Search results for: bloodstream expression site
3508 Waste Generation in Iranian Building Industry: Addressing a Theory
Authors: Golnaz Moghimi, Alireza Afsharghotli, Alireza Rezaei
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Construction waste has been gradually increased as a result of upsizing construction projects which are occurred within the lifecycle of buildings. Since waste management is a major priority and has profound impacts on the volume of waste generated in construction stage, the majority of efforts have been attempted to reuse, recycle and reduce waste. However, there is still room to study on lack of sufficient knowledge about waste management in construction industry. This paper intends to provide an insight into the effect of project management knowledge areas on waste management solely on construction stage. To this end, a survey among Iranian building construction industry contractors was conducted to identify the effectiveness of project management knowledge areas on three jobsite key factors including ‘Site activity’, ‘Training’, and ‘Awareness’. As a result, four management disciplines were identified as most influential ones on amount of construction waste. These disciplines were Project Cost Management, Quality Management, Human Resource Management, and Integration Management. Based on the research findings, a new model was presented to develop effective construction waste strategies.Keywords: awareness, PMBOK, site activity, training, waste management
Procedia PDF Downloads 3603507 A New Developed Formula to Determine the Shear Buckling Stress in Welded Aluminum Plate Girders
Authors: Badr Alsulami, Ahmed S. Elamary
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This paper summarizes and presents main results of an in-depth numerical analysis dealing with the shear buckling resistance of aluminum plate girders. The studies conducted have permitted the development of a simple design expression to determine the critical shear buckling stress in aluminum web panels. This expression takes into account the effects of reduction of strength in aluminum alloys due to the welding process. Ultimate shear resistance (USR) of plate girders can be obtained theoretically using Cardiff theory or Hӧglund’s theory. USR of aluminum alloy plate girders predicted theoretically using BS8118 appear inconsistent when compared with test data. Theoretical predictions based on Hӧglund’s theory, are more realistic. Cardiff theory proposed to predict the USR of steel plate girders only. Welded aluminum alloy plate girders studied experimentally by others; the USR resulted from tests are reviewed. Comparison between the test results with the values obtained from Hӧglund’s theory, BS8118 design method, and Cardiff theory performed theoretically. Finally, a new equation based on Cardiff tension-field theory proposed to predict theoretically the USR of aluminum plate girders.Keywords: shear resistance, aluminum, Cardiff theory, Hӧglund's theory, plate girder
Procedia PDF Downloads 4263506 21st Century Computer Technology for the Training of Early Childhood Teachers: A Study of Second-Year Education Students Challenged with Building a Kindergarten Website
Authors: Yonit Nissim, Eyal Weissblueth
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This research is the continuation of a process that began in 2010 with the goal of redesigning the training program for future early childhood teachers at the Ohalo College, to integrate technology and provide 21st-century skills. The article focuses on a study of the processes involved in developing a special educational unit which challenged students with the task of designing, planning and building an internet site for kindergartens. This project was part of their second-year studies in the early childhood track of an interdisciplinary course entitled 'Educating for the Future.' The goal: enabling students to gain experience in developing an internet site specifically for kindergartens, and gain familiarity with Google platforms, the acquisition and use of innovative skills and the integration of technology in pedagogy. Research questions examined how students handled the task of building an internet site. The study explored whether the guided process of building a site helped them develop proficiency in creativity, teamwork, evaluation and learning appropriate to the 21st century. The research tool was a questionnaire constructed by the researchers and distributed online to the students. Answers were collected from 50-course participants. Analysis of the participants’ responses showed that, along with the significant experience and benefits that students gained from building a website for kindergarten, ambivalence was shown toward the use of new, unfamiliar and complex technology. This attitude was characterized by unease and initial emotional distress triggered by the departure from routine training to an island of uncertainty. A gradual change took place toward the adoption of innovation with the help of empathy, training, and guidance from the instructors, leading to the students’ success in carrying out the task. Initial success led to further successes, resulting in a quality product and a feeling of personal competency among the students. A clear and extreme emotional shift was observed on the spectrum from a sense of difficulty and dissatisfaction to feelings of satisfaction, joy, competency and cognitive understanding of the importance of facing a challenge and succeeding. The findings of this study can contribute to increased understanding of the complex training process of future kindergarten teachers, coping with a changing world, and pedagogy that is supported by technology.Keywords: early childhood teachers, educating for the future, emotions, kindergarten website
Procedia PDF Downloads 1563505 In Vitro and in Vivo Evaluation of Nano Collagen Molecules to Enhance Mesenchymal Stem Cells Differentiate into Insulin Producing Cells
Authors: Chin-Tsu Ma, Yi-Jhen Wu, Hsia Ying Cheng, Han Hsiang Huang, Shyh Ming Kuo
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The use of specific molecules including nutrients and pharmacological agents has been tried in modulation of stem cells differentiation (MSCs) to insulin producing cells. The aim of this study is to investigate the ability of nano collagen molecules (nutrient or scaffold) to enhance the MSCs differentiation into insulin-producing cells in combination with nicotinamide and exendin-4 (pharmacological agents) in vitro and in vivo. The results demonstrated that the cells exhibit morphologically islet-like clusters after treatment with nano collagen molecules, nicotinamide and exendin-4. MSCs extra treated with nano collagen molecules showed significant increases in Nkx6.1 and insulin mRNA expression at 14-d and 21-d culture compared with those merely treated with nicotinamide and exendin-4. Early 7-day elevation in PDX-1 mRNA expression was observed. Furthermore, the MSCs exposed to nano collagen molecules produced the highest secretion of insulin (p < 0.05). Type-2 diabetes induced by high-fat diet and low dose of streptozotocin in rat model was built in this study. This rat exhibited higher food intake, water intake, lower glucose tolerance, lower-insulin tolerance, and higher HbA1C (significant increases, p < 0.01) as compared with the normal rat that demonstrated the model of type-2 diabetes was successfully built. Biopsy examinations also showed that obvious destruction of islet. After injection of differentiated MSCs into the destructed pancreas of diabetes rat, more regenerated islet were observed at the rats that treated with nano collagen molecules and exhibited much lower HbA1C as compared with the normal rat and diabetes rat after 4 weeks (significant deceases, p < 0.001). These results indicate that the culturing MSCs with nano collagen molecules, nicotinamide, and exendin-4 are beneficial for MSCs differentiation into islet-like cells. These nano collagen molecules may lead to alternations or up-regulation of gene expression and influence the differentiated outcomes induced by nicotinamide and exendin-4.Keywords: nano collagen molecules, nicotinamide, MSCs, diabetes
