor effect of nanocapsules. Moreover, the nanocapsules presented antitumor effect while not affecting normal cells. Magnetic properties of the nanocapsules were investigated by means of dc magnetization, ac susceptibility and electron spin resonance (ESR) measurements. The nanocapsules presented a typical superparamagnetic behavior around room temperature manifested itself by the split between zero field-cooled/field-cooled (ZFC/FC) magnetization curves and the absence of hysteresis on the field-dependent magnetization curve above the blocking temperature. Moreover, the blocking temperature decreased with increasing applied magnetic field. The superparamagnetic character of the nanocapsules was also confirmed by the occurrence of a maximum in temperature dependences of both real ′(T) and imaginary ′′ (T) components of the ac magnetic susceptibility, which shifted towards higher temperatures with increasing frequency. Additionally, upon decreasing the temperature the ESR signal shifted to lower fields and gradually broadened following closely the predictions for the ESR of superparamagnetoc nanoparticles. The observed superparamagnetic properties of nanocapsules enable their simple manipulation by means of magnetic field gradient, after introduction into the blood stream, which is a necessary condition for their use as magnetic drug carriers. The observed anticancer and superparamgnetic properties show that the magnetic nanocapsules loaded with organic selenium (IV) compounds should be considered as an effective material system for magnetic drug delivery and magnetohyperthermia inductor in antitumor therapy.

Keywords: cancer treatment, magnetic drug delivery system, nanomaterials, nanotechnology

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Authors: Priyanka Jain, Ashish K. Sharma, Esha Shukla, K. J. Mukherjee

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We propose a paradigm shift in our approach to design improved platforms for recombinant protein production, by addressing system level issues rather than the individual steps associated with recombinant protein synthesis like transcription, translation, etc. We demonstrate that by controlling and modulating the cellular stress response (CSR), which is responsible for feedback control of protein synthesis, we can generate hyper-producing strains. We did transcriptomic profiling of post-induction cultures, expressing different types of protein, to analyze the nature of this cellular stress response. We found significant down-regulation of substrate utilization, translation, and energy metabolism genes due to generation CSR inside the host cell. However, transcription profiling has also shown that many genes are up-regulated post induction and their role in modulating the CSR is unclear. We hypothesized that these up-regulated genes trigger signaling pathways, generating the CSR and concomitantly reduce the recombinant protein yield. To test this hypothesis, we knocked out the up-regulated genes, which did not have any downstream regulatees, and analyzed their impact on cellular health and recombinant protein expression. Two model proteins i.e., GFP and L-Asparaginase were chosen for this analysis. We observed a significant improvement in expression levels, with some knock-outs showing more than 7-fold higher expression compared to control. The 10 best single knock-outs were chosen to make 45 combinations of all possible double knock-outs. A further increase in expression was observed in some of these double knock- outs with GFP levels being highest in a double knock-out ΔyhbC + ΔelaA. However, for L-Asparaginase which is a secretory protein, the best results were obtained using a combination of ΔelaA+ΔcysW knock-outs. We then tested all the knock outs for their ability to enhance the expression of a 'difficult-to-express' protein. The Rubella virus E1 protein was chosen and tagged with sfGFP at the C-terminal using a linker peptide for easy online monitoring of expression of this fusion protein. Interestingly, the highest increase in Rubella-sGFP levels was obtained in the same double knock-out ΔelaA + ΔcysW (5.6 fold increase in expression yield compared to the control) which gave the highest expression for L-Asparaginase. However, for sfGFP alone, the ΔyhbC+ΔmarR knock-out gave the highest level of expression. These results indicate that there is a fair degree of commonality in the nature of the CSR generated by the induction of different proteins. Transcriptomic profiling of the double knock out showed that many genes associated with the translational machinery and energy biosynthesis did not get down-regulated post induction, unlike the control where these genes were significantly down-regulated. This confirmed our hypothesis of these genes playing an important role in the generation of the CSR and allowed us to design a strategy for making better expression hosts by simply knocking out key genes. This strategy is radically superior to the previous approach of individually up-regulating critical genes since it blocks the mounting of the CSR thus preventing the down-regulation of a very large number of genes responsible for sustaining the flux through the recombinant protein production pathway.

Keywords: cellular stress response, GFP, knock-outs, up-regulated genes

Procedia PDF Downloads 228

Authors: Wenfa Yu, Thuva Gnanasampanthan, John Finlay, Jessica Clarke, Charlotte Anderson, Tony Clare, Axel Rosenhahn

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Marine biofouling is a worldwide problem at vast economic and ecological costs. Historically it was combated with toxic coatings such as tributyltin. As those coatings being banned nowadays, finding environmental friendly antifouling solution has become an urgent topic. In this study antifouling coatings consisted of natural occurring polysaccharides hyaluronic acid (HA), alginic acid (AA), chitosan (Ch) and polyelectrolyte polyethylenimine (PEI) are constructed into polyelectrolyte multilayers (PEMs) in a Layer-by-Layer (LbL) method. LbL PEM construction is a straightforward way to assemble biomacromolecular coatings on surfaces. Advantages about PEM include ease of handling, highly diverse PEM composition, precise control over the thickness and so on. PEMs have been widely employed in medical application and there are numerous studies regarding their protein adsorption, elasticity and cell adhesive properties. With the adjustment of coating composition, termination layer charge, coating morphology and cross-linking method, it is possible to prepare low marine biofouling coatings with PEMs. In this study, using spin coating technology, PEM construction was achieved at smooth multilayers with roughness as low as 2nm rms and highly reproducible thickness around 50nm. To obtain stability in sea water, the multilayers were covalently cross-linked either thermally or chemically. The cross-linking method affected surface energy, which was reflected in water contact angle, thermal cross-linking led to hydrophobic surfaces and chemical cross-linking generated hydrophilic surfaces. The coatings were then evaluated regarding its protein resistance and biological species resistance. While the hydrophobic thermally cross-linked PEM had low resistance towards proteins, the resistance of chemically cross-linked PEM strongly depended on the PEM termination layer and the charge of the protein, opposite charge caused high adsorption and same charge low adsorption, indicating electrostatic interaction plays a crucial role in the protein adsorption processes. Ulva linza was chosen as the biological species for antifouling performance evaluation. Despite of the poor resistance towards protein adsorption, thermally cross-linked PEM showed good resistance against Ulva spores settlement, the chemically cross-linked multilayers showed poor resistance regardless of the termination layer. Marine species adhesion is a complex process, although it involves proteins as bioadhesives, protein resistance its own is not a fully indicator for its antifouling performance. The species will pre select the surface, responding to cues like surface energy, chemistry, or charge and so on. Thus making it difficult for one single factors to determine its antifouling performance. Preparing PEM coating is a comprehensive work involving choosing polyelectrolyte combination, determining termination layer and the method for cross-linking. These decisions will affect PEM properties such as surface energy, charge, which is crucial, since biofouling is a process responding to surface properties in a highly sensitive and dynamic way.

Keywords: hyaluronic acid, polyelectrolyte multilayers, protein resistance, Ulva linza zoospores

Procedia PDF Downloads 164

Authors: Pierobon Enrico, Missiaggia Marta, Castelluzzo Michele, Tommasino Francesco, Ricci Leonardo, Scifoni Emanuele, Vincezo Monaco, Boscardin Maurizio, La Tessa Chiara

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Clinical outcomes collected over the past three decades have suggested that ion therapy has the potential to be a treatment modality superior to conventional radiation for several types of cancer, including recurrences, as well as for other diseases. Although the results have been encouraging, numerous treatment uncertainties remain a major obstacle to the full exploitation of particle radiotherapy. To overcome therapy uncertainties optimizing treatment outcome, the best possible radiation quality description is of paramount importance linking radiation physical dose to biological effects. Microdosimetry was developed as a tool to improve the description of radiation quality. By recording the energy deposition at the micrometric scale (the typical size of a cell nucleus), this approach takes into account the non-deterministic nature of atomic and nuclear processes and creates a direct link between the dose deposited by radiation and the biological effect induced. Microdosimeters measure the spectrum of lineal energy y, defined as the energy deposition in the detector divided by most probable track length travelled by radiation. The latter is provided by the so-called “Mean Chord Length” (MCL) approximation, and it is related to the detector geometry. To improve the characterization of the radiation field quality, we define a new quantity replacing the MCL with the actual particle track length inside the microdosimeter. In order to measure this new quantity, we propose a two-stage detector consisting of a commercial Tissue Equivalent Proportional Counter (TEPC) and 4 layers of Low Gain Avalanche Detectors (LGADs) strips. The TEPC detector records the energy deposition in a region equivalent to 2 um of tissue, while the LGADs are very suitable for particle tracking because of the thickness thinnable down to tens of micrometers and fast response to ionizing radiation. The concept of HDM has been investigated and validated with Monte Carlo simulations. Currently, a dedicated readout is under development. This two stages detector will require two different systems to join complementary information for each event: energy deposition in the TEPC and respective track length recorded by LGADs tracker. This challenge is being addressed by implementing SoC (System on Chip) technology, relying on Field Programmable Gated Arrays (FPGAs) based on the Zynq architecture. TEPC readout consists of three different signal amplification legs and is carried out thanks to 3 ADCs mounted on a FPGA board. LGADs activated strip signal is processed thanks to dedicated chips, and finally, the activated strip is stored relying again on FPGA-based solutions. In this work, we will provide a detailed description of HDM geometry and the SoC solutions that we are implementing for the readout.

