Search results for: knock out mutant

Authors: Ravi Raghav Sonani, Niraj Kumar Singh, Jitendra Kumar, Datta Madamwar

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Phycobiliproteins (PBPs) are light harvesting proteins showing very strong absorbance and fluorescence in the visible range of the solar spectrum. Phycoerythrin (PE) and phycocyanin (PC) are majorly found PBPs in the cyanobacteria and red algae. In the present study, we have investigated the reactive oxygen species (ROS)-averting capacity of purified PE and PC of cyanobacterial origin. Furthermore, the possibility - whether the ROS-averting potential of PBPs can be explored in the therapeutics of oxidative stress associated physiological anomalies including aging and neurodegenerative diseases. The nematode Caenorhabditis elegans has been used as model organism in this study. PE and PC treatment moderated normal aging and associated physiological functionalities like pharyngeal pumping and locomotion of C. elegans. Moreover, PE-treatment enhanced the stress (oxidative and heat) tolerance upon PE and PC treatment. Specifically, PE treatment was also noted to moderate the progression of Alzheimer’s disease in transgenic C. elegans CL4176. However, PC-treatment curtailed the polyQ aggregation mediated proteotoxicity in C. elegans AM141 (Huntington disease model) under stressed (paraquat stress) as well as normal conditions. The effectiveness of PE and PC in expanding the lifespan of mutant C. elegans knockout for some up- (daf 16) and down- (daf-2 and age-1) stream regulators of insulin/IGF-1 signalling (IIS) shows the independency of their effects from DAF-2–AGE-1–DAF-16 signalling pathway. In conclusion, the present report demonstrates the anti-aging and neuro-protective potential of cyanobacterial PE and PC.

Keywords: phycobiliproteins, aging, alzheimer, huntington, C. elegans

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Authors: Çiğdem Avcı Karataş

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Mitigation of structural damage caused by earthquake and reduction of fatality is one of the main concerns of engineers in seismic prone zones of the world. To achieve this aim many technologies have been developed in the last decades and applied in construction and retrofit of structures. On the one hand Turkey is well-known a country of high level of seismicity; on the other hand steel-composite structures appear competitive today in this country by comparison with other types of structures, for example only-steel or concrete structures. Composite construction is the dominant form of construction for the multi-storey building sector. The reason why composite construction is often so good can be expressed in one simple way - concrete is good in compression and steel is good in tension. By joining the two materials together structurally these strengths can be exploited to result in a highly efficient design. The reduced self-weight of composite elements has a knock-on effect by reducing the forces in those elements supporting them, including the foundations. The floor depth reductions that can be achieved using composite construction can also provide significant benefits in terms of the costs of services and the building envelope. The scope of this paper covers analysis, materials take-off, cost analysis and economic comparisons of a multi-storey building with composite and steel frames. The aim of this work is to show that designing load carrying systems as composite is more economical than designing as steel. Design of the nine stories building which is under consideration is done according to the regulation of the 2007, Turkish Earthquake Code and by using static and dynamic analysis methods. For the analyses of the steel and composite systems, plastic analysis methods have been used and whereas steel system analyses have been checked in compliance with EC3 and composite system analyses have been checked in compliance with EC4. At the end of the comparisons, it is revealed that composite load carrying systems analysis is more economical than the steel load carrying systems analysis considering the materials to be used in the load carrying system and the workmanship to be spent for this job.