Procedia PDF Downloads 4103504 Early Cell Cultures Derived from Human Prostate Cancer Tissue Express Tissue-Specific Epithelial and Cancer Markers
Authors: Vladimir Ryabov, Mikhail Baryshevs, Mikhail Voskresenskey, Boris Popov
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The human prostate gland (PG) samples were obtained from patients who had undergone radical prostatectomy for prostate cancer (PC) and used to extract total RNA and prepare the prostate stromal cell cultures (PSCC) and patients-derived organoids (PDO). Growth of the cell cultures was accessed under microscopic evaluation in transmitted light and the marker expression by reverse polymerase chain reaction (RT-PCR), immunofluorescence, and immunoblotting. Some PCR products from prostate tissue, PSCC, and PDO were cloned and sequenced. We found that the cells of early and late passages of PSCC and corresponding PDO expressed luminal (androgen receptor, AR; cytokeratin 18, CK18) and basal (CK5, p63) epithelial markers, the production of which decreased or disappeared in late PSCC and PDO. The PSCC and PDO of early passages from cancer tissue additionally produced cancer markers AMACR, TMPRSS2-ERG, and Ezh2. The expression of TMPRSS2-ERG fusion transcripts was verified by cloning and sequencing the PCR products. The results obtained suggest that early passages of PSCC might be used as a pre-clinical model for the evaluation of early markers of prostate cancer.Keywords: localized prostate cancer, prostate epithelial markers, prostate cancer markers, AMACR, TMPRSS2-ERG, prostate stromal cell cultures, PDO
Procedia PDF Downloads 1103503 Synthesis, Inhibitory Activity, and Molecular Modelling of 2-Hydroxy-3-Oxo-3-Phenylpropionate Derivatives as HIV-1-Integrase Inhibitors
Authors: O. J. Jesumoroti, Faridoon, R. Klein, K. A. Iobb, D. Mnkadhla, H. C. Hoppe, P. T. Kaye
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The 1, 3-aryl diketo acids (DKA) based agents represent an important class of HIV integrase (IN) strand transfer inhibitors. In other to study the chelating role of the divalent metal ion in the inhibition of IN strand transfer, we designed and synthesized a series of 2-hydroxy-3-oxo-3-phenyl propionate derivatives with the notion that such compounds could interact with the divalent ion in the active site of IN. The synthetic sequence to the desired compounds involves the concept of Doebner knoevenagel condensation, Fischer esterification and ketohydroxylation using neuclophilic re-oxidant; compounds were characterized by their IR, IHNMR, 13CNMR, HRMS spectroscopic data and melting point determination. Also, molecular docking was employed in this study and it was revealed that there is interaction with the active site of the enzyme. However, there is disparity in the corresponding anti-HIV activity determined by the experimental bioassay. These compounds lack potency at low micromolar concentration when compared to the results of the docking studies. Nevertheless, the results of the study suggest modification of the aryl ring with one or two hydroxyl groups to improve the inhibitory activity.Keywords: anti-HIV-1 integrase, ketohydroxylation, molecular docking, propionate derivatives
Procedia PDF Downloads 1953502 On-Site Coaching on Freshly-Graduated Nurses to Improves Quality of Clinical Handover and to Avoid Clinical Error
Authors: Sau Kam Adeline Chan
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World Health Organization had listed ‘Communication during Patient Care Handovers’ as one of its highest 5 patient safety initiatives. Clinical handover means transfer of accountability and responsibility of clinical information from one health professional to another. The main goal of clinical handover is to convey patient’s current condition and treatment plan accurately. Ineffective communication at point of care is globally regarded as the main cause of the sentinel event. Situation, Background, Assessment and Recommendation (SBAR), a communication tool, is extensively regarded as an effective communication tool in healthcare setting. Nonetheless, just by scenario-based program in nursing school or attending workshops on SBAR would not be enough for freshly graduated nurses to apply it competently in a complex clinical practice. To what extend and in-depth of information should be conveyed during handover process is not easy to learn. As such, on-site coaching is essential to upgrade their expertise on the usage of SBAR and ultimately to avoid any clinical error. On-site coaching for all freshly graduated nurses on the usage of SBAR in clinical handover was commenced in August 2014. During the preceptorship period, freshly graduated nurses were coached by the preceptor. After that, they were gradually assigned to take care of a group of patients independently. Nurse leaders would join in their shift handover process at patient’s bedside. Feedback and support were given to them accordingly. Discrepancies on their clinical handover process were shared with them and documented for further improvement work. Owing to the constraint of manpower in nurse leader, about coaching for 30 times were provided to a nurse in a year. Staff satisfaction survey was conducted to gauge their feelings about the coaching and look into areas for further improvement. Number of clinical error avoided was documented as well. The nurses reported that there was a significant improvement particularly in their confidence and knowledge in clinical handover process. In addition, the sense of empowerment was developed when liaising with senior and experienced nurses. Their proficiency in applying SBAR was enhanced and they become more alert to the critical criteria of an effective clinical handover. Most importantly, accuracy of transferring patient’s condition was improved and repetition of information was avoided. Clinical errors were prevented and quality patient care was ensured. Using SBAR as a communication tool looks simple. The tool only provides a framework to guide the handover process. Nevertheless, without on-site training, loophole on clinical handover still exists, patient’s safety will be affected and clinical error still happens.Keywords: freshly graduated nurse, competency of clinical handover, quality, clinical error
Procedia PDF Downloads 1503501 Functional Characterization of Transcriptional Regulator WhiB Proteins of Mycobacterium Tuberculosis
Authors: Sonam Kumari
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Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, possesses a remarkable feature of entering into and emerging from a persistent state. The mechanism by which Mtb switches from the dormant state to the replicative form is still poorly characterized. Proteome studies have given us an insight into the role of certain proteins in giving stupendous virulence to Mtb, but numerous dotsremain unconnected and unaccounted. The WhiB family of proteins is one such protein that is associated with developmental processes in actinomycetes.Mtb has seven such proteins (WhiB1 to WhiB7).WhiB proteins are transcriptional regulators; their conserved C-terminal HTH motif is involved in DNA binding. They regulate various essential genes of Mtbby binding to their promoter DNA. Biophysical Analysis of the effect of DNA binding on WhiB proteins has not yet been appropriately characterized. Interaction with DNA induces conformational changes in the WhiB proteins, confirmed by steady-state fluorescence and circular dichroism spectroscopy. ITC has deduced thermodynamic parameters and the binding affinity of the interaction. Since these transcription factors are highly unstable in vitro, their stability and solubility were enhanced by the co-expression of molecular chaperones. The present study findings help determine the conditions under which the WhiB proteins interact with their interacting partner and the factors that influence their binding affinity. This is crucial in understanding their role in regulating gene expression in Mtbandin targeting WhiB proteins as a drug target to cure TB.Keywords: tuberculosis, WhiB proteins, mycobacterium tuberculosis, nucleic acid binding