Keywords: particle tracking, ion therapy, low gain avalanche diode, tissue equivalent proportional counter, microdosimetry

Procedia PDF Downloads 175

Authors: K. A.Veselova, N. V.Gromova, I. N.Antonova, I. N. Kalakutskii

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There are many diseases and incidents that may result facial defects and deformities: cancer, trauma, burns, congenital anomalies, and autoimmune diseases. In some cases, patient may acquire atypically extensive facial defect, including more than one anatomical region or, by contrast, atypically small defect (e.g. partial auricular defect). The anaplastology gives us opportunity to help patient with facial disfigurement in cases when plastic surgery is contraindicated. Using of implant retention for facial prosthesis is strongly recommended because improves both aesthetic and functional results and makes using of the prosthesis more comfortable. Prostheticly oriented fixture position is extremely important for aesthetic and functional long-term result; however, the optimal site for fixture placement is not clear in cases with atypical configuration of facial defect. The objective of this report is to demonstrate challenges in fixture position determination we have faced with and offer the solution. In this report, four cases of implant-supported facial prosthesis are described. Extra-oral implants with four millimeter length were used in all cases. The decision regarding the quantity of surgical stages was based on anamnesis of disease. Facial prostheses were manufactured according to conventional technique. Clinical and technological difficulties and mistakes are described, and prostheticly oriented approach for determination of fixture position is demonstrated. The case with atypically large combined orbital and nasal defect resulting after arteriovenous malformation is described: the correct positioning of artificial eye was impossible due to wrong position of the fixture (with suprastructure) located in medial aspect of supraorbital rim. The suprastructure was unfixed and this fixture wasn`t used for retention in order to achieve appropriate artificial eye placement and better aesthetic result. In other case with small partial auricular defect (only helix and antihelix were absent) caused by squamoized cell carcinoma T1N0M0 surgical template was used to avoid the difficulties. To achieve the prostheticly oriented fixture position in case of extremely small defect the template was made on preliminary cast using vacuum thermoforming method. Two radiopaque markers were incorporated into template in preferable for fixture placement positions taking into account future prosthesis configuration. The template was put on remaining ear and cone-beam CT was performed to insure, that the amount of bone is enough for implant insertion in preferable position. Before the surgery radiopaque markers were extracted and template was holed for guide drill. Fabrication of implant-retained facial prostheses gives us opportunity to improve aesthetics, retention and patients’ quality of life. But every inaccuracy in planning leads to challenges on surgery and prosthetic stages. Moreover, in cases with atypically small or extended facial defects prostheticly oriented approach for determination of fixture position is strongly required. The approach including surgical template fabrication is effective, easy and cheap way to avoid mistakes and unpredictable result.

Keywords: anaplastology, facial prosthesis, implant-retained facial prosthesis., maxillofacil prosthese

Procedia PDF Downloads 114

Authors: Imen Soltani, Takoua Soltani, Taoufik Aguili

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Metamaterials crossover classic physical boundaries and gives rise to new phenomena and applications in the domain of beam steering and shaping. Where electromagnetic near and far field manipulations were achieved in an accurate manner. In this sense, 3D imaging is one of the beneficiaries and in particular Denis Gabor’s invention: holography. But, the major difficulty here is the lack of a suitable recording medium. So some enhancements were essential, where the 2D version of bulk metamaterials have been introduced the so-called metasurface. This new class of interfaces simplifies the problem of recording medium with the capability of tuning the phase, amplitude, and polarization at a given frequency. In order to achieve an intelligible wavefront control, the electromagnetic properties of the metasurface should be optimized by means of solving Maxwell’s equations. In this context, integral methods are emerging as an important method to study electromagnetic from microwave to optical frequencies. The method of moment presents an accurate solution to reduce the problem of dimensions by writing its boundary conditions in the form of integral equations. But solving this kind of equations tends to be more complicated and time-consuming as the structural complexity increases. Here, the use of equivalent circuit’s method exhibits the most scalable experience to develop an integral method formulation. In fact, for allaying the resolution of Maxwell’s equations, the method of Generalised Equivalent Circuit was proposed to convey the resolution from the domain of integral equations to the domain of equivalent circuits. In point of fact, this technique consists in creating an electric image of the studied structure using discontinuity plan paradigm and taken into account its environment. So that, the electromagnetic state of the discontinuity plan is described by generalised test functions which are modelled by virtual sources not storing energy. The environmental effects are included by the use of an impedance or admittance operator. Here, we propose a tunable metasurface composed of graphene-based elements which combine the advantages of reflectarrays concept and graphene as a pillar constituent element at Terahertz frequencies. The metasurface’s building block consists of a thin gold film, a dielectric spacer SiO₂ and graphene patch antenna. Our electromagnetic analysis is based on the method of moment combined with generalised equivalent circuit (MoM-GEC). We begin by restricting our attention to study the effects of varying graphene’s chemical potential on the unit cell input impedance. So, it was found that the variation of complex conductivity of graphene allows controlling the phase and amplitude of the reflection coefficient at each element of the array. From the results obtained here, we were able to determine that the phase modulation is realized by adjusting graphene’s complex conductivity. This modulation is a viable solution compared to tunning the phase by varying the antenna length because it offers a full 2π reflection phase control.

Keywords: graphene, method of moment combined with generalised equivalent circuit, reconfigurable metasurface, reflectarray, terahertz domain

Procedia PDF Downloads 176

Authors: Mariam Khaled, Noha Farag, Ghada Mohamed Abdel Salam, Khaled Abu-Aisha, Mohamed El-Azizi

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Gastric and colorectal cancers are among the most frequent causes of cancer-associated mortalities in Africa. They have been considered as a global public health concern, as nearly one million new cases are reported per year. IL-1β is a pro-inflammatory cytokine-produced by activated macrophages and monocytes- and a member of the IL-1 family. The inactive IL-1β precursor is cleaved and activated by caspase-1 enzyme, which itself is activated by the assembly of intracellular structures defined as NLRP3 (Nod Like receptor P3) inflammasomes. Activated IL-1β stimulates the Interleukin-1 receptor type-1 (IL-1R1), which is responsible for the initiation of a signal transduction pathway leading to cell proliferation. It has been proven that the IL-1β gene is a highly polymorphic gene in which single nucleotide polymorphisms (SNPs) may affect its expression. It has been previously reported that SNPs including base transitions between C and T at positions, -511 (C-T; dbSNP: rs16944) and -31 (C-T; dbSNP: rs1143627), from the transcriptional start site, contribute to the pathogenesis of gastric and colorectal cancers by affecting IL-1β levels. Altered production of IL-1β due to such polymorphisms is suspected to stimulate an amplified inflammatory response and promote Epithelial Mesenchymal Transition leading to malignancy. Allele frequency distribution of the IL-1β-31 and -511 SNPs, in different populations, and their correlation to the incidence of gastric and colorectal cancers, has been intriguing to researchers worldwide. The current study aims to investigate allele distributions of the IL-1β SNPs among gastric and colorectal cancers Egyptian patients. In order to achieve to that, 89 Biopsy and surgical specimens from the antrum and corpus mucosa of chronic gastritis subjects and gastric and colorectal carcinoma patients was collected for DNA extraction followed by restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR). The amplified PCR products of IL-1β-31C > T and IL-1β-511T > C were digested by incubation with the restriction endonuclease enzymes ALu1 and Ava1. Statistical analysis was carried out to determine the allele frequency distribution in the three studied groups. Also, the effect of the IL-1β -31 and -511 SNPs on nuclear factor binding was analyzed using Fluorescence Electrophoretic Mobility Shift Assay (EMSA), preceded by nuclear factor extraction from gastric and colorectal tissue samples and LPS stimulated monocytes. The results of this study showed that a significantly higher percentage of Egyptian gastric cancer patients have a homozygous CC genotype at the IL-1β-31 position and a heterozygous TC genotype at the IL-1β-511 position. Moreover, a significantly higher percentage of the colorectal cancer patients have a homozygous CC genotype at the IL-1β-31 and -511 positions as compared to the control group. In addition, the EMSA results showed that IL-1β-31C/T and IL-1β-511T/C SNPs do not affect nuclear factor binding. Results of this study suggest that the IL-1β-31 C/T and IL-1β-511 T/C may be correlated to the incidence of gastric cancer in Egyptian patients; however, similar findings couldn’t be proven in the colorectal cancer patients group for the IL-1β-511 T/C SNP. This is the first study to investigate IL-1β -31 and -511 SNPs in the Egyptian population.

Keywords: colorectal cancer, Egyptian patients, gastric cancer, interleukin-1β, single nucleotide polymorphisms

Procedia PDF Downloads 140

Authors: Umara Nissar Rafiqi, Irum Gul, Nazima Nasrullah, Monica Saifi, Malik Z. Abdin

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Background: Stevia rebaudiana, a member of Asteraceae family is an important medicinal plant and produces a commercially used non-caloric natural sweetener, which is also an alternate herbal cure for diabetes. Steviol glycosides are the main sweetening compounds present in these plants. Secondary metabolites are crucial to the adaption of plants to the environment and its overcoming stress conditions. In agricultural procedures, the abiotic stresses like salinity, high metal toxicity and drought, in particular, are responsible for the majority of the reduction that differentiates yield potential from harvestable yield. Salt stress and heavy metal toxicity lead to increased production of reactive oxygen species (ROS). To avoid oxidative damage due to ROS and osmotic stress, plants have a system of anti-oxidant enzymes along with several stress induced enzymes. This helps in scavenging the ROS and relieve the osmotic stress in different cell compartments. However, whether stress induced toxicity modulates the activity of these enzymes in Stevia rebaudiana is poorly understood. Aim: The present study focussed on the effect of salinity, heavy metal toxicity (lead and mercury) on physiological traits and transcriptional profiling of Stevia rebaudiana. Method: Stevia rebaudiana plants were collected from the Central Institute of Medicinal and Aromatic plants (CIMAP), Patnagar, India and maintained under controlled conditions in a greenhouse at Hamdard University, Delhi, India. The plants were subjected to different concentrations of salt (0, 25, 50 and 75 mM respectively) and heavy metals, lead and mercury (0, 100, 200 and 300 µM respectively). The physiological traits such as shoot length, root numbers, leaf growth were evaluated. The samples were collected at different developmental stages and analysed for transcription profiling by RT-PCR. Transcriptional studies in stevia rebaudiana involves important antioxidant enzymes: catalase (CAT), superoxide dismutase (SOD), cytochrome P450 monooxygenase (CYP) and stress induced aquaporin (AQU), auxin repressed protein (ARP-1), Ndhc gene. The data was analysed using GraphPad Prism and expressed as mean ± SD. Result: Low salinity and lower metal toxicity did not affect the fresh weight of the plant. However, this was substantially decreased by 55% at high salinity and heavy metal treatment. With increasing salinity and heavy metal toxicity, the values of all studied physiological traits were significantly decreased. Chlorosis in treated plants was also observed which could be due to changes in Fe:Zn ratio. At low concentrations (upto 25 mM) of NaCl and heavy metals, we did not observe any significant difference in the gene expressions of treated plants compared to control plants. Interestingly, at high salt concentration and high metal toxicity, a significant increase in the expression profile of stress induced genes was observed in treated plants compared to control (p < 0.005). Conclusion: Stevia rebaudiana is tolerant to lower salt and heavy metal concentration. This study also suggests that with the increase in concentrations of salt and heavy metals, harvest yield of S. rebaudiana was hampered.