Keywords: composite analysis, earthquake, steel, multi-storey building

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Authors: Yan-Shiang Chiou, Yi-Huang Hsueh

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Bacillus cereus is a foodborne pathogen that causes two types of foodborne illness, the emetic and diarrheal syndromes. B. cereus consistently ranks among the top three among bacterial foodborne outbreaks in the ten years of 2001 to 2010 in Taiwan. Foodborne outbreak caused by B. cereus has been increased, and recently it ranks second foodborne pathogen after Vibrio parahaemolyticus. This pathogen is difficult to control due to its ubiquitousness in the environment, the psychrotrophic nature of many strains, and the heat resistance of their spores. Because complete elimination of biofilms is difficult, a better understanding of the molecular mechanisms of biofilm formation by B. cereus will help to develop better strategies to control this pathogen. Surface translocation can be an important factor in biofilm formation. In B. cereus, NprR is a quorum sensor, and its apo NprR is a dimer and changes to a tetramer in the presence of NprX. The small peptide NprX may induce conformational change allowing the apo dimer to switch to an active tetramer specifically recognizing target DNA sequences. Our result showed that mutation of nprRX causes surface spreading deficiency. Mutation of flagella, pili and surfactant genes (flgAB, bcpAB, krsABC), did not abolish spreading motility. Under nprRX mutant, mutation of spo0A restored the spreading deficiency. This suggests that spreading motility is not related surfactant, pili and flagella but other unknown mechanism and Spo0A, a sporulation initiation protein, inhibits spreading motility.

Keywords: Bacillus cereus, nprRX, spo0A, spreading motility

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Authors: Muhasin Koyiloth, Sathyanarayana N. Gummadi

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Biological membranes are ordered association of lipids, proteins, and carbohydrates. Lipids except sterols possess asymmetric distribution across the bilayer. Eukaryotic membranes possess a group of lipid translocators called scramblases that disrupt phospholipid asymmetry. Their action is implicated in cell activation during wound healing and phagocytic clearance of apoptotic cells. Cholesterol is one of the major membrane lipids distributed evenly on both the leaflet and can directly influence the membrane fluidity through the ordering effect. The fluidity has an impact on the activity of several membrane proteins. The palmitoylated phospholipid scramblases localized to the lipid raft which is characterized by a higher number of sterols. Here we propose that cholesterol can interact with scramblases through putative CRAC motif and can modulate their activity. To prove this, we reconstituted phospholipid scramblase 1 of C. elegans (SCRM-1) in proteoliposomes containing different amounts of cholesterol (Liquid ordered/Lo). We noted that the presence of cholesterol reduced the scramblase activity of wild-type SCRM-1. The interaction between SCRM-1 and cholesterol was confirmed by fluorescence spectroscopy using NBD-Chol. Also, we observed loss of such interaction when one of I273 in the CRAC motif mutated to Asp. Interestingly, the point mutant has partially retained scramblase activity in Lo vesicles. The current study elucidated the important interaction between cholesterol and SCRM-1 to fine-tune its activity in artificial membranes.

Keywords: artificial membranes, CRAC motif, plasma membrane, PL scramblase

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Authors: Sarita Puri, Tapan K. Chaudhuri

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Chaperonin GroEL is a tetradecameric Escherichia coli protein having identical subunits of 57 kDa. The elucidation of thermodynamic parameters related to stability for the native GroEL is not feasible as it undergoes irreversible unfolding because of its large size (800kDa) and multimeric nature. Nevertheless, it is important to determine the thermodynamic stability parameters for the highly stable GroEL protein as it helps in folding and holding of many substrate proteins during many cellular stresses. Properly folded monomers work as building-block for the formation of native tetradecameric GroEL. Spontaneous refolding behavior of monomeric GroEL makes it suitable for protein-denaturant interactions and thermodynamic stability based studies. The urea mediated unfolding is a three state process which means there is the formation of one intermediate state along with native and unfolded states. The heat mediated denaturation is a two-state process. The unfolding process is reversible as observed by the spontaneous refolding of denatured protein in both urea and head mediated refolding processes. Analysis of folding/unfolding data provides a measure of various thermodynamic stability parameters for the monomeric GroEL. The proposed mechanism of unfolding of monomeric GroEL is a three state process which involves formation of one stable intermediate having folded apical domain and unfolded equatorial, intermediate domains. Research in progress is to demonstrate the importance of specific residues in stability and oligomerization of GroEL protein. Several mutant versions of GroEL are under investigation to resolve the above mentioned issue.