Procedia PDF Downloads 1073500 Detection of Viral-Plant Interaction Using Some Pathogenesis Related Protein Genes to Identify Resistant Genes against Potato LeafRoll Virus and Potato Virus Y in Egyptian Isolates
Authors: Dalia. G. Aseel, E. E. Hafez, S. M. Hammad
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Viral RNAs of both potato leaf roll virus (PLRV) and potato virus Y (PVY) were extracted from infected potato leaves collected from different Egyptian regions. Differential Display Polymerase Chain Reaction (DD-PCR) using (Endogluconase, β-1,3-glucanases, Chitinase, Peroxidase and Polyphenol oxidase) primers (forward strand) for was performed. The obtained data revealed different banding patterns depending on the viral type and the region of infection. Regarding PLRV, a 58 up regulated and 19 down regulated genes were detected, while, 31 up regulated and 14 down regulated genes were observed in case of PVY. Based on the nucleotide sequencing, variable phylogenetic relationships were reported for the three sequenced genes coding for: Induced stolen tip protein, Disease resistance RPP-like protein and non-specific lipid-transfer protein. In a complementary approach, using the quantitative Real-time PCR, the expressions of PRs genes understudy were estimated in the infected leaves by PLRV and PVY of three potato cultivars (Spunta, Diamont and Cara). The infection with both viruses inhibited the expressions of the five PRs genes. On the contrary, infected leaves by PLRV or PVY elevated the expression of some defense genes. This interaction also may be enhanced and/or inhibited the expression of some genes responsible for the plant defense mechanisms.Keywords: PLRV, PVY, PR genes, DD-PCR, qRT-PCR, sequencing
Procedia PDF Downloads 3403499 Anatomical Features of Internal Pudendal Artery
Authors: Adel Yasky, Waseem Al-Talalwah, Shorok Al Dorazi, Roger Soames
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The internal pudendal artery is a standard branch of the anterior division of the internal iliac artery. The current study includes 41 cadavers to investigate the origin and branches of the internal pudendal artery and its clinical significances. The internal pudendal artery arose directly from the anterior division of the internal iliac artery in 48.3% while it arose indirectly in 48.5%. However, the internal pudendal artery arose from the posterior division of internal iliac artery in 1.6%. Moreover, it arose internal iliac artery bifurcation site in 1.6%. Further, the internal pudendal artery supplied the urinary bladder in 17.1%. Also, the internal pudendal artery supplied the rectum 33.5% respectively. It gave uterine and vaginal arteries in 9.4% and 7.8% respectively. Finally, it supplied the sciatic nerve via giving lateral sacral branch in 1.6%. Internists, surgeons and radiologists have to be aware of the variability to decrease iatrogenic injury. Therefore, unnecessary proximal ligation should be avoided at the site of indirect origin of the internal pudendal artery to prevent sciatic neuropathy. Further, intrapelvic bleeding as result of laceration of internal pudendal branches during hysterectomy, prostatectomy or proctectomy should be expected. Therefore, this study increases the awareness of surgeons leading to minimize iatrogenic faults,Keywords: internal pudendal artery, inferior gluteal artery, superior gluteal artery, internal iliac artery, impotence, decreased libido
Procedia PDF Downloads 3563498 Soil Bioremediation Monitoring Systems Powered by Microbial Fuel Cells
Authors: András Fülöp, Lejla Heilmann, Zsolt Szabó, Ákos Koós
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Microbial fuel cells (MFCs) present a sustainable biotechnological solution to future energy demands. The aim of this study was to construct soil based, single cell, membrane-less MFC systems, operated without treatment to continuously power on-site monitoring and control systems during the soil bioremediation processes. Our Pseudomonas aeruginosa 541 isolate is an ideal choice for MFCs, because it is able to produce pyocyanin which behaves as electron-shuttle molecule, furthermore, it also has a significant antimicrobial effect. We tested several materials and structural configurations to obtain long term high power output. Comparing different configurations, a proton exchange membrane-less, 0.6 m long with 0.05 m diameter MFC tubes offered the best long-term performances. The long-term electricity production were tested from starch, yeast extract (YE), carboxymethyl cellulose (CMC) with humic acid (HA) as a mediator. In all cases, 3 kΩ external load have been used. The two best-operated systems were the Pseudomonas aeruginosa 541 containing MFCs with 1 % carboxymethyl cellulose and the MFCs with 1% yeast extract in the anode area and 35% hydrogel in the cathode chamber. The first had 3.3 ± 0.033 mW/m2 and the second had 4.1 ± 0.065 mW/m2 power density values. These systems have operated for 230 days without any treatment. The addition of 0.2 % HA and 1 % YE referred to the volume of the anode area resulted in 1.4 ± 0.035 mW/m2 power densities. The mixture of 1% starch with 0.2 % HA gave 1.82 ± 0.031 mW/m2. Using CMC as retard carbon source takes effect in the long-term bacterial survivor, thus enable the expression of the long term power output. The application of hydrogels in the cathode chamber significantly increased the performance of the MFC units due to their good water retention capacity.Keywords: microbial fuel cell, bioremediation, Pseudomonas aeruginosa, biotechnological solution
Procedia PDF Downloads 2933497 Spatial Assessment of Soil Contamination from Informal E-Waste Recycling Site in Agbogbloshie, Ghana
Authors: Kyere Vincent Nartey, Klaus Greve, Atiemo Sampson
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E-waste is discarded electrical electronic equipment inclusive of all components, sub-assemblies and consumables which are part of the product at the time of discarding and known to contain both hazardous and valuable fractions. E-waste is recycled within the proposed ecological restoration of the Agbogbloshie enclave using crude and rudimental recycling procedures such as open burning and manual dismantling which result in pollution and contamination of soil, water and air. Using GIS, this study was conducted to examine the spatial distribution and extent of soil contamination by heavy metals from the e-waste recycling site in Agbogbloshie. From the month of August to November 2013, 146 soil samples were collected in addition to their coordinates using GPS. Elemental analysis performed on the collected soil samples using X-Ray fluorescence revealed over 30 elements including, Ni, Cr, Zn, Cu, Pb and Mn. Using geostatistical techniques in ArcGIS 10.1 spatial assessment and distribution maps were generated. Mathematical models or equations were used to estimate the degree of contamination and pollution index. Results from soil analysis from the Agbogbloshie enclave showed that levels of measured or observed elements were significantly higher than the Canadian EPA and Dutch environmental standards.Keywords: e-waste, geostatistics, soil contamination, spatial distribution
Procedia PDF Downloads 5153496 Fasted and Postprandial Response of Serum Physiological Response, Hepatic Antioxidant Abilities and Hsp70 Expression in M. amblycephala Fed Different Dietary Carbohydrate
Authors: Chuanpeng Zhou