Keywords: Stevia rebaudiana, natural sweetener, salinity, heavy metal toxicity

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Authors: Hülya Torun, Ondřej Novák, Jaromír Mikulík, Miroslav Strnad, Faik A. Ayaz

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World climate is changing. Millions of people in the world still face chronic undernourishment for conducting a healthy life and the world’s population is growing steadily. To meet this growing demand, agriculture and food systems must adapt to the adverse effects of climate change and become more resilient, productive and sustainable. From this perspective, to determine tolerant cultivars for undesirable environmental conditions will be necessary food production for sustainable development. Among abiotic stresses, soil salinity is one of the most detrimental global fact restricting plant sources. Development of salt-tolerant lines is required in order to increase the crop productivity and quality in salt-treated lands. Therefore, the objective of this study was to investigate the morphological and physiological responses of barley cultivars accessions to salinity stress by NaCl. For this purpose, it was aimed to determine the crosstalk between some endogenous phytohormones and exogenous salicylic acid (SA) in two different vegetative parts (leaves and roots) of barley (Hordeum vulgare L.; Poaceae; 2n=14; Ince-04) which is detected salt-tolerant. The effects of SA on growth parameters, leaf relative water content (RWC), endogenous phytohormones; including indole-3-acetic acid (IAA), cytokinins (CKs), abscisic acid (ABA), jasmonic acid (JA) and ethylene were investigated in barley cultivars under salinity stress. SA was applied to 17-day-old seedlings of barley in two different ways including before (pre-treated for 24 h) and simultaneously with NaCl stress treatment. NaCl (0, 150, 300 mM) exposure in the hydrophonic system was associated with a rapid decrease in growth parameters and RWC, which is an indicator of plant water status, resulted in a strong up-regulation of ABA as a stress indicator. Roots were more dramatically affected than leaves. Water conservation in 150 mM NaCl treated-barley plants did not change, but decreased in 300 mM NaCl treated plants. Pre- and simultaneously treatment of SA did not significantly alter growth parameters and RWC. ABA, JA and ethylene are known to be related with stress. In the present work, ethylene also increased, similarly to ABA, but not with the same intensity. While ABA and ethylene increased by the increment of salt concentrations, JA levels rapidly decreased especially in roots. Both pre- and simultaneously SA applications alleviated salt-induced decreases in 300 mM NaCl resulted in the increment of ABA levels. CKs and IAA are related to cell growth and development. At high salinity (300 mM NaCl), CKs (cZ+cZR) contents increased in both vegetative organs while IAA levels stayed at the same level with control groups. However, IAA increased and cZ+cZR rapidly decreased in leaves of barley plants with SA treatments before salt applications (in pre- SA treated groups). Simultaneously application of SA decreased CKs levels in both leaves and roots of the cultivar. Due to increasing concentrations of NaCl in association with decreasing ABA, JA and ethylene content and increments in CKs and IAA were recorded with SA treatments. As results of the study, in view of all the phytohormones that we tested, exogenous SA induced greater tolerance to salinity particularly when applied before salinity stress.

Keywords: Barley, Hordeum vulgare, phytohormones, salicylic acid, salinity

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Authors: T. Heiman, D. Olenik-Shemesh

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Over the past decade, with the rapid growth of electronic communication, the internet and, in particular, social networking has become an inseparable part of people's daily lives. Along with its benefits, a new type of online aggression has emerged, defined as cyber bullying, a form of interpersonal aggressive behavior that takes place through electronic means. Cyber-bullying is characterized by repetitive behavior over time of maladaptive authority and power usage using computers and cell phones via sending insulting messages and hurtful pictures. Preliminary findings suggest that the prevalence of involvement in cyber-bullying among higher education students varies between 10 and 35%. As to date, universities are facing an uphill effort in trying to restrain online misbehavior. As no studies examined the relationships between cyber-bullying involvement with personal aspects, and its impacts on academic achievement and work functioning, this present study examined the nature of cyber-bullying involvement among 1,052 undergraduate students (mean age = 27.25, S.D = 4.81; 66.2% female), coping with, as well as the effects of social support, perceived self-efficacy, well-being, and body-perception, in relation to cyber-victimization. We assume that students in higher education are a vulnerable population and at high risk of being cyber-victims. We hypothesize that social support might serve as a protective factor and will moderate the relationships between the socio-emotional variables and the occurrence of cyber- victimization. The findings of this study will present the relationships between cyber-victimization and the social-emotional aspects, which constitute risk and protective factors. After receiving approval from the Ethics Committee of the University, a Google Drive questionnaire was sent to a random sample of students, studying in the various University study centers. Students' participation was voluntary, and they completed the five questionnaires anonymously: Cyber-bullying, perceived self-efficacy, subjective well-being, social support and body perception. Results revealed that 11.6% of the students reported being cyber-victims during last year. Examining the emotional and behavioral reactions to cyber-victimization revealed that female emotional and behavioral reactions were significantly greater than the male reactions (p < .001). Moreover, females reported on a significant higher social support compared to men; male reported significantly on a lower social capability than female; and men's body perception was significantly more positive than women's scores. No gender differences were observed for subjective well-being scale. Significant positive correlations were found between cyber-victimization and fewer friends, lower grades, and work ineffectiveness (r = 0.37- .40, p < 0 .001). The results of the Hierarchical regression indicated significantly that cyber-victimization can be predicted by lower social support, lower body perception, and gender (female), that explained 5.6% of the variance (R2 = 0.056, F(5,1047) = 12.47, p < 0.001). The findings deepen our understanding of the students' involvement in cyber-bullying, and present the relationships of the social-emotional and academic aspects on cyber-victim students. In view of our findings, higher education policy could help facilitate coping with cyber-bullying incidents, and student support units could develop intervention programs aimed at reducing cyber-bullying and its impacts.

Keywords: academic and personal factors, cyber-victimization, social support, higher education

Procedia PDF Downloads 289

Authors: Chang-Hui Shen, Michelle Esposito, Andrew J. Shen, Michael Adejokun, Diana Laterman

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The packaging of DNA into nucleosomes is critical to genomic compaction, yet it can leave gene promoters inaccessible to activator proteins or transcription machinery and thus prevents transcriptional initiation. Both chromatin remodelers and histone acetylases (HATs) are the two main transcription co-activators that can reconfigure chromatin structure for transcriptional activation. Ino80p is the core component of the INO80 remodeling complex. Recently, it was shown that Ino80p dissociates from the yeast INO1 promoter after induction. However, when certain HATs were deleted or mutated, Ino80p accumulated at the promoters during gene activation. This suggests a link between HATs’ presence and Ino80p’s dissociation. However, it has yet to be demonstrated that Ino80p can be acetylated. To determine if Ino80p can be acetylated, wild-type Saccharomyces cerevisiae cells carrying Ino80p engineered with a double FLAG tag (MATa INO80-FLAG his3∆200 leu2∆0 met15∆0 trp1∆63 ura3∆0) were grown to mid log phase, as were non-tagged wild type (WT) (MATa his3∆200 leu2∆0 met15∆0 trp1∆63 ura3∆0) and ino80∆ (MATa ino80∆::TRP1 his3∆200 leu2∆0 met15∆0 trp1∆63 ura3∆0) cells as controls. Cells were harvested, and the cell lysates were subjected to immunoprecipitation (IP) with α-FLAG resin to isolate Ino80p. These eluted IP samples were subjected to SDS-PAGE and Western blot analysis. Subsequently, the blots were probed with the α-FLAG and α-acetyl lysine antibodies, respectively. For the blot probed with α-FLAG, one prominent band was shown in the INO80-FLAG cells, but no band was detected in the IP samples from the WT and ino80∆ cells. For the blot probed with the α-acetyl lysine antibody, we detected acetylated Ino80p in the INO80-FLAG strain while no bands were observed in the control strains. As such, our results showed that Ino80p can be acetylated. This acetylation can explain the co-activator’s recruitment patterns observed in current gene activation models. In yeast INO1, it has been shown that Ino80p is recruited to the promoter during repression, and then dissociates from the promoter once de-repression begins. Histone acetylases, on the other hand, have the opposite pattern of recruitment, as they have an increased presence at the promoter as INO1 de-repression commences. This Ino80p recruitment pattern significantly changes when HAT mutant strains are studied. It was observed that instead of dissociating, Ino80p accumulates at the promoter in the absence of functional HATs, such as Gcn5p or Esa1p, under de-repressing processes. As such, Ino80p acetylation may be required for its proper dissociation from the promoters. The remodelers’ dissociation mechanism may also have a wide range of implications with respect to transcriptional initiation, elongation, or even repression as it allows for increased spatial access to the promoter for the various transcription factors and regulators that need to bind in that region. Our findings here suggest a previously uncharacterized interaction between Ino80p and other co-activators recruited to promoters. As such, further analysis of Ino80p acetylation not only will provide insight into the role of epigenetic modifications in transcriptional activation, but also gives insight into the interactions occurring between co-activators at gene promoters during gene regulation.

Keywords: acetylation, chromatin remodeler, epigenetic modification, Ino80p

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Authors: Miroslava Cuperlovic-Culf, Lipu Wang, Ketty Boyle, Nadine Makley, Ian Burton, Anissa Belkaid, Mohamed Touaibia, Marc E. Surrette

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Metabolic changes are one of the major factors in the development of a variety of diseases in various species. Metabolism of agricultural plants is altered the following infection with pathogens sometimes contributing to resistance. At the same time, pathogens use metabolites for infection and progression. In humans, metabolism is a hallmark of cancer development for example. Quantified metabolomics data combined with other omics or clinical data and analyzed using various unsupervised and supervised methods can lead to better diagnosis and prognosis. It can also provide information about resistance as well as contribute knowledge of compounds significant for disease progression or prevention. In this work, different methods for metabolomics quantification and analysis from Nuclear Magnetic Resonance (NMR) measurements that are used for investigation of disease development in wheat and human cells will be presented. One-dimensional 1H NMR spectra are used extensively for metabolic profiling due to their high reliability, wide range of applicability, speed, trivial sample preparation and low cost. This presentation will describe a new method for metabolite quantification from NMR data that combines alignment of spectra of standards to sample spectra followed by multivariate linear regression optimization of spectra of assigned metabolites to samples’ spectra. Several different alignment methods were tested and multivariate linear regression result has been compared with other quantification methods. Quantified metabolomics data can be analyzed in the variety of ways and we will present different clustering methods used for phenotype determination, network analysis providing knowledge about the relationships between metabolites through metabolic network as well as biomarker selection providing novel markers. These analysis methods have been utilized for the investigation of fusarium head blight resistance in wheat cultivars as well as analysis of the effect of estrogen receptor and carbonic anhydrase activation and inhibition on breast cancer cell metabolism. Metabolic changes in spikelet’s of wheat cultivars FL62R1, Stettler, MuchMore and Sumai3 following fusarium graminearum infection were explored. Extensive 1D 1H and 2D NMR measurements provided information for detailed metabolite assignment and quantification leading to possible metabolic markers discriminating resistance level in wheat subtypes. Quantification data is compared to results obtained using other published methods. Fusarium infection induced metabolic changes in different wheat varieties are discussed in the context of metabolic network and resistance. Quantitative metabolomics has been used for the investigation of the effect of targeted enzyme inhibition in cancer. In this work, the effect of 17 β -estradiol and ferulic acid on metabolism of ER+ breast cancer cells has been compared to their effect on ER- control cells. The effect of the inhibitors of carbonic anhydrase on the observed metabolic changes resulting from ER activation has also been determined. Metabolic profiles were studied using 1D and 2D metabolomic NMR experiments, combined with the identification and quantification of metabolites, and the annotation of the results is provided in the context of biochemical pathways.