Keywords: equilibrium unfolding, monomeric GroEl, spontaneous refolding, thermodynamic stability

Procedia PDF Downloads 251

Authors: Adel Alblihy, Reem Ali, Mashael Algethami, Ahmed Shoqafi, Michael S. Toss, Juliette Brownlie, Natalie J. Tatum, Ian Hickson, Paloma Ordonez Moran, Anna Grabowska, Jennie N. Jeyapalan, Nigel P. Mongan, Emad A. Rakha, Srinivasan Madhusudan

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Platinum resistance is a clinical challenge in ovarian cancer. Platinating agents induce DNA damage which activate Mre11 nuclease directed DNA damage signalling and response (DDR). Upregulation of DDR may promote chemotherapy resistance. Here we have comprehensively evaluated Mre11 in epithelial ovarian cancers. In clinical cohort that received platinum- based chemotherapy (n=331), Mre11 protein overexpression was associated with aggressive phenotype and poor progression free survival (PFS) (p=0.002). In the ovarian cancer genome atlas (TCGA) cohort (n=498), Mre11 gene amplification was observed in a subset of serous tumours (5%) which correlated highly with Mre11 mRNA levels (p<0.0001). Altered Mre11 levels was linked with genome wide alterations that can influence platinum sensitivity. At the transcriptomic level (n=1259), Mre11 overexpression was associated with poor PFS (p=0.003). ROC analysis showed an area under the curve (AUC) of 0.642 for response to platinum-based chemotherapy. Pre-clinically, Mre11 depletion by gene knock down or blockade by small molecule inhibitor (Mirin) reversed platinum resistance in ovarian cancer cells and in 3D spheroid models. Importantly, Mre11 inhibition was synthetically lethal in platinum sensitive XRCC1 deficient ovarian cancer cells and 3D-spheroids. Selective cytotoxicity was associated with DNA double strand break (DSB) accumulation, S-phase cell cycle arrest and increased apoptosis. We conclude that pharmaceutical development of Mre11 inhibitors is a viable clinical strategy for platinum sensitization and synthetic lethality in ovarian cancer.

Keywords: MRE11; XRCC1, ovarian cancer, platinum sensitization, synthetic lethality

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Authors: Samuel Mejorado

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C. Reinahrdtii serves as one of the most promising organisms from which to obtain biological hydrogen. However, its production catalyst, [FeFe]-hydrogenase, is largely inhibited by the presence of oxygen. In recent years, researchers have identified a Proton Gradient Regulation 5 (PGR5) deficient mutant, which shows enhanced respiration and lower accumulations of oxygen within the system. In this research, we investigated the effects of varying nutrient conditions on PGR5 mutants' ability to produce hydrogen. After growing PGR5 mutants in varying nutrient conditions under 55W fluorescent lamps at 30℃ with constant stirring at 200 rpm, a common water displacement method was utilized to obtain a definitive volumetric reading of hydrogen produced by these mutants over a period of 12 days. After the trials, statistical t-tests and ANOVAs were performed to better determine the effect which nutrient conditions have on PGR5 mutants' ability to produce hydrogen. In this, we report that conditions of sulfur deprivation most optimally enhanced hydrogen production within these mutants, with groups grown under these conditions demonstrating the highest production capacity over the entire 12-day period. Similarly, it was found that when grown under conditions of nitrogen deprivation, a favorable shift towards carbon fixation and overall lipid/starch metabolism was observed. Overall, these results demonstrate that PGR5-deficient mutants stand as a promising source of biohydrogen when grown under conditions of sulfur deprivation. To date, photochemical characteristics of [FeFe]-hydrogenase in these mutants have yet to be investigated under conditions of sulfur deprivation.