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The effect of dietary carbohydrate (CHO) level on serum physiological response, hepatic antioxidant abilities and heat shock protein 70 (HSP70) expression of Wuchang bream (Megalobrama amblycephala) was studied. Two isonitrogenous (28.56% crude protein) and isolipidic (5.28% crude lipid) diets were formulated to contain 30% or 53% wheat starch. Diets were fed for 90 days to fish in triplicate tanks (28 fish per tank). At the end of feeding trial, significantly higher serum triglyceride level, insulin level, cortisol level, malondialdehyde (MDA) content were observed in fish fed the 53% CHO diet, while significantly lower serum total protein content, alkaline phosphatase (AKP) activity, superoxide dismutase (SOD) activity and total antioxidative capacity (T-AOC) were found in fish fed the 53% CHO diet compared with those fed the 30% diet. The relative level of hepatic heat shock protein 70 mRNA was significantly higher in the 53% CHO group than that in the 30% CHO at 6, 12, and 48 h after feeding. The results of this study indicated that ingestion of 53% dietary CHO impacted the nonspecific immune ability and caused metabolic stress of Megalobrama amblycephala.Keywords: Megalobrama amblycephala, carbohydrate, fasted and postprandial response, immunity, Hsp70
Procedia PDF Downloads 4593495 Functional Cell Surface Display Using Ice Nucleation Protein from Erwina ananas on Escherischia coli
Authors: Mei Yuin Joanne Wee, Rosli Md. Illias
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Cell surface display is the expression of a protein with an anchoring motif on the surface of the cell. This approach offers advantages when used in bioconversion in terms of easier purification steps and more efficient enzymatic reaction. A surface display system using ice nucleation protein (InaA) from Erwina ananas as an anchoring motif has been constructed to display xylanase (xyl) on the surface of Escherischia coli. The InaA was truncated so that it is made up of the N- and C-terminal domain (INPANC-xyl) and it has successfully directed xylanase to the surface of the cell. A study was also done on xylanase fused to two other ice nucleation proteins, InaK (INPKNC-xyl) and InaZ (INPZNC-xyl) from Pseudomonas syringae KCTC 1832 and Pseudomonas syringae S203 respectively. Surface localization of the fusion protein was verified using SDS-PAGE and Western blot on the cell fractions and all anchoring motifs were successfully displayed on the outer membrane of E. coli. Upon comparison, whole-cell activity of INPANC-xyl was more than six and five times higher than INPKNC-xyl and INPZNC-xyl respectively. Furthermore, the expression of INPANC-xyl on the surface of E. coli did not inhibit the growth of the cell. This is the first report of surface display system using ice nucleation protein, InaA from E. ananas. From this study, this anchoring motif offers an attractive alternative to the current surface display systems.Keywords: cell surface display, Escherischia coli, ice nucleation protein, xylanase
Procedia PDF Downloads 3903494 Surgical Site Infections Post Ventriculoperitoneal (VP) Shunting: A Matched Healthcare Cost and Length of Stay Study
Authors: Issa M. Hweidi, Saba W. Al-Ibraheem
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This study aimed to assess the increased hospital length of stay and healthcare costs associated with SSIs among ventriculoperitoneal shunting surgery patients in Jordan. This study adopted a retrospective and nested 1:1 matched case-control design. A non-probability convenient sample of 48 VP shunt patients was recruited for the purpose of the study. The targeted groups of the study basically used to cross-match the variables investigated to minimize the risk of confounding. Information was extracted from the text of patients' electronic health records. As compared to the non-SSI group, the SSI group had an extra mean healthcare cost of $13,696.53 (p=0.001) and longer hospital length of stay (22.64 mean additional days). Furthermore, Acinetobacter baumannii and Klebsiella pneumonia were identified as being the most predominant causative agents of SSIs. The results of this study may provide baseline data for national and regional benchmarking to evaluate the quality of care provided to likewise patients. Adherence to infection control strategies and protocols considering new surveillance methods of SSIs is encouraged.Keywords: ventriculoperitoneal shunt, health care cost, length of stay, neurosurgery, surgical site infections
Procedia PDF Downloads 773493 Developing Customizable Scaffolds With Antimicrobial Properties for Vascular Tissue Regeneration Using Low Temperature Plasma
Authors: Komal Vig, Syamala Soumyakrishnan, Yadav Baral
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Bypass surgery, using the autologous vein has been one of the most effective treatments for cardiovascular diseases (CVD). More recently tissue engineering including engineered vascular grafts to synthesize blood vessels is gaining usage. Dacron and ePTFE has been employed for vascular grafts, however, these does not work well for small diameter grafts (<6 mm) due to intimal hyperplasia and thrombosis. In the present study PTFE was treated with LTP to improve the endothelialization of intimal surface of graft. Scaffolds were also modified with polyvinylpyrrolidone coated silver nanoparticles (Ag-PVP) and the antimicrobial peptides, p753 and p359. Human umbilical vein endothelial cells (HUVEC) were plated on the developed scaffolds and cell proliferation was determined by the MTT assay. Cells attachment on scaffolds was visualized by microscopy. mRNA expressions levels of different cell markers were investigated using quantitative real-time PCR (qPCR). X ray photoelectron spectroscopic confirmed the introduction of oxygenated functionalities from LTP air plasma. Microscopic and MTT assays indicated increase in cell viability in LTP treated scaffolds. Gene expression studies shows enhanced expression of cell adhesion marker Integrin- α 5 gene after LTP treatment. The KB test displayed a zone of inhibition for Ag-PVP, p753 and p359 of 19mm, 14mm, and 12mm respectively. To determine toxicity of antimicrobial agents to cells, MTT Assay was performed using HEK293 cells. MTT Assay exhibited that Ag-PVP and the peptides were non-toxic to cells at 100μg/mL and 50μg/mL, respectively. Live/dead analysis and plate count of treated bacteria exhibited bacterial inhibition on develop scaffold compared to non-treated scaffold. SEM was performed to analyze the structural changes of bacteria after treatment with antimicrobial agents. Gene expression studies were conducted on RNA from bacteria treated with Ag-PVP and peptides using qRT-PCR. Based on our initial results, more scaffolds alternatives will be developed and investigated for cell growth and vascularization studies.Keywords: low temperature plasma, vascular graft, HUVEC cells, antimicrobial
Procedia PDF Downloads 2453492 Using Contingency Valuation Approaches to Assess Community Benefits through the Use of Great Zimbabwe World Heritage Site as a Tourism Attraction
Authors: Nyasha Agnes Gurira, Patrick Ngulube