Keywords: metabolic biomarkers, metabolic network, metabolomics, multivariate linear regression, NMR quantification, quantified metabolomics, spectral alignment

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Authors: Mirosław Krzemieniewski, Marcin Zieliński, Marcin Dębowski

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Electrokinetic disintegration is one of the high-voltage electric methods. The design of systems is exceptionally simple. Biomass flows through a system of pipes with alongside mounted electrodes that generate an electric field. Discharges in the electric field deform cell walls and lead to their successive perforation, thereby making their contents easily available to bacteria. The spark-over occurs between electrode surface and pipe jacket which is the second pole and closes the circuit. The value of voltage ranges from 10 to 100kV. Electrodes are supplied by normal “power grid” monophase electric current (230V, 50Hz). Next, the electric current changes into direct current of 24V in modules serving for particular electrodes, and this current directly feeds the electrodes. The installation is completely safe because the value of generated current does not exceed 250mA and because conductors are grounded. Therefore, there is no risk of electric shock posed to the personnel, even in the case of failure or incorrect connection. Low values of the electric current mean small energy consumption by the electrode which is extremely low – only 35W per electrode – compared to other methods of disintegration. Pipes with electrodes with diameter of DN150 are made of acid-proof steel and connected from both sides with 90º elbows ended with flanges. The available S and U types of pipes enable very convenient fitting with system construction in the existing installations and rooms or facilitate space management in new applications. The system of pipes for electrokinetic disintegration may be installed horizontally, vertically, askew, on special stands or also directly on the wall of a room. The number of pipes and electrodes is determined by operating conditions as well as the quantity of substrate, type of biomass, content of dry matter, method of disintegration (single or circulatory), mounting site etc. The most effective method involves pre-treatment of substrate that may be pumped through the disintegration system on the way to the fermentation tank or recirculated in a buffered intermediate tank (substrate mixing tank). Biomass structure destruction in the process of electrokinetic disintegration causes shortening of substrate retention time in the tank and acceleration of biogas production. A significant intensification of the fermentation process was observed in the systems operating in the technical scale, with the greatest increase in biogas production reaching 18%. The secondary, but highly significant for the energetic balance, effect is a tangible decrease of energy input by agitators in tanks. It is due to reduced viscosity of the biomass after disintegration, and may result in energy savings reaching even 20-30% of the earlier noted consumption. Other observed phenomena include reduction in the layer of surface scum, reduced sewage capability for foaming and successive decrease in the quantity of bottom sludge banks. Considering the above, the system for electrokinetic disintegration seems a very interesting and valuable solutions meeting the offer of specialist equipment for the processing of plant biomass, including Virginia fanpetals, before the process of methane fermentation.

Keywords: electrokinetic disintegration, biomass, biogas production, fermentation, Virginia fanpetals

Procedia PDF Downloads 377

Authors: Atin Adhikari, Sushma Kurella, Pratik Banerjee, Nabanita Mukherjee, Yamini M. Chandana Gollapudi, Bushra Shah

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Different sharp instruments, drilling machines, and high speed rotary instruments are routinely used in dental clinics during dental cleaning. Therefore, these cleaning procedures release a lot of oral microorganisms including bacteria in clinic air and may cause significant occupational bioaerosol exposure risks for dentists, dental hygienists, patients, and dental clinic employees. Two major goals of this study were to quantify volumetric airborne concentrations of bacteria and to assess overall microbial activity in this type of occupational environment. The study was conducted in several dental clinics of southern Georgia and 15 dental cleaning procedures were targeted for sampling of airborne bacteria and testing of overall microbial activity in settled dusts over clinic floors. For air sampling, a Biostage viable cascade impactor was utilized, which comprises an inlet cone, precision-drilled 400-hole impactor stage, and a base that holds an agar plate (Tryptic soy agar). A high-flow Quick-Take-30 pump connected to this impactor pulls microorganisms in air at 28.3 L/min flow rate through the holes (jets) where they are collected on the agar surface for approx. five minutes. After sampling, agar plates containing the samples were placed in an ice chest with blue ice and plates were incubated at 30±2°C for 24 to 72 h. Colonies were counted and converted to airborne concentrations (CFU/m3) followed by positive hole corrections. Most abundant bacterial colonies (selected by visual screening) were identified by PCR amplicon sequencing of 16S rRNA genes. For understanding overall microbial activity in clinic floors and estimating a general cleanliness of the clinic surfaces during or after dental cleaning procedures, ATP levels were determined in swabbed dust samples collected from 10 cm2 floor surfaces. Concentration of ATP may indicate both the cell viability and the metabolic status of settled microorganisms in this situation. An ATP measuring kit was used, which utilized standard luciferin-luciferase fluorescence reaction and a luminometer, which quantified ATP levels as relative light units (RLU). Three air and dust samples were collected during each cleaning procedure (at the beginning, during cleaning, and immediately after the procedure was completed (n = 45). Concentrations at the beginning, during, and after dental cleaning procedures were 671±525, 917±1203, and 899±823 CFU/m3, respectively for airborne bacteria and 91±101, 243±129, and 139±77 RLU/sample, respectively for ATP levels. The concentrations of bacteria were significantly higher than typical indoor residential environments. Although an increasing trend for airborne bacteria was observed during cleaning, the data collected at three different time points were not significantly different (ANOVA: p = 0.38) probably due to high standard deviations of data. The ATP levels, however, demonstrated a significant difference (ANOVA: p <0.05) in this scenario indicating significant change in microbial activity on floor surfaces during dental cleaning. The most common bacterial genera identified were: Neisseria sp., Streptococcus sp., Chryseobacterium sp., Paenisporosarcina sp., and Vibrio sp. in terms of frequencies of occurrences, respectively. The study concluded that bacterial exposure in dental clinics could be a notable occupational biohazard, and appropriate respiratory protections for the employees are urgently needed.

Keywords: bioaerosols, hospital hygiene, indoor air quality, occupational biohazards

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Authors: K. Rhea Devaiah, B. S. Premalatha

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Background: Tonsillar Lingual sulcus is the area between the tonsils and the base of the tongue. The surgical resection of the lesions in the head and neck results in changes in speech and swallowing functions. The severity of the speech and swallowing problem depends upon the site and extent of the lesion, types and extent of surgery and also the flexibility of the remaining structures. Need of the study: This paper focuses on the importance of speech and swallowing rehabilitation in an individual with the lesion in the Tonsillar Lingual Sulcus and post-operative functions. Aim: Evaluating the speech and swallow functions post-intensive speech and swallowing rehabilitation. The objectives are to evaluate the speech intelligibility and swallowing functions after intensive therapy and assess the quality of life. Method: The present study describes a report of an individual aged 47years male, with the diagnosis of basaloid squamous cell carcinoma, left tonsillar lingual sulcus (pT2n2M0) and underwent wide local excision with left radical neck dissection with PMMC flap reconstruction. Post-surgery the patient came with a complaint of reduced speech intelligibility, and difficulty in opening the mouth and swallowing. Detailed evaluation of the speech and swallowing functions were carried out such as OPME, articulation test, speech intelligibility, different phases of swallowing and trismus evaluation. Self-reported questionnaires such as SHI-E(Speech handicap Index- Indian English), DHI (Dysphagia handicap Index) and SESEQ -K (Self Evaluation of Swallowing Efficiency in Kannada) were also administered to know what the patient feels about his problem. Based on the evaluation, the patient was diagnosed with pharyngeal phase dysphagia associated with trismus and reduced speech intelligibility. Intensive speech and swallowing therapy was advised weekly twice for the duration of 1 hour. Results: Totally the patient attended 10 intensive speech and swallowing therapy sessions. Results indicated misarticulation of speech sounds such as lingua-palatal sounds. Mouth opening was restricted to one finger width with difficulty chewing, masticating, and swallowing the bolus. Intervention strategies included Oro motor exercise, Indirect swallowing therapy, usage of a trismus device to facilitate mouth opening, and change in the food consistency to help to swallow. A practice session was held with articulation drills to improve the production of speech sounds and also improve speech intelligibility. Significant changes in articulatory production and speech intelligibility and swallowing abilities were observed. The self-rated quality of life measures such as DHI, SHI and SESE Q-K revealed no speech handicap and near-normal swallowing ability indicating the improved QOL after the intensive speech and swallowing therapy. Conclusion: Speech and swallowing therapy post carcinoma in the tonsillar lingual sulcus is crucial as the tongue plays an important role in both speech and swallowing. The role of Speech-language and swallowing therapists in oral cancer should be highlighted in treating these patients and improving the overall quality of life. With intensive speech-language and swallowing therapy post-surgery for oral cancer, there can be a significant change in the speech outcome and swallowing functions depending on the site and extent of lesions which will thereby improve the individual’s QOL.

Keywords: oral cancer, speech and swallowing therapy, speech intelligibility, trismus, quality of life

Procedia PDF Downloads 112

Authors: Adrienn Kiss, Karoly Zauer, Gyorgy Keglevich, Rita Molnarne Bernath

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Ginger (Zingiber officinale) is a perennial plant from Southeast Asia, widely used as a spice, herb, and medicine for many illnesses since its beneficial health effects were observed thousands of years ago. Among the compounds found in ginger, zingerone [4-hydroxy-3- methoxyphenyl-2-butanone] deserves special attention: it has an anti-inflammatory and antispasmodic effect, it can be used in case of diarrheal disease, helps to prevent the formation of blood clots, has antimicrobial properties, and can also play a role in preventing the Alzheimer's disease. Ferulic acid [(E)-3-(4-hydroxy-3-methoxyphenyl)-prop-2-enoic acid] is another cinnamic acid derivative in ginger, which has promising properties. Like many phenolic compounds, ferulic acid is also an antioxidant. Based on the results of animal experiments, it is assumed to have a direct antitumoral effect in lung and liver cancer. It also deactivates free radicals that can damage the cell membrane and the DNA and helps to protect the skin against UV radiation. The aim of this work was to synthesize these two compounds by new methods. A few of the reactions were based on the hydrogenation of dehydrozingerone [4-(4-Hydroxy-3-methoxyphenyl)-3-buten-2-one] to zingerone. Dehydrozingerone can be synthesized by a relatively simple method from acetone and vanillin with good yield (80%, melting point: 41 °C). Hydrogenation can be carried out chemically, for example by the reaction of zinc and acetic acid, or Grignard magnesium and ethyl alcohol. Another way to complete the reduction is the electrochemical pathway. The electrolysis of dehydrozingerone without diaphragm in aqueous media was attempted to produce ferulic acid in the presence of sodium carbonate and potassium iodide using platinum electrodes. The electrolysis of dehydrozingerone in the presence of potassium carbonate and acetic acid to prepare zingerone was carried out similarly. Ferulic acid was expected to be converted to dihydroferulic acid [3-(4-Hydroxy-3-methoxyphenyl)propanoic acid] in potassium hydroxide solution using iron electrodes, separating the anode and cathode space with a Soxhlet paper sheath impregnated with saturated magnesium chloride solution. For this reaction, ferulic acid was synthesized from vanillin and malonic acid in the presence of pyridine and piperidine (yield: 88.7%, melting point: 173°C). Unfortunately, in many cases, the expected transformations did not happen or took place in low conversions, although gas evolution occurred. Thus, a deeper understanding of these experiments and optimization are needed. Since both compounds are found in different plants, they can also be obtained by alkaline extraction or steam distillation from distinct plant parts (ferulic acid from ground bamboo shoots, zingerone from grated ginger root). The products of these reactions are rich in several other organic compounds as well; therefore, their separation must be solved to get the desired pure material. The products of the reactions described above were characterized by infrared spectral data and melting points. The use of these two simple methods may be informative for the formation of the products. In the future, we would like to study the ferulic acid and zingerone content of other plants and extract them efficiently. The optimization of electrochemical reactions and the use of other test methods are also among our plans.