Keywords: biofuel, biohydrogen, [FeFe]-hydrogenase, algal biofuel

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Authors: Hainan Chen, Jina Qing, Xiao Zhu, Ling Gao, Ampadu O. Jackson, Min Zhang, Kai Yin

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Objective: Editing macrophage activation to dampen inflammatory diseases by promoting the repolarization of inflammatory (M1) macrophages to anti-inflammatory (M2) macrophages is highly associated with mitochondrial respiration. Recent studies have suggested that mitochondrial apolipoprotein A-1 binding protein (APOA1BP) was essential for the cellular metabolite NADHX repair to NADH, which is necessary for the mitochondrial function. The exact role of APOA1BP in the repolarization of M1 to M2, however, is uncertain. Material and method: THP-1-derived macrophages were incubated with LPS (10 ng/ml) or/and IL-4 (100 U/ml) for 24 hours. Biochemical parameters of oxidative phosphorylation and M1/M2 markers were analyzed after overexpression of APOA1BP in cells. Results: Compared with control and IL-4-exposed M2 cells, APOA1BP was downregulated in M1 macrophages. APOA1BP restored the decline in mitochondrial function to improve metabolic and phenotypic reprogramming of M1 to M2 macrophages. Blocking oxidative phosphorylation by oligomycin blunts the effects of APOA1BP on M1 to M2 repolarization. Mechanistically, LPS triggered the hydration of NADH and increased its hydrate NADHX which inhibit cellular NADH dehydrogenases, a key component of electron transport chain for oxidative phosphorylation. APOA1BP decreased the level of NADHX via converting R-NADHX to biologically useful S-NADHX. The mutant of APOA1BP aspartate188, the binding site of NADHX, fail to repair oxidative phosphorylation, thereby preventing repolarization. Conclusions: Restoring mitochondrial function by increasing mitochondrial APOA1BP might be useful to improve the reprogramming of inflammatory macrophages into anti-inflammatory cells to control inflammatory diseases.

Keywords: inflammatory diseases, macrophage repolarization, mitochondrial respiration, apolipoprotein A-1 binding protein, NADHX, NADH

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Authors: H. Nabahat, E. Amiri, F. AzaditalabDavoudabadi, N. Zaeri

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Tooth decay is one of the most common forms of oral and dental illness in the world, which causes huge costs of treatment, especially in high-risk groups such as people with oral dry mouth, prevention and control of it are very important. The use of traditional treatments such as extraction of drugs from medicinal plants is of paramount importance to Iran and the international community as well. The present study was conducted with the aim of investigating the antibacterial effect of the extract of Salvia officinalis and Mentha pulegium, which are the most commonly used drugs in the treatment of oral and teeth bacterial (Streptococcus mutant, Lactobacillus rhamnosis, and Actinomyces viscosis) in vitro method. In this experimental study, two herbs of Salvia and Mentha were prepared by maceration of hydroalcoholic extract, and the antibacterial effect was evaluated by broth macro dilution on streptococcal mutagen bacteria, lactobacillus rhamnosis, and viscose actinomycosis. The results were analyzed by the Whitney Mann test (P > 0.05). The results showed that the minimum inhibitory concentration (MIC) of the salmonella extract for Streptococcus mutan were 6.25 and 12.5 μg/ml, respectively, for lactobacillus of 1.56 and 3.12 μg/ml, respectively, and for actinomycosis viscose, The order of 12.5 and 100 μg/ml was obtained. As a result, broth macro dilution showed that both extracts of Salvia and Mentha had an inhibitory effect on all three species of bacteria. This effect for Salvia was significantly (P < 0.05) more than Mentha and was within the concentration range of both the extracts and had a bactericidal effect on all three bacteria.