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Heritage as an asset can be used to achieve cultural and socio-economic development through its careful use as a tourist attraction. Cultural heritage sites, especially those listed as World Heritage sites generate a lot of revenue through their use as tourist attractions. According to article 5(a) of the World Heritage Convention, World Heritage Sites (WHS) must serve a function in the life of the communities. This is further stressed by the International Council on Monuments and Sites (ICOMOS) charter on cultural heritage tourism which recognizes the positive effects of tourism on cultural heritage and underlines that domestic and international tourism is among the foremost vehicles for cultural exchange, conservation should thus provide for responsible and well-managed opportunities for local communities. The inclusion of communities in the world heritage agenda identifies them as the owners of the heritage and partners in the management planning process. This reiterates the need to empower communities and enable them to participate in the decisions which relate to the use of their heritage divorcing from the ideals of viewing communities as beneficiaries from the heritage resource. It recognizes community ownership rights to cultural heritage an element enshrined in Zimbabwe’ national constitution. Through the use of contingency valuation approaches, by assessing the Willingness to pay for visitors at the site the research determined the tourism use value of Great Zimbabwe (WHS). It assessed the extent to which the communities at Great Zimbabwe (WHS) have been developed through the tourism use of the WHS. Findings show that the current management mechanism in place regards communities as stakeholders in the management of the WHS, their ownership and property rights are not fully recognized. They receive indirect benefits from the tourism use of the WHS. This paper calls for a shift in management approach where community ownership rights are fully recognized and more inclusive approaches are adopted to ensure that the goal of sustainable development is achieved. Pro-poor benefits of tourism are key to enhancing the livelihoods of communities and can only be achieved if their rights are recognized and respected.Keywords: communities, cultural heritage tourism, development, property ownership rights, pro-poor benefits, sustainability, world heritage site
Procedia PDF Downloads 2593491 Scheduling Building Projects: The Chronographical Modeling Concept
Authors: Adel Francis
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Most of scheduling methods and software apply the critical path logic. This logic schedule activities, apply constraints between these activities and try to optimize and level the allocated resources. The extensive use of this logic produces a complex an erroneous network hard to present, follow and update. Planning and management building projects should tackle the coordination of works and the management of limited spaces, traffic, and supplies. Activities cannot be performed without the resources available and resources cannot be used beyond the capacity of workplaces. Otherwise, workspace congestion will negatively affect the flow of works. The objective of the space planning is to link the spatial and temporal aspects, promote efficient use of the site, define optimal site occupancy rates, and ensures suitable rotation of the workforce in the different spaces. The Chronographic scheduling modelling belongs to this category and models construction operations as well as their processes, logical constraints, association and organizational models, which help to better illustrate the schedule information using multiple flexible approaches. The model defined three categories of areas (punctual, surface and linear) and four different layers (space creation, systems, closing off space, finishing, and reduction of space). The Chronographical modelling is a more complete communication method, having the ability to alternate from one visual approach to another by manipulation of graphics via a set of parameters and their associated values. Each individual approach can help to schedule a certain project type or specialty. Visual communication can also be improved through layering, sheeting, juxtaposition, alterations, and permutations, allowing for groupings, hierarchies, and classification of project information. In this way, graphic representation becomes a living, transformable image, showing valuable information in a clear and comprehensible manner, simplifying the site management while simultaneously utilizing the visual space as efficiently as possible.Keywords: building projects, chronographic modelling, CPM, critical path, precedence diagram, scheduling
Procedia PDF Downloads 1553490 DNpro: A Deep Learning Network Approach to Predicting Protein Stability Changes Induced by Single-Site Mutations
Authors: Xiao Zhou, Jianlin Cheng
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A single amino acid mutation can have a significant impact on the stability of protein structure. Thus, the prediction of protein stability change induced by single site mutations is critical and useful for studying protein function and structure. Here, we presented a deep learning network with the dropout technique for predicting protein stability changes upon single amino acid substitution. While using only protein sequence as input, the overall prediction accuracy of the method on a standard benchmark is >85%, which is higher than existing sequence-based methods and is comparable to the methods that use not only protein sequence but also tertiary structure, pH value and temperature. The results demonstrate that deep learning is a promising technique for protein stability prediction. The good performance of this sequence-based method makes it a valuable tool for predicting the impact of mutations on most proteins whose experimental structures are not available. Both the downloadable software package and the user-friendly web server (DNpro) that implement the method for predicting protein stability changes induced by amino acid mutations are freely available for the community to use.Keywords: bioinformatics, deep learning, protein stability prediction, biological data mining
Procedia PDF Downloads 4713489 Analysis of Differentially Expressed Genes in Spontaneously Occurring Canine Melanoma
Authors: Simona Perga, Chiara Beltramo, Floriana Fruscione, Isabella Martini, Federica Cavallo, Federica Riccardo, Paolo Buracco, Selina Iussich, Elisabetta Razzuoli, Katia Varello, Lorella Maniscalco, Elena Bozzetta, Angelo Ferrari, Paola Modesto
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Introduction: Human and canine melanoma have common clinical, histologic characteristics making dogs a good model for comparative oncology. The identification of specific genes and a better understanding of the genetic landscape, signaling pathways, and tumor–microenvironmental interactions involved in the cancer onset and progression is essential for the development of therapeutic strategies against this tumor in both species. In the present study, the differential expression of genes in spontaneously occurring canine melanoma and in paired normal tissue was investigated by targeted RNAseq. Material and Methods: Total RNA was extracted from 17 canine malignant melanoma (CMM) samples and from five paired normal tissues stored in RNA-later. In order to capture the greater genetic variability, gene expression analysis was carried out using two panels (Qiagen): Human Immuno-Oncology (HIO) and Mouse-Immuno-Oncology (MIO) and the miSeq platform (Illumina). These kits allow the detection of the expression profile of 990 genes involved in the immune response against tumors in humans and mice. The data were analyzed through the CLCbio Genomics Workbench (Qiagen) software using the Canis lupus familiaris genome as a reference. Data analysis were carried out both comparing the biologic group (tumoral vs. healthy tissues) and comparing neoplastic tissue vs. paired healthy tissue; a Fold Change greater than two and a p-value less than 0.05 were set as the threshold to select interesting genes. Results and Discussion: Using HIO 63, down-regulated genes were detected; 13 of those were also down-regulated comparing neoplastic sample vs. paired healthy tissue. Eighteen genes were up-regulated, 14 of those were also down-regulated comparing neoplastic sample vs. paired healthy tissue. Using the MIO, 35 down regulated-genes were detected; only four of these were down-regulated, also comparing neoplastic sample vs. paired healthy tissue. Twelve genes were up-regulated in both types of analysis. Considering the two kits, the greatest variation in Fold Change was in up-regulated genes. Dogs displayed a greater genetic homology with humans than mice; moreover, the results have shown that the two kits are able to detect different genes. Most of these genes have specific cellular functions or belong to some enzymatic categories; some have already been described to be correlated to human melanoma and confirm the validity of the dog as a model for the study of molecular aspects of human melanoma.Keywords: animal model, canine melanoma, gene expression, spontaneous tumors, targeted RNAseq