Keywords: ferulic acid, ginger, synthesis, zingerone

Procedia PDF Downloads 175

Authors: M. P. S. Tomar, Neelam Bansal

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Histomorphological, histochemical and scanning electron microscopic observations were recorded in developing cornea of buffalo fetuses. The samples from fetal cornea were collected in appropriate fixative from slaughter house and Veterinary Clinics, GADVASU, Ludhiana. The microscopic slides were stained for detailed histomorphological and histochemical studies. The scanning electron microscopic studies were performed at Electron microscopy & Nanobiology Lab, PAU Ludhiana. In present study, it was observed that, in 36 days (d) fetus, the corneal epithelium was well marked single layered structure which was placed on stroma mesenchyme. Cornea appeared as the continuation of developing sclera. The thickness of cornea and its epithelium increased as well as the epithelium started becoming double layered in 47d fetus at corneo-scleral junction. The corneal thickness in this stage suddenly increased thus easily distinguished from developing sclera. The separation of corneal endothelium from stroma was evident as a single layered epithelium. The stroma possessed numerous fibroblasts in 49d stage eye. Descemet’s membrane was appeared at 52d stage. The limbus area was separated by a depression from the developing cornea in 61d stage. In 65d stage, the Bowman’s layer was more developed. Fibroblasts were arranged parallel to each other as well as parallel to the surface of developing cornea in superficial layers. These fibroblasts and fibers were arranged in wavy pattern in the center of stroma. Corneal epithelium started to be stratified as a double layered epithelium was present in this age of fetal eye. In group II (>120 Days), the corneal epithelium was stratified towards a well marked irido-corneal angle. The stromal fibroblasts followed a complete parallel arrangement in its entire thickness. In full term fetuses, a well developed cornea was observed. It was a fibrous layer which had five distinct layers. From outside to inwards were described as the outer most layer was the 7-8 layered corneal epithelial, subepithelial basement membrane (Bowman’s membrane), substantia propria or stroma, posterior limiting membrane (Descemet’s membrane) and the posterior epithelium (corneal endothelium). The corneal thickness and connective tissue elements were continued to be increased. It was 121.39 + 3.73µ at 36d stage which increased to 518.47 + 4.98 µ in group III fetuses. In fetal life, the basement membrane of corneal epithelium and endothelium depicted strong to intense periodic Acid Schiff’s (PAS) reaction. At the irido-corneal angle, the endothelium of blood vessels was also positive for PAS activity. However, cornea was found mild positive for alcian blue reaction. The developing cornea showed strong reaction for basic proteins in outer epithelium and the inner endothelium layers. Under low magnification scanning electron microscope, cornea showed two types of cells viz. light cells and dark cells. The light cells were smaller in size and had less number of microvilli in their surface than in the dark cells. Despite these surface differences between light and dark cells, the corneal surface showed the same general pattern of microvilli studding all exposed surfaces out to the cell margin. which were long (with variable height), slight tortuous slender and possessed a micro villus shaft with a very prominent knob.

Keywords: buffalo, cornea, eye, fetus, ontogeny, scanning electron microscopy

Procedia PDF Downloads 150

Authors: Mei Chen, Yingping Hou, Michelle Hao, Soheil Aghamohammadzadeh, Esteban Terzo, Roger Clark, Vijay Modur

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Background: Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a genetic skin condition characterized by skin tearing and unremitting blistering upon minimal trauma. Repeated blistering, fibrosis, and scarring lead to aggressive squamous cell carcinoma later in life. RDEB is caused by mutations in the COL7A1 gene encoding collagen type VII (C7), the major component of anchoring fibrils mediating epidermis-dermis adherence. Nonsense mutations in the COL7A1 gene of a subset of RDEB patients leads to premature termination codons (PTC). Similarly, most Junctional Epidermolysis Bullosa (JEB) cases are caused by nonsense mutations in the LAMB3 gene encoding the β3 subunit of laminin 332. Currently, there is an unmet need for the treatment of RDEB and JEB. Zikani Therapeutics has discovered an array of macrocyclic compounds with ring structures similar to macrolide antibiotics that can facilitate readthrough activity of nonsense mutations in the COL7A1 and LAMB3 genes by acting as Ribosome Modulating Agents (RMAs). The medicinal chemistry synthetic advancements of these macrocyclic compounds have allowed targeting the human ribosome while preserving the structural elements responsible for the safety and pharmacokinetic profile of clinically used macrolide antibiotics. Methods: C7 expression was used as a measure of readthrough activity by immunoblot assays in two primary human fibroblasts from RDEB patients (R578X/R578X and R163X/R1683X-COL7A1). Similarly, immunoblot assays in C325X/c.629-12T > A-LAMB3 keratinocytes were used to measure readthrough activity for JEB. The relative readthrough activity of each compound was measured relative to Gentamicin. An imaging-based fibroblast migration assay was used as an assessment of C7 functionality in RDEB-fibroblasts over 16-20 hrs. The incubation period for the above experiments was 48 hrs for RDEB fibroblasts and 72 hours for JEB keratinocytes. Results: 9 RMAs demonstrated increased protein expression in both patient RDEB fibroblasts. The highest readthrough activity at tested concentrations without cytotoxicities increased protein expression up to 179% of Gentamicin (400 µg/ml), with favored readthrough activity in R163X/R1683X-COL7A1 fibroblasts. Concurrent with protein expression, fibroblast hypermotility phenotype observed in RDEB was rescued by reducing motility by ~35% to WT levels (the same level as 690 µM Gentamicin treated cells). Laminin β3 expression was also shown to be increased by 6 RMAs in keratinocytes to 33-83% of (400 µg/ml) Gentamicin. Conclusions: To date, 9 RMAs have been identified that enhance the expression of functional C7 in a mutation-dependent manner in two different RDEB patient fibroblast backgrounds (R578X/R578X and R163X/R1683X-COL7A1). A further 6 RMAs have been identified that enhance the readthrough of C325X-LAMB3 in JEB patient keratinocytes. Based on the clinical trial conducted by us with topical gentamycin in 2017, Zikani’s RMAs achieve clinically significant levels of read-through for the treatment of recessive dystrophic and Junctional Epidermolysis Bullosa.

Keywords: epidermolysis bullosa, nonsense mutation, readthrough, ribosome modulation

Procedia PDF Downloads 98

Authors: Suikinai Nobre Santos, Ranko Gacesa, Paul F. Long, Itamar Soares de Melo

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Extremophilic microorganism are adapted to biotopes combining several stress factors (temperature, pressure, radiation, salinity and pH), which indicate the richness valuable resource for the exploitation of novel biotechnological processes and constitute unique models for investigations their biomolecules (1, 2). The above information encourages us investigate bioprospecting synthesized compounds by a noval actinomycete, designated thermotolerant Streptomyces caatingaensis CMAA 1322, isolated from sample soil tropical dry forest (Caatinga) in the Brazilian semiarid region (3-17°S and 35-45°W). This set of constrating physical and climatic factores provide the unique conditions and a diversity of well adapted species, interesting site for biotechnological purposes. Preliminary studies have shown the great potential in the production of cytotoxic, pesticidal and antimicrobial molecules (3). Thus, to extend knowledge of the genes clusters responsible for producing biosynthetic pathways of natural products in strain CMAA1322, whole-genome shotgun (WGS) DNA sequencing was performed using paired-end long sequencing with PacBio RS (Pacific Biosciences). Genomic DNA was extracted from a pure culture grown overnight on LB medium using the PureLink genomic DNA kit (Life Technologies). An approximately 3- to 20-kb-insert PacBio library was constructed and sequenced on an 8 single-molecule real-time (SMRT) cell, yielding 116,269 reads (average length, 7,446 bp), which were allocated into 18 contigs, with 142.11x coverage and N50 value of 20.548 bp (BioProject number PRJNA288757). The assembled data were analyzed by Rapid Annotations using Subsystems Technology (RAST) (4) the genome size was found to be 7.055.077 bp, comprising 6167 open reading frames (ORFs) and 413 subsystems. The G+C content was estimated to be 72 mol%. The closest-neighbors tool, available in RAST through functional comparison of the genome, revealed that strain CMAA1322 is more closely related to Streptomyces hygroscopicus ATCC 53653 (similarity score value, 537), S. violaceusniger Tu 4113 (score value, 483), S. avermitilis MA-4680 (score value, 475), S. albus J1074 (score value, 447). The Streptomyces sp. CMAA1322 genome contains 98 tRNA genes and 135 genes copies related to stress response, mainly osmotic stress (14), heat shock (16), oxidative stress (49). Functional annotation by antiSMASH version 3.0 (5) identified 41 clusters for secondary metabolites (including two clusters for lanthipeptides, ten clusters for nonribosomal peptide synthetases [NRPS], three clusters for siderophores, fourteen for polyketide synthetase [PKS], six clusters encoding a terpene, two clusters encoding a bacteriocin, and one cluster encoding a phenazine). Our work provide in comparative analyse of genome and extract produced (data no published) by lineage CMAA1322, revealing the potential of microorganisms accessed from extreme environments as Caatinga” to produce a wide range of biotechnological relevant compounds.