Keywords: antibacterial effect, dental bacteria, herbal extracts , salvia officinalis, mentha pulegium

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Authors: Grace McCambridge, Alanna Keady, Madhur Agrawal, Dequina Nicholas Alvarado, Barbara Nikolajczyk, Leena Panneerseelan-Bharath

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Age is a non-modifiable risk factor for the inflammation that underlies pathologies such as type 2 diabetes mellitus (T2DM). Inflammation, as indicated by circulating cytokines, rises in aging, but mechanisms that promote this ‘inflammaging’ remain poorly defined. Furthermore, downstream consequences of inflammaging, including the development of an inflammatory profile that predicts comorbidities like T2DM, remain speculative. We tested the possibility that natural aging-associated changes in autophagy, a process that is compromised in both aging and T2DM, regulates inflammatory profiles in older subjects. Our data showed that circulating CD4⁺ T cells from older compared to younger subjects have (i) defects in autophagy; (ii) higher mitochondria accumulation; (iii) a failure to metabolically shift from oxidative phosphorylation to anaerobic glycolysis upon αCD3/CD28 activation; (iv) more reactive oxygen species (ROS) accumulation; and (v) a cytokine profile that recapitulates the Th17 profile that predicts T2DM. ROS scavenging in cells from older subjects restored mitochondrial mass and membrane potential (indicators of improved autophagy) and reduced Th17 cytokines to amounts made by T cells from younger subjects. Knock-down of the autophagy protein Atg3 in T cells from younger subjects increased mitochondrial accumulation and Th17 cytokines. To begin translating these findings to clinical practice, we showed that physiological concentrations of the diabetes drug metformin (100 µM) added in vitro enhanced autophagy, prevented mitochondria and ROS accumulation, increased anaerobic glycolysis, and decreased Th17 cytokines in activated CD4⁺ T cells from older subjects. Metformin therefore improves autophagy and multiple downstream pro-inflammatory mechanisms CD4⁺ T cells from older subjects. We conclude that autophagy improvement ameliorates the development of a T2DM-predictive Th17 profile in aging, and thus holds promise for delay or prevention of aging-associated metabolic decline.

Keywords: autophagy, mitochondrial turnover, ROS, glycolysis

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Authors: Charalampos N. Bompas, Eleni Poulou-Sidiropoulou, Martina Samiotaki, Alexios Vlamis-Gardikas

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Ιn all living organisms, antioxidant defenses are orchestrated by the thioredoxin (Trx) and glutaredoxin (Grx) systems. The Trx system of Escherichia coli (E. coli) is comprised of Trx1 and Trx2, both reduced by thioredoxin reductase (TrxR). The Grx system consists of four Grxs (Grx1, Grx2, Grx3, and Grx4), all reduced by glutathione (GSH) except for Grx4, which is reduced by TrxR. Under normal conditions, the GSH reductase of the Grx system keeps GSH at its reduced state. NADPH+ provides the electrons for all reductions in the Trx and Grx systems. Although the role of the E. coli Trx system is widely known, the function of the Grx system reflects the main property of Grx1, which is the reduction of ribonucleotide reductase Ia (RRIa). E. coli Grx3 (encoded by grxC) may also reduce RRIa in vitro but with slow kinetics. The molecule may account for up to 0.4% of total soluble protein and has been the subject of extensive structural studies. Its biological function, however, remains unknown. Herein, affinity chromatography with monothiol Grx3 serving as bait was used to detect the interactions of Grx3 with other proteins. Different types of interactions were identified (covalent, weak, and strong non-covalent) that suggested novel functions for Grx3. In silico approaches were employed to validate selected interactions. In addition, total protein extracts from the null mutant for grxC and the wild-type strain were compared. The overall findings suggest that Grx3 is involved in various metabolic processes, protein synthesis, and stress responses, expanding the recognized functions of Grx3 beyond the possible reduction of RRIa.