Procedia PDF Downloads 2003488 BIM-based Construction Noise Management Approach With a Focus on Inner-City Construction
Authors: Nasim Babazadeh
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Growing demand for a quieter dwelling environment has turned the attention of construction companies to reducing the propagated noise of their project. In inner-city constructions, close distance between the construction site and surrounding buildings lessens the efficiency of passive noise control methods. Dwellers of the nearby areas may file complaints and lawsuits against the construction companies due to the emitted construction noise, thereby leading to the interruption of processes, compensation costs, or even suspension of the project. Therefore, construction noise should be predicted along with the project schedule. The advantage of managing the noise in the pre-construction phase is two-fold. Firstly, changes in the time plan and construction methods can be applied more flexibly. Thus, the costs related to rescheduling can be avoided. Secondly, noise-related legal problems are expected to be reduced. To implement noise mapping methods for the mentioned prediction, the required detailed information (such as the location of the noisy process, duration of the noisy work) can be exported from the 4D BIM model. The results obtained from the noise maps would be used to help the planners to define different work scenarios. The proposed approach has been applied for the foundation and earthwork of a site located in a residential area, and the obtained results are discussed.Keywords: building information modeling, construction noise management, noise mapping, 4D BIM
Procedia PDF Downloads 1863487 Identification of a Novel Maize Dehydration-Responsive Gene with a Potential Role in Improving Maize Drought Tolerance
Authors: Kyle Phillips, Ndiko Ludidi
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Global climate change has resulted in altered rainfall patterns, which has resulted in annual losses in maize crop yields due to drought. Therefore it is important to produce maize cultivars that are more drought-tolerant, which is not an easily accomplished task as plants have a plethora of physical and biochemical adaptation methods. One such mechanism is the drought-induced expression of enzymatic and non-enzymatic proteins which assist plants to resist the effects of drought on their growth and development. One of these proteins is AtRD22 which has been identified in Arabidopsis thaliana. Using an in silico approach, a maize protein with 48% sequence homology to AtRD22 has been identified. This protein appears to be localized in the extracellular matrix, similarly to AtRD22. Promoter analysis of the encoding gene reveals cis-acting elements suggestive of induction of the gene’s expression by abscisic acid (ABA). Semi-quantitative transcriptomic analysis of the putative maize RD22 has revealed an increase in transcript levels after the exposure to drought. Current work elucidates the effect of up-regulation and silencing of the maize RD22 gene on the tolerance of maize to drought. The potential role of the maize RD22 gene in maize drought tolerance can be used as a tool to improve food security.Keywords: abscisic acid, drought-responsive cis-acting elements, maize drought tolerance, RD22
Procedia PDF Downloads 4673486 TNF Modulation of Cancer Stem Cells in Renal Clear Cell Carcinoma
Authors: Rafia S. Al-lamki, Jun Wang, Simon Pacey, Jordan Pober, John R. Bradley
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Tumor necrosis factor alpha (TNF), signaling through TNFR2, may act an autocrine growth factor for renal tubular epithelial cells. Clear cell renal carcinomas (ccRCC) contain cancer stem cells (CSCs) that give rise to progeny which form the bulk of the tumor. CSCs are rarely in cell cycle and, as non-proliferating cells, resist most chemotherapeutic agents. Thus, recurrence after chemotherapy may result from the survival of CSCs. Therapeutic targeting of both CSCs and the more differentiated bulk tumor populations may provide a more effective strategy for treatment of RCC. In this study, we hypothesized that TNFR2 signaling will induce CSCs in ccRCC to enter cell cycle so that treatment with ligands that engage TNFR2 will render CSCs susceptible to chemotherapy. To test this hypothesis, we have utilized wild-type TNF (wtTNF) or specific muteins selective for TNFR1 (R1TNF) or TNFR2 (R2TNF) to treat either short-term organ cultures of ccRCC and adjacent normal kidney (NK) tissue or cultures of CD133+ cells isolated from ccRCC and adjacent NK, hereafter referred to as stem cell-like cells (SCLCs). The effect of cyclophosphamide (CP), currently an effective anticancer agent, was tested on CD133+SCLCs from ccRCC and NK before and after R2TNF treatment. Responses to TNF were assessed by flow cytometry (FACS), immunofluorescence, and quantitative real-time PCR, TUNEL, and cell viability assays. Cytotoxic effect of CP was analyzed by Annexin V and propidium iodide staining with FACS. In addition, we assessed the effect of TNF on isolated SCLCs differentiation using a three-dimensional (3D) culture system. Clinical samples of ccRCC contain a greater number SCLCs compared to NK and the number of SCSC increases with higher tumor grade. Isolated SCLCs show expression of stemness markers (oct4, Nanog, Sox2, Lin28) but not differentiation markers (cytokeratin, CD31, CD45, and EpCAM). In ccRCC organ cultures, wtTNF and R2TNF increase CD133 and TNFR2 expression and promote cell cycle entry whereas wtTNF and R1TNF increase TNFR1 expression and promote cell death of SCLCs. Similar findings are observed in SCLCs isolated from NK but the effect was greater in SCLCs isolated from ccRCC. Application of CP distinctly triggered apoptotic and necrotic cell death in SLCSs pre-treatment with R2TNF as compared to CP treatment alone, with SCLCs from ccRCC more sensitive to CP compared to SLCS from NK. Furthermore, TNF promotes differentiation of SCLCs to an epithelial phenotype in 3D cultures, confirmed by cytokeratin expression and loss of stemness markers Nanog and Sox2. The differentiated cells show positive expression of TNF and TNFR2. These findings provide evidence that selective engagement of TNFR2 drive CSCs to cell proliferation/differentiation, and targeting of cycling cells with TNFR2 agonist in combination with anti-cancer agents may be a potential therapy for RCC.Keywords: cancer stem cells, ccRCC, cell cycle, cell death, TNF, TNFR1, TNFR2, CD133
Procedia PDF Downloads 2633485 Infusion Pump Historical Development, Measurement and Parts of Infusion Pump
Authors: Samuel Asrat