Keywords: caatinga, streptomyces, environmental stresses, biosynthetic pathways

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Authors: Tim Bolle, Franziska Kreimendahl, Thomas Gries, Stefan Jockenhoevel

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The therapeutic treatment of extensive, deep wounds is limited. Autologous split-skin grafts are used as a so-called ‘gold standard’. Most common deficits are the defects at the donor site, the risk of scarring as well as the limited availability and quality of the autologous grafts. The aim of this project is a tissue engineered dermal-epidermal skin replacement to overcome the limitations of the gold standard. A key requirement for the development of such a three-dimensional implant is the formation of a functional capillary-like network inside the implant to ensure a sufficient nutrient and gas supply. Tailored three-dimensional warp knitted spacer fabrics are used to reinforce the mechanically week fibrin gel-based scaffold and further to create a directed in vitro pre-vascularization along the parallel-oriented pile yarns within a co-culture. In this study various three-dimensional warp knitted spacer fabrics were developed in a factorial design to analyze the influence of the machine parameters such as the stitch density and the pattern of the fabric on the scaffold performance and further to determine suitable parameters for a successful fibrin gel-incorporation and a physiological performance of the scaffold. The fabrics were manufactured on a Karl Mayer double-bar raschel machine DR 16 EEC/EAC. A fine machine gauge of E30 was used to ensure a high pile yarn density for sufficient nutrient, gas and waste exchange. In order to ensure a high mechanical stability of the graft, the fabrics were made of biocompatible PVDF yarns. Key parameters such as the pore size, porosity and stress/strain behavior were investigated under standardized, controlled climate conditions. The influence of the input parameters on the mechanical and morphological properties as well as the ability of fibrin gel incorporation into the spacer fabric was analyzed. Subsequently, the pile yarns of the spacer fabrics were colonized with Human Umbilical Vein Endothelial Cells (HUVEC) to analyze the ability of the fabric to further function as a guiding structure for a directed vascularization. The cells were stained with DAPI and investigated using fluorescence microscopy. The analysis revealed that the stitch density and the binding pattern have a strong influence on both the mechanical and morphological properties of the fabric. As expected, the incorporation of the fibrin gel was significantly improved with higher pore sizes and porosities, whereas the mechanical strength decreases. Furthermore, the colonization trials revealed a high cell distribution and density on the pile yarns of the spacer fabrics. For a tailored reinforcing structure, the minimum porosity and pore size needs to be evaluated which still ensures a complete incorporation of the reinforcing structure into the fibrin gel matrix. That will enable a mechanically stable dermal graft with a dense vascular network for a sufficient nutrient and oxygen supply of the cells. The results are promising for subsequent research in the field of reinforcing mechanically weak biological scaffolds and develop functional three-dimensional scaffolds with an oriented pre-vascularization.

Keywords: fibrin-gel, skin replacement, spacer fabric, pre-vascularization

Procedia PDF Downloads 257

Authors: J. Ticona Chambi, E. A. De Almeida, C. A. Andrade Raymundo Gaiotto, A. M. Do Espírito Santo, L. Infantes, S. L. Cuffini

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The solubility of active pharmaceutical ingredients (APIs) is challenging for the pharmaceutical industry. The new multicomponent crystalline forms as cocrystal and solvates present an opportunity to improve the solubility of APIs. Commonly, the procedure to obtain multicomponent crystalline forms of a drug starts by screening the drug molecule with the different coformers/solvents. However, it is necessary to develop methods to obtain multicomponent forms in an efficient way and with the least possible environmental impact. The Hansen Solubility Parameters (HSPs) is considered a tool to obtain theoretical knowledge of the solubility of the target compound in the chosen solvent. H-Bond Propensity (HBP), Molecular Complementarity (MC), Coordination Values (CV) are tools used for statistical prediction of cocrystals developed by the Cambridge Crystallographic Data Center (CCDC). The HSPs and the CCDC tools are based on inter- and intra-molecular interactions. The curcumin (Cur), target molecule, is commonly used as an anti‐inflammatory. The demethoxycurcumin (Demcur) and bisdemethoxycurcumin (Bisdcur) are natural analogues of Cur from turmeric. Those target molecules have differences in their solubilities. In this way, the work aimed to analyze and compare different tools for multicomponent forms prediction (solvates) of Cur, Demcur and Biscur. The HSP values were calculated for Cur, Demcur, and Biscur using the chemical group contribution methods and the statistical optimization from experimental data. The HSPmol software was used. From the HSPs of the target molecules and fifty solvents (listed in the HSP books), the relative energy difference (RED) was determined. The probability of the target molecules would be interacting with the solvent molecule was determined using the CCDC tools. A dataset of fifty molecules of different organic solvents was ranked for each prediction method and by a consensus ranking of different combinations: HSP, CV, HBP and MC values. Based on the prediction, 15 solvents were selected as Dimethyl Sulfoxide (DMSO), Tetrahydrofuran (THF), Acetonitrile (ACN), 1,4-Dioxane (DOX) and others. In a starting analysis, the slow evaporation technique from 50°C at room temperature and 4°C was used to obtain solvates. The single crystals were collected by using a Bruker D8 Venture diffractometer, detector Photon100. The data processing and crystal structure determination were performed using APEX3 and Olex2-1.5 software. According to the results, the HSPs (theoretical and optimized) and the Hansen solubility sphere for Cur, Demcur and Biscur were obtained. With respect to prediction analyses, a way to evaluate the predicting method was through the ranking and the consensus ranking position of solvates already reported in the literature. It was observed that the combination of HSP-CV obtained the best results when compared to the other methods. Furthermore, as a result of solvent selected, six new solvates, Cur-DOX, Cur-DMSO, Bicur-DOX, Bircur-THF, Demcur-DOX, Demcur-ACN and a new Biscur hydrate, were obtained. Crystal structures were determined for Cur-DOX, Biscur-DOX, Demcur-DOX and Bicur-Water. Moreover, the unit-cell parameter information for Cur-DMSO, Biscur-THF and Demcur-ACN were obtained. The preliminary results showed that the prediction method is showing a promising strategy to evaluate the possibility of forming multicomponent. It is currently working on obtaining multicomponent single crystals.

Keywords: curcumin, HSPs, prediction, solvates, solubility

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Authors: Francesca Gullo, Paola Palmero, Massimo Messori

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Transparent wood is considered a promising structural material for the development of environmentally friendly, energy-efficient engineered components. To obtain transparent wood from natural wood materials two approaches can be used: i) bottom-up and ii) top-down. Through the second method, the color of natural wood samples is lightened through a chemical bleaching process that acts on chromophore groups of lignin, such as the benzene ring, quinonoid, vinyl, phenolics, and carbonyl groups. These chromophoric units form complex conjugate systems responsible for the brown color of wood. There are two strategies to remove color and increase the whiteness of wood: i) lignin removal and ii) lignin bleaching. In the lignin removal strategy, strong chemicals containing chlorine (chlorine, hypochlorite, and chlorine dioxide) and oxidizers (oxygen, ozone, and peroxide) are used to completely destroy and dissolve the lignin. In lignin bleaching methods, a moderate reductive (hydrosulfite) or oxidative (hydrogen peroxide) is commonly used to alter or remove the groups and chromophore systems of lignin, selectively discoloring the lignin while keeping the macrostructure intact. It is, therefore, essential to manipulate nanostructured wood by precisely controlling the nanopores in the cell walls by monitoring both chemical treatments and process conditions, for instance, the treatment time, the concentration of chemical solutions, the pH value, and the temperature. The elimination of wood light scattering is the second step in the fabrication of transparent wood materials, which can be achieved through two-step approaches: i) the polymer impregnation method and ii) the densification method. For the polymer impregnation method, the wood scaffold is treated with polymers having a corresponding refractive index (e.g., PMMA and epoxy resins) under vacuum to obtain the transparent composite material, which can finally be pressed to align the cellulose fibers and reduce interfacial defects in order to have a finished product with high transmittance (>90%) and excellent light-guiding. However, both the solution-based bleaching and the impregnation processes used to produce transparent wood generally consume large amounts of energy and chemicals, including some toxic or pollutant agents, and are difficult to scale up industrially. Here, we report a method to produce optically transparent wood by modifying the lignin structure with a chemical reaction at room temperature using small amounts of hydrogen peroxide in an alkaline environment. This method preserves the lignin, which results only deconjugated and acts as a binder, providing both a strong wood scaffold and suitable porosity for infiltration of biobased polymers while reducing chemical consumption, the toxicity of the reagents used, polluting waste, petroleum by-products, energy and processing time. The resulting transparent wood demonstrates high transmittance and low thermal conductivity. Through the combination of process efficiency and scalability, the obtained materials are promising candidates for application in the field of construction for modern energy-efficient buildings.

Keywords: bleached wood, energy-efficient components, hydrogen peroxide, transparent wood, wood composites

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Authors: Vincent Oghenekevbe Olughor, Olusoji Mayowa Ige

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Except for a preexisting liver disease and malnutrition, there are no predilections for low serum albumin (SA) levels in humans. At normal reference levels (4.0-6.0g/dl) SA is a universal marker for mortality and morbidity risks assessments where depletion by 1.0g/dl increases mortality risk by 137% and morbidity by 89%.It has 40 known functions contributing significantly to the sustenance of human life. A depletion in SA to <2.2g/dl, in most clinical settings worldwide, leads to loss of oncotic pressure of blood causing clinical manifestations of bipedal Oedema, in which the patients remain conscious. SA also contributes significantly to buffering of blood to a life-sustaining pH of 7.35-7.45. A drop in blood pH to <6.9 will lead to instant coma and death, which can occur after SA continues to deplete after manifestations of bipedal Oedema. In an intervention study conducted in 2014 following the discovery that “SA is depleted during malaria fever”, a Nutraceutical formulated for use as treatment adjunct to prevent SA depletions during malaria to <2.4g/dl after Efficacy testing was found to be satisfactory. There are five known types of Malaria caused by Apicomplexan parasites, Plasmodium: the most lethal being that caused by Plasmodium falciparum causing malignant tertian malaria, in which the fever was occurring every 48 hours coincides with the dumping of malaria-toxins (Hemozoin) into blood, causing contamination: blood must remain sterile. Other Apicomplexan parasites, Toxoplasma and Cryptosporidium, are opportunistic infections of HIV. Separate studies showed SA depletions in MDRTB (multidrug resistant TB), and MDRTB-HIV patients by the same mechanism discovered with malaria and such depletions will be further complicated whenever Apicomplexan parasitic infections co-exist. Both Apicomplexan parasites and the TB parasite belong to the Obligate-group of Parasites, which are parasites that replicate only inside its host; and most of them have capacities to over-consume host nutrients during parasitaemia. In MDRTB patients the body attempts repeatedly to prevent depletions in SA to critical levels in the presence of adequate nutrients and only for a while in MDRTB-HIV patients. These groups of patients will, therefore, benefit from the already tested Nutraceutical in malaria patients. The Nutraceutical bio-Powder was formulated (to BP 1988 specification) from twelve nature-based food-grade nutrients containing all dedicated nutrients for ensuring improved synthesis of Albumin by the liver. The Nutraceutical was administered daily for 38±2days in 23 children, in a prospective phase-2 clinical trial, and its impact on body weight and core blood parameters were documented at the start and end of efficacy testing period. Sixteen children who did not experience malaria-induced depletions of SA had significant SA increase; seven children who experienced malaria-induced depletions of SA had insignificant SA decrease. The Packed Cell Volume Percentage (PCV %), a measure of the Oxygen carrying capacity of blood and the amount of nutrients the body can absorb, increased in both groups. The total serum proteins (SA+ Globulins) increased or decreased within the continuum of normal. In conclusion, MDRTB and MDRTB-HIV patients will benefit from a variant of this Nutraceutical when used as treatment adjunct.