Keywords: escherichia coli, glutaredoxin 3, interactome, thiol-disulfide oxidoreductase

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Authors: Kyung-Tae Lee, Li Li Wang, Kwang-Woo Jung, Yong-Sun Bahn

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Microtubules are involved in mechanical support, cytoplasmic organization as well as in a number of cellular processes by interacting with diverse microtubule-associated proteins (MAPs), such as plus-end tracking proteins, motor proteins, and tubulin-folding cofactors. A common feature of these proteins is the presence of a cytoskeleton-associated protein-glycine-rich (CAP-Gly) domain, which is evolutionarily conserved and generally considered to bind to α-tubulin to regulate functions of microtubules. However, there has been a dearth of research on CAP-Gly proteins in fungal pathogens, including Cryptococcus neoformans, which causes fatal meningoencephalitis globally. In this study, we identified five CAP-Gly proteins encoding genes in C. neoformans. Among these, Cgp1, encoded by CNAG_06352, has a unique domain structure that has not been reported before in other eukaryotes. Supporting the role of Cpg1 in microtubule-related functions, we demonstrate that deletion or overexpression of CGP1 alters cellular susceptibility to thiabendazole, a microtubule destabilizer, and Cgp1 is co-localized with cytoplasmic microtubules. Related to the cellular functions of microtubules, Cgp1 also governs maintenance of membrane stability and genotoxic stress responses. Furthermore, we demonstrate that Cgp1 uniquely regulates sexual differentiation of C. neoformans with distinct roles in the early and late stage of mating. Our domain analysis reveals that the CAP-Gly domain plays major roles in all the functions of Cgp1. Finally, the cgp1Δ mutant is attenuated in virulence. In conclusion, this novel CAP-Gly protein, Cgp1, has pleotropic roles in regulating growth, stress responses, differentiation and pathogenicity of C. neoformans.

Keywords: human fungal pathogen, CAP-Glycine protein, microtubule, meningoencephalitis

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Authors: Aminah Muchdar

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To increase the genetic diversity of soybean in order to adapt to agroecology in Indonesia conducted ways including introduction, cross, mutation and genetic transformation. The purpose of this research is to obtain early maturity soybean mutant lines, large seed tolerant to drought with high yield potential. This study consisted of two stages: the first is sensitivity of gamma rays carried out in the Laboratory BATAN. The genetic variety used is Anjasmoro. The method seeds irradiated with gamma rays at a rate of activity with the old ci 1046.16976 irradiation 0-71 minutes. Irradiation doses of 0, 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1000gy. The results indicated all seeds irradiated with doses of 0 - 1000gy, just a dose of 200 and 300gy are able to show the percentage of germination, plant height, number of leaves, number of normal sprouts and green leaves of the best and can be continued for a second trial in order to assemble and to get mutants which is expected. The result of second stage of soybean M2 Population irradiated with diversity Gamma Irradiation performed that in the form of soybean planting, the seed planted is the first derivative of the M2 irradiated seeds. The result after the age of 30ADP has already showing growth and development of plants that vary when compared to its parent, both in terms of plant height, number of leaves, leaf shape and leaf forage level. In the generative phase, a plant that has been irradiated 200 and 300 gy seen some plants flower form packs, but not formed pods, there is also a form packs of flowers, but few pods produce soybean morphological characters such as plant height, number of branches, pods, days to flowering, harvesting, seed weight and seed number.

Keywords: gamma ray, genetic mutation, irradiation, soybean

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Authors: Gajanan M. Sonwane

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Nowadays, the entire pharmaceutical industry is facing the challenge of increasing efficiency and innovation. The major hurdles are the growing cost of research and development and a concurrent stagnating number of new chemical entities (NCEs). Hence, the challenge is to select the most druggable targets and to search the equivalent drug-like compounds, which also possess specific pharmacokinetic and toxicological properties that allow them to be developed as drugs. The present research work includes the studies of developing new anticancer heterocycles by using molecular modeling techniques. The heterocycles synthesized through such methodology are much effective as various physicochemical parameters have been already studied and the structure has been optimized for its best fit in the receptor. Hence, on the basis of the literature survey and