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Infusion pumps have become indispensable tools in modern healthcare, allowing for precise and controlled delivery of fluids, medications, and nutrients to patients. This paper provides an overview of the historical development, measurement, and parts of infusion pumps. The historical development of infusion pumps can be traced back to the early 1960s when the first rudimentary models were introduced. These early pumps were large, cumbersome, and often unreliable. However, advancements in technology and engineering over the years have led to the development of smaller, more accurate, and user-friendly infusion pumps. Measurement of infusion pumps involves assessing various parameters such as flow rate, volume delivered, and infusion duration. Flow rate, typically measured in milliliters per hour (mL/hr), is a critical parameter that determines the rate at which fluids or medications are delivered to the patient. Accurate measurement of flow rate is essential to ensure the proper administration of therapy and prevent adverse effects. Infusion pumps consist of several key parts, including the pump mechanism, fluid reservoir, tubing, and control interface. The pump mechanism is responsible for generating the necessary pressure to push fluids through the tubing and into the patient's bloodstream. The fluid reservoir holds the medication or solution to be infused, while the tubing serves as the conduit through which the fluid travels from the reservoir to the patient. The control interface allows healthcare providers to program and adjust the infusion parameters, such as flow rate and volume. In conclusion, infusion pumps have evolved significantly since their inception, offering healthcare providers unprecedented control and precision in delivering fluids and medications to patients. Understanding the historical development, measurement, and parts of infusion pumps is essential for ensuring their safe and effective use in clinical practice. Procedia PDF Downloads 703484 The Environmental Impact Assessment of Land Use Planning (Case Study: Tannery Industry in Al-Garma District)
Authors: Husam Abdulmuttaleb Hashim
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The environmental pollution problems represent a great challenge to the world, threatening to destroy all the evolution that mankind has reached, the organizations and associations that cares about environment are trying to warn the world from the forthcoming danger resulted from excessive use of nature resources and consuming it without looking to the damage happened as a result of unfair use of it. Most of the urban centers suffers from the environmental pollution problems and health, economic, and social dangers resulted from this pollution, and while the land use planning is responsible for distributing different uses in urban centers and controlling the interactions between these uses to reach a homogeneous and perfect state for the different activities in cities, the occurrence of environmental problems in the shade of existing land use planning operation refers to the disorder or insufficiency in this operation which leads to presence of such problems, and this disorder lays in lack of sufficient importance to the environmental considerations during the land use planning operations and setting up the master plan, so the research start to study this problem and finding solutions for it, the research assumes that using accurate and scientific methods in early stages of land use planning operation will prevent occurring of environmental pollution problems in the future, the research aims to study and show the importance of the environmental impact assessment method (EIA) as an important planning tool to investigate and predict the pollution ranges of the land use that has a polluting pattern in land use planning operation. This research encompasses the concept of environmental assessment and its kinds and clarifies environmental impact assessment and its contents, the research also dealt with urban planning concept and land use planning, it also dealt with the current situation of the case study (Al-Garma district) and the land use planning in it and explain the most polluting use on the environment which is the industrial land use represented in the tannery industries and then there was a stating of current situation of this land use and explaining its contents and environmental impacts resulted from it, and then we analyzed the tests applied by the researcher for water and soil, and perform environmental evaluation through applying environmental impact assessment matrix using the direct method to reveal the pollution ranges on the ambient environment of industrial land use, and we also applied the environmental and site limits and standards by using (GIS) and (AUTOCAD) to select the site of the best alternative of the industrial region in Al-Garma district after the research approved the unsuitability of its current site location for the environmental and site limitations, the research conducted some conclusions and recommendations regard clarifying the concluded facts and to set the proper solutions.Keywords: EIA, pollution, tannery industry, land use planning
Procedia PDF Downloads 4503483 Teaching the Tacit Nuances of Japanese Onomatopoeia through an E-Learning System: An Evaluation Approach of Narrative Interpretation
Authors: Xiao-Yan Li, Takashi Hashimoto, Guanhong Li, Shuo Yang
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In Japanese, onomatopoeia is an important element in the lively expression of feelings and experiences. It is very difficult for students of Japanese to acquire onomatopoeia, especially, its nuances. In this paper, based on traditional L2 learning theories, we propose a new method to improve the efficiency of teaching the nuances – both explicit and tacit - to non-native speakers of Japanese. The method for teaching the tacit nuances of onomatopoeia consists of three elements. First is to teach the formal rules representing the explicit nuances of onomatopoeic words. Second is to have the students create new onomatopoeic words by utilizing those formal rules. The last element is to provide feedback by evaluating the onomatopoeias created. Our previous study used five-grade relative estimation. However students were confused about the five-grade system, because they could not understand the evaluation criteria only based on a figure. In this new system, then, we built an evaluation database through native speakers’ narrative interpretation. We asked Japanese native speakers to describe their awareness of the nuances of onomatopoeia in writing. Then they voted on site and defined priorities for showing to learners on the system. To verify the effectiveness of the proposed method and the learning system, we conducted a preliminary experiment involving two groups of subjects. While Group A got feedback about the appropriateness of their onomatopoeic constructions from the native speakers’ narrative interpretation, Group B got feedback just in the form of the five-grade relative estimation. A questionnaire survey administered to all of the learners clarified our learning system availability and also identified areas that should be improved. Repetitive learning of word-formation rules, creating new onomatopoeias and gaining new awareness from narrative interpretation is the total process used to teach the explicit and tacit nuances of onomatopoeia.Keywords: onomatopoeia, tacit nuance, narrative interpretation, e-learning system, second language teaching
Procedia PDF Downloads 3973482 Citrullinated Myelin Basic Protein Mediated Inflammation in Astrocytes
Authors: Lali Shanshiashvili, Marika Chikviladze, Nino Mamulashvili, Maia Sepashvili, Nana Narmania, David Mikeladze