Keywords: antitrypsin-free Nutraceutical, apicomplexan parasites, no predilections for low serum albumin, toxoplasmosis

Procedia PDF Downloads 288

Authors: B. Voucko, M. Benkovic, N. Cukelj, S. Drakula, D. Novotni, S. Balbino, D. Curic

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Oxidative stress is considered as one of the causes leading to metabolic disorders in humans. Therefore, the ability of antioxidants to inhibit free radical production is their primary role in the human organism. Antioxidants originating from cereals, especially flavonoids and polyphenols, are mostly bound and indigestible. Micronization damages the cell wall which consecutively results in bioactive material to be more accessible in vivo. In order to ensure complete fragmentation, micronization is often combined with high temperatures (e.g., for bran 200°C) which can lead to degradation of bioactive compounds. The innovative non-thermal technology of cryo-milling is an ultra-fine micronization method that uses liquid nitrogen (LN2) at a temperature of 195°C to freeze and cool the sample during milling. Freezing at such low temperatures causes the material to become brittle which ensures the generation of fine particles while preserving the bioactive content of the material. The aim of this research was to determine if production of ultra-fine material with cryo-milling will result in the augmentation of available bioactive compounds of buckwheat and pumpkin seed cake. For that reason, buckwheat and pumpkin seed cake were ground in a ball mill (CryoMill, Retch, Germany) with and without the use of LN2 for 8 minutes, in a 50 mL stainless steel jar containing one grinding ball (Ø 25 mm) at an oscillation frequency of 30 Hz. The cryo-milled samples were cooled with LN2 for 2 minutes prior to milling, followed by the first cycle of milling (4 minutes), intermediary cooling (2 minutes), and finally the second cycle of milling (further 4 minutes). A continuous process of milling was applied to the samples ground without freezing with LN2. Particle size distribution was determined using the Scirocco 2000 dry dispersion unit (Malvern Instruments, UK). Antioxidant activity was determined by 2,2-Diphenyl-1-picrylhydrazyl (DPPH) test and ferric reducing antioxidant power (FRAP) assay, while the total phenol content was determined using the Folin Ciocalteu method, using the ultraviolet-visible spectrophotometer (Specord 50 Plus, Germany). The content of the free phenolic acids, rutin in buckwheat, tyrosol in pumpkin seed cake, was determined with an HPLC-PDA method (Agilent 1200 series, Germany). Cryo-milling resulted in 11 times smaller size of buckwheat particles, and 3 times smaller size of pumpkin seed particles than milling without the use of LN2, but also, a lower uniformity of the particle size distribution. Lack of freezing during milling of pumpkin seed cake caused a formation of agglomerates due to its high-fat content (21 %). Cryo-milling caused augmentation of buckwheat flour antioxidant activity measured by DPPH test (23,9%) and an increase in available rutin content (14,5%). Also, it resulted in an augmentation of the total phenol content (36,9%) and available tyrosol content (12,5%) of pumpkin seed cake. Antioxidant activity measured with the FRAP test, as well as the content of phenolic acids remained unchanged independent of the milling process. The results of this study showed the potential of cryo-milling for complete raw material utilization in the food industry, as well as a tool for extraction of aimed bioactive components.

Keywords: bioactive, ball-mill, buckwheat, cryo-milling, pumpkin seed cake

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Authors: Prachi Buche Marathe

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Karanja Laad is a city located in the Vidarbha region in the state of Maharashtra, India. It has a huge amount of tangible and intangible heritage in the form of monuments, precincts, a group of structures, festivals and procession route, which is neglected and lost with time. Three different religions Hinduism, Islam and Jainism along with associations of being a birthplace of Swami Nrusinha Saraswati, an exponent of Datta Sampradaya sect and the British colonial layer have shaped the culture and society of the place over the period. The architecture of the town Karanja Laad has enhanced its unique historic and cultural value with a combination of all these historic layers. Karanja Laad is also a traditional trading historic town with unique hybrid architectural style and has a good potential for developing as a tourist place along with the present image of a pilgrim destination of Datta Sampradaya. The aim of the research is to prepare a conservation proposal for the historic town along with the management framework. Objectives of the research are to study the evolution of Karanja town, to identify the cultural resources along with issues of the historic core of the city, to understand Datta sampradaya, and contribution of Saint Nrusinha Saraswati in the religious sect and his association as an important personality with Karanja. The methodology of the research is site visits to the Karanja city, making field surveys for documentation and discussions and questionnaires with the residents to establish heritage and identify potential and issues within the historic core thereby establishing a case for conservation. Field surveys are conducted for town level study of land use, open spaces, occupancy, ownership, traditional commodity and community, infrastructure, streetscapes, and precinct activities during the festival and non-festival period. Building level study includes establishing various typologies like residential, institutional commercial, religious, and traditional infrastructure from the mythological references like waterbodies (kund), lake and wells. One of the main issues is that the loss of the traditional footprint as well as the traditional open spaces which are getting lost due to the new illegal encroachments and lack of guidelines for the new additions to conserve the original fabric of the structures. Traditional commodities are getting lost since there is no promotion of these skills like pottery and painting. Lavish bungalows like Kannava mansion, main temple Wada (birthplace of the saint) have a huge potential to be developed as a museum by adaptive re-use which will, in turn, attract many visitors during festivals which will boost the economy. Festival procession routes can be identified and a heritage walk can be developed so as to highlight the traditional features of the town. Overall study has resulted in establishing a heritage map with 137 heritage structures identified as potential. Conservation proposal is worked out on the town level, precinct level and building level with interventions such as developing construction guidelines for further development and establishing a heritage cell consisting architects and engineers for the upliftment of the existing rich heritage of the Karanja city.

Keywords: built heritage, conservation, Datta Sampradaya, Karanja Laad, Swami Nrusinha Saraswati, procession route

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Authors: Christine Wiesenthal

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This paper asks how to read a particularly vexed and complicated life writing text. For over half a century, the wartime journals of Ruth Andreas Friedrich (1901-1977) circulated as among a handful of more or less authoritative and “authentic” first-hand accounts of German resistance under Hitler. A professional journalist, Andreas Friedrich is remembered today largely through her publications at the war’s end, which appeared in English as Berlin Underground (published by Henry Holt in 1947), just before their publication in Germany as Der Schattenmann “The Shadow Man” (also in 1947). A British edition by the now obscure Latimer House Limited (London) followed in 1948; it is based closely on but is not identical to, the Henry Holt American edition, which in turn differs significantly from its German counterpart. Both Berlin Underground and Der Schattenmann figure Andreas-Friedrich as a key figure in an anti-fascist cell that operated in Berlin under the code name “Uncle Emil,” and provide a riveting account of political terror, opportunism, and dissent under the Nazi regime. Recent scholars have, however, begun to raise fascinating and controversial questions about Andreas-Friedrich’s own writing/reconstruction process in compiling the journals and about her highly selective curatorial role and claims. The apparent absence of any surviving original manuscript for Andreas-Friedrich’s journals amplifies the questions around them. Crucially, so too does the role of the translator of the English editions of Berlin Underground, the enigmatic June Barrows Mussey, a subject that has thus far gone virtually unnoticed and which this paper will focus on. Mussey, a prolific American translator, simultaneously cultivated a career as a professional magician, publishing a number of books on that subject under the alias Henry Hay. While the record indicates that Mussey attempted to compartmentalize his professional life, research into the publishing and translation history of Berlin Underground suggests that the two roles converge in the fact of the translator’s invisibility, by effacing the traces of his own hand and leaving unmarked his own significant textual interventions, Mussey, in effect, edited, abridged, and altered Andreas Friedrich’s journals for the second time. In fact, it could be said that while the fictitious “Uncle Emil” is positioned as “the shadow man” of the German edition, Mussey himself also emerges as precisely that in the English rendering of the journals. The implications of Mussey’s translation of Andreas Friedrich’s journals are one of the most important un-examined gaps in the shadowy publishing history of Berlin Underground, a history full of “tricks” (Mussey’s words) and illusions of transparency. Based largely on archival research of unpublished materials and methods of close reading and comparative analysis, this study will seek to convey some preliminary insights and exploratory work and frame questions toward what is ultimately envisioned as an experimental project in poetic historiography. As this work is still in the early stages, it would be especially welcome to have the opportunity provided by this conference to connect with a community of life writing colleagues who might help think through some of the challenges and possibilities that lie ahead.