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Purpose: During demyelinating inflammatory diseases and after the damage of the myelin sheet, myelin-derived proteins, including myelin basic protein (MBP), are secreted into the extracellular space. MBP shows extensive post-translational modifications, including the deimination of arginine residues. Deiminated MBP is structurally less ordered, susceptible to proteolytic attack, and more immunogenic than the unmodified one. It is hypothesized that MBP could change the inflammatory response in astrocytes. Methods: MBP was isolated and purified from bovine brain white matter. Primary astrocyte cultures were prepared from whole brains of 2-day-old Wistar rats. For evaluation of glutamate uptake/release in astrocytes following treatment of cells with MBP charge isomers, Glutamate Assay Kit was used. The expression of EAAT-2 (excitatory amino acid transporters), peroxisome proliferator-activated receptor gamma (PPAR- γ), inhibitor of nuclear factor kappa B (IkB), and high mobility group protein B1 (HMGB1) in astrocytes were assayed by Western Blot analysis. Results: This study investigated the action of deiminated isomer (C8) on the cultured primary astrocytes and compared its effects with the effects of unmodified C1 isomers. The study found that C8 and C1 MBP differently act on the uptake and release of glutamate in astrocytes: nonmodified C1 MBP increases the uptake of glutamate and does not change the release, whereas C8 decreases the release of glutamate but does not alter the uptake. Nevertheless, both isomers increased the expression of PPAR-γ and EAAT2 in the same intensity. However, immunostaining and Western Blots of cell lysates showed a decrease of IkB and increased expression of HMGB1 after the treatment of astrocytes by C8. Moreover, in the presence of C8, astrocytes release more nitric oxide than unmodified C1 isomers. Conclusion: These data suggest that the deiminated isomer of MBP evokes an inflammatory response and enhances the ability of astrocytes to release proinflammatory mediators through activation of NF-kB after the breakdown of myelin sheets. Acknowledgment: This research was supported by the SRNSF Georgia RF17_534 grant.Keywords: myelin basic protein, glutamate, deimination, astrocytes, inflammation
Procedia PDF Downloads 2063481 The Multiple Sclerosis and the Role of Human Herpesvirus 6 in Its Progression
Authors: Sina Mahdavi
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Background and Objective: Multiple sclerosis (MS) is an inflammatory autoimmune disease of the CNS that affects the myelination process in the central nervous system (CNS). Complex interactions of various "environmental or infectious" factors may act as triggers in autoimmunity and disease progression. The association between viral infections, especially Human Herpesvirus 6 (HHV-6), and MS is one potential cause that is not well understood. In this study, we aim to summarize the available data on HHV-6 infection in MS disease progression. Materials and Methods: For this study, the keywords "Multiple sclerosis", " Human Herpesvirus 6 ", and "central nervous system" in the databases PubMed and Google Scholar between 2017 and 2022 were searched, and 12 articles were chosen, studied, and analyzed. Results: HHV 6 tends towards TCD 4+ lymphocytes and enters the CNS due to the weakening of the blood-brain barrier due to inflammatory damage. Following the observation that the HHV-6 U24 protein has a seven amino acid sequence with myelin basic protein, which is one of the main components of the myelin sheath, it could cause a molecular mimicry mechanism followed by cross-reactivity. Reactivation of HHV-6 in the CNS can cause the release of proinflammatory cytokines, including TNF-α, leading to immune-mediated demyelination in patients with MS. Conclusion: There is a high expression of endogenous retroviruses during the course of MS, which indicates the relationship between HHV-6 and MS, and that this virus can play a role in the development of MS by creating an inflammatory state. Therefore, measures to modulate the expression of HHV-6 may be effective in reducing inflammatory processes in demyelinated areas of MS patients.Keywords: multiple sclerosis, human herpesvirus 6, central nervous system, autoimmunity
Procedia PDF Downloads 1123480 Geotechnical Investigation of Soil Foundation for Ramps of Dawar El-Tawheed Bridge in Jizan City, Kingdom of Saudi Arabia
Authors: Ali H. Mahfouz, Hossam E. M. Sallam, Abdulwali Wazir, Hamod H. Kharezi
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The soil profile at site of the bridge project includes soft fine grained soil layer located between 5.0 m to 11.0 m in depth, it has high water content, low SPT no., and low bearing capacity. The clay layer induces high settlement due to surcharge application of earth embankment at ramp T1, ramp T2, and ramp T3 especially at heights from 9m right 3m. Calculated settlement for embankment heights less than 3m may be accepted regarding Saudi Code for soil and foundation. The soil and groundwater at the project site comprise high contents of sulfates and chlorides of high aggressively on concrete and steel bars, respectively. Regarding results of the study, it has been recommended to use stone column piles or new technology named PCC piles as soil improvement to improve the bearing capacity of the weak layer. The new technology is cast in-situ thin wall concrete pipe piles (PCC piles), it has economically advantageous and high workability. The technology can save time of implementation and cost of application is almost 30% of other types of piles.Keywords: soft foundation soil, bearing capacity, bridge ramps, soil improvement, geogrid, PCC piles
Procedia PDF Downloads 3993479 Absolute Quantification of the Bexsero Vaccine Component Factor H Binding Protein (fHbp) by Selected Reaction Monitoring: The Contribution of Mass Spectrometry in Vaccinology
Authors: Massimiliano Biagini, Marco Spinsanti, Gabriella De Angelis, Sara Tomei, Ilaria Ferlenghi, Maria Scarselli, Alessia Biolchi, Alessandro Muzzi, Brunella Brunelli, Silvana Savino, Marzia M. Giuliani, Isabel Delany, Paolo Costantino, Rino Rappuoli, Vega Masignani, Nathalie Norais
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The gram-negative bacterium Neisseria meningitidis serogroup B (MenB) is an exclusively human pathogen representing the major cause of meningitides and severe sepsis in infants and children but also in young adults. This pathogen is usually present in the 30% of healthy population that act as a reservoir, spreading it through saliva and respiratory fluids during coughing, sneezing, kissing. Among surface-exposed protein components of this diplococcus, factor H binding protein is a lipoprotein proved to be a protective antigen used as a component of the recently licensed Bexsero vaccine. fHbp is a highly variable meningococcal protein: to reflect its remarkable sequence variability, it has been classified in three variants (or two subfamilies), and with poor cross-protection among the different variants. Furthermore, the level of fHbp expression varies significantly among strains, and this has also been considered an important factor for predicting MenB strain susceptibility to anti-fHbp antisera. Different methods have been used to assess fHbp expression on meningococcal strains, however, all these methods use anti-fHbp antibodies, and for this reason, the results are affected by the different affinity that antibodies can have to different antigenic variants. To overcome the limitations of an antibody-based quantification, we developed a quantitative Mass Spectrometry (MS) approach. Selected Reaction Monitoring (SRM) recently emerged as a powerful MS tool for detecting and quantifying proteins in complex mixtures. SRM is based on the targeted detection of ProteoTypicPeptides (PTPs), which are unique signatures of a protein that can be easily detected and quantified by MS. This approach, proven to be highly sensitive, quantitatively accurate and highly reproducible, was used to quantify the absolute amount of fHbp antigen in total extracts derived from 105 clinical isolates, evenly distributed among the three main variant groups and selected to be representative of the fHbp circulating subvariants around the world. We extended the study at the genetic level investigating the correlation between the differential level of expression and polymorphisms present within the genes and their promoter sequences. The implications of fHbp expression on the susceptibility of the strain to killing by anti-fHbp antisera are also presented. To date this is the first comprehensive fHbp expression profiling in a large panel of Neisseria meningitidis clinical isolates driven by an antibody-independent MS-based methodology, opening the door to new applications in vaccine coverage prediction and reinforcing the molecular understanding of released vaccines.Keywords: quantitative mass spectrometry, Neisseria meningitidis, vaccines, bexsero, molecular epidemiology
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