Keywords: women’s wartime diaries, translation studies, auto/biographical theory, politics of life writing

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Authors: Evgenia Iakovleva, Juha Koivisto, Pasi Karppinen, J. Inkinen, Mikko Alava

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Preventing the spread of infectious diseases throughout the worldwide is one of the most important tasks of modern health care. Infectious diseases not only account for one fifth of the deaths in the world, but also cause many pathological complications for the human health. Touch surfaces pose an important vector for the spread of infections by varying microorganisms, including antimicrobial resistant organisms. Further, antimicrobial resistance is reply of bacteria to the overused or inappropriate used of antibiotics everywhere. The biggest challenges in bacterial detection by existing methods are non-direct determination, long time of analysis, the sample preparation, use of chemicals and expensive equipment, and availability of qualified specialists. Therefore, a high-performance, rapid, real-time detection is demanded in rapid practical bacterial detection and to control the epidemiological hazard. Among the known methods for determining bacteria on the surfaces, Hyperspectral methods can be used as direct and rapid methods for microorganism detection on different kind of surfaces based on fluorescence without sampling, sample preparation and chemicals. The aim of this study was to assess the relevance of such systems to remote sensing of surfaces for microorganisms detection to prevent a global spread of infectious diseases. Bacillus subtilis and Escherichia coli with different concentrations (from 0 to 10x8 cell/100µL) were detected with hyperspectral camera using different filters as visible visualization of bacteria and background spots on the steel plate. A method of internal standards was applied for monitoring the correctness of the analysis results. Distances from sample to hyperspectral camera and light source are 25 cm and 40 cm, respectively. Each sample is optically imaged from the surface by hyperspectral imaging system, utilizing a JAI CM-140GE-UV camera. Light source is BeamZ FLATPAR DMX Tri-light, 3W tri-colour LEDs (red, blue and green). Light colors are changed through DMX USB Pro interface. The developed system was calibrated following a standard procedure of setting exposure and focused for light with λ=525 nm. The filter is ThorLabs KuriousTM hyperspectral filter controller with wavelengths from 420 to 720 nm. All data collection, pro-processing and multivariate analysis was performed using LabVIEW and Python software. The studied human eye visible and invisible bacterial stains clustered apart from a reference steel material by clustering analysis using different light sources and filter wavelengths. The calculation of random and systematic errors of the analysis results proved the applicability of the method in real conditions. Validation experiments have been carried out with photometry and ATP swab-test. The lower detection limit of developed method is several orders of magnitude lower than for both validation methods. All parameters of the experiments were the same, except for the light. Hyperspectral imaging method allows to separate not only bacteria and surfaces, but also different types of bacteria, such as Gram-negative Escherichia coli and Gram-positive Bacillus subtilis. Developed method allows skipping the sample preparation and the use of chemicals, unlike all other microbiological methods. The time of analysis with novel hyperspectral system is a few seconds, which is innovative in the field of microbiological tests.

Keywords: Escherichia coli, Bacillus subtilis, hyperspectral imaging, microorganisms detection

Procedia PDF Downloads 224

Authors: Ellana Boada, Eric Santos-Clotas, Alba Cabrera-Codony, Maria Martin, Lluis Baneras, Frederic Gich

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Volatile methylsiloxanes (VMS) are a group of manmade silicone compounds widely used in household and industrial applications that end up on the biogas produced through the anaerobic digestion of organic matter in landfills and wastewater treatment plants. The presence of VMS during the biogas energy conversion can cause damage on the engines, reducing the efficiency of this renewable energy source. Non regenerative adsorption onto activated carbon is the most widely used technology to remove siloxanes from biogas, while new trends point out that biotechnology offers a low-cost and environmentally friendly alternative to conventional technologies. The first objective of this research was to enrich, isolate and identify bacterial species able to grow using siloxane molecules as a sole carbon source: anoxic wastewater sludge was used as initial inoculum in liquid anoxic enrichments, adding D4 (as representative siloxane compound) previously adsorbed on activated carbon. After several months of acclimatization, liquid enrichments were plated onto solid media containing D4 and thirty-four bacterial isolates were obtained. 16S rRNA gene sequencing allowed the identification of strains belonging to the following species: Ciceribacter lividus, Alicycliphilus denitrificans, Pseudomonas aeruginosa and Pseudomonas citronellolis which are described to be capable to degrade toxic volatile organic compounds. Kinetic assays with 8 representative strains revealed higher cell growth in the presence of D4 compared to the control. Our second objective was to characterize the community composition and diversity of the microbial community present in the enrichments and to elucidate whether the isolated strains were representative members of the community or not. DNA samples were extracted, the 16S rRNA gene was amplified (515F & 806R primer pair), and the microbiome analyzed from sequences obtained with a MiSeq PE250 platform. Results showed that the retrieved isolates only represented a minor fraction of the microorganisms present in the enrichment samples, which were represented by Alpha, Beta, and Gamma proteobacteria as dominant groups in the category class thus suggesting that other microbial species and/or consortia may be important for D4 biodegradation. These results highlight the need of additional protocols for the isolation of relevant D4 degraders. Currently, we are developing molecular tools targeting key genes involved in siloxane biodegradation to identify and quantify the capacity of the isolates to metabolize D4 in batch cultures supplied with a synthetic gas stream of air containing 60 mg m⁻³ of D4 together with other volatile organic compounds found in the biogas mixture (i.e. toluene, hexane and limonene). The isolates were used as inoculum in a biotrickling filter containing lava rocks and activated carbon to assess their capacity for siloxane removal. Preliminary results of biotrickling filter performance showed 35% of siloxane biodegradation in a contact time of 14 minutes, denoting that biological siloxane removal is a promising technology for biogas upgrading.

Keywords: bacterial cultivation, biogas upgrading, microbiome, siloxanes

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Authors: Ivana Cabarkapa, Zorica Tomicic, Olivera Duragic

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Antimicrobial resistance represents one of the major challenges facing humanity in the last decades. Increasing antibiotic-resistant pathogens indicates the need for the development of alternative antibacterial drugs and new treatment strategies. One of the innovative emerging treatments in overcoming multidrug-resistant pathogens certainly represents the inhibition anti-quorum sensing system. For most of the food-borne pathogens, the expression of the virulence depends on their capability communication with other members of the population by means of quorum sensing (QS). QS represents a specific way of bacterial intercellular communication, which enabled owing to their ability to detect and to respond to cell population density by gene regulation. QS mechanisms are responsible for controls the pathogenesis, virulence luminescence, motility, sporulation and biofilm formation of many organisms by regulating gene expression. Therefore, research in this field is being an attractive target for the development of new natural antibacterial agents. Anti-QS compounds are known to have the ability to prohibit bacterial pathogenicity. Considering the importance of quorum sensing during bacterial pathogenesis, this research has been focused on evaluation anti - QS properties of four essential oils (EOs) Origanum heracleoticum, Origanum vulgare, Thymus vulgare, and Thymus serpyllum, using biomonitor strain of Chromobacterium violaceum CV026. Tests conducted on Luria Bertani agar supplemented with N hexanol DL homoserine lacton (HHL) 10µl/50ml of agar. The anti-QS potential of the EOs was assayed in a range of concentrations of 200 – 0.39 µl/ml using the disc diffusion method. EOs of Th. vulgaris and T. serpyllum were exhibited anti-QS activity indicated by a non- pigmented ring with a dilution-dependent manner. The lowest dilution of EOs T. vulgaris and T. serpyllum in which they exhibited visually detectable inhibition of violacein synthesis was 6.25 µl/ml for both tested EOs. EOs of O. heracleoticum and O. vulgare were displayed different active principles, i.e., antimicrobial activity indicated by the inner clear ring and anti-QS activity indicated by the outer non-pigmented ring, in a concentration-dependent manner. The lowest dilution of EOs of O. heracleoticum and O. vulgare in which exhibited visually detectable inhibition of violacein synthesis was 1.56 and 3.25 µl/ml, respectively. Considering that, the main constituents of the tested EOs represented by monoterpenes (carvacrol, thymol, γ-terpinene, and p-cymene), anti - QS properties of tested EOs can be mainly attributed to their activity. In particular, from the scientific literature, carvacrol and thymol show a sub-inhibitory effect against foodborne pathogens. Previous studies indicated that sub-lethal concentrations of carvacrol reduced the mobility of bacteria due to the ability of interference using QS mechanism between the bacterial cells, and thereby reducing the ability of biofilm formation The precise mechanism by which carvacrol inhibits biofilm formation is still not fully understood. Our results indicated that EOs displayed different active principles, i.e., antimicrobial activity indicated by the inner clear ring and anti-QS activity indicated by an outer non- pigmented ring with visually detectable inhibition of violacein. Preliminary results suggest that EOs represent a promising alternative for effective control of the emergence and spread of resistant pathogens.

Keywords: anti-quorum sensing activity, Chromobacterium violaceum, essential oils, violacein

Procedia PDF Downloads 138

Authors: Banu Pradheepa Kamarajan, Muthusamy Ananthasubramanian

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Biofilm forming Pseudomonads occupy the top third position in causing hospital acquired infections. P. aeruginosa is notoriously known for its tendency to develop drug resistance. Major classes of drug such as β-lactams, aminoglycosides, quinolones, and polymyxins are found ineffective against multi-drug resistance Pseudomonas. To combat the infections, rather than administration of a single antibiotic, use of combinations (tobramycin and essential oils from plants and/or silver nanoparticles, chitosan, nitric oxide, cis-2-decenoic acid) in single formulation are suggested to control P. aeruginosa biofilms. Conventional techniques to prevent hospital-acquired implant infections such as coatings with antibiotics, controlled release of antibiotics from the implant material, contact-killing surfaces, coating the implants with functional DNase I and, coating with glycoside hydrolase are being followed. Coatings with bioactive components besides having limited shelf-life, require cold-chain and, are likely to fail when bacteria develop resistance. Recently identified nano-scale physical architectures on the insect wings are expected to have potential bactericidal property. Nanopillars are bactericidal to Staphylococcus aureus, Bacillus subtilis, K. pnuemoniae and few species of Pseudomonas. Our study aims to investigate the survival rate of biofilm forming Pseudomonas aeruginosa strain over non-biofilm forming strain on the nanopillar architecture of dragonfly (Pantala flavescens) wing. Dragonflies were collected near house-hold areas and, insect identification was carried out by the Department of Entomology, Tamilnadu Agricultural University, Coimbatore, India. Two strains of P. aeruginosa such as PAO1 (potent biofilm former) and MTCC 1688 (non-weak biofilm former) were tested against the glass coverslip (control) and wings of dragonfly (test) for 48 h. The wings/glass coverslips were incubated with bacterial suspension in 48-well plate. The plates were incubated at 37 °C under static condition. Bacterial attachment on the nanopillar architecture of the wing surface was visualized using FESEM. The survival rate of P. aeruginosa was tested using colony counting technique and flow cytometry at 0.5 h, 1 h, 2 h, 7 h, 24 h, and 48 h post-incubation. Cell death was analyzed using propidium iodide staining and DNA quantification. The results indicated that the survival rate of non-biofilm forming P. aeruginosa is 0.2 %, whilst that of biofilm former is 45 % on the dragonfly wings at the end of 48 h. The reduction in the survival rate of biofilm and non-biofilm forming P. aeruginosa was 20% and 40% respectively on the wings compared to the glass coverslip. In addition, Fourier Transformed Infrared Radiation was used to study the modification in the surface chemical composition of the wing during bacterial attachment and, post-sonication. This result indicated that the chemical moieties are not involved in the bactericidal property of nanopillars by the conserved characteristic peaks of chitin pre and post-sonication. The nanopillar architecture of the dragonfly wing efficiently deters the survival of non-biofilm forming P. aeruginosa, but not the biofilm forming strain. The study highlights the ability of biofilm formers to survive on wing architecture. Understanding this survival strategy will help in designing the architecture that combats the colonization of biofilm forming pathogens.

Keywords: biofilm, nanopillars, Pseudomonas aeruginosa, survival rate

Procedia PDF Downloads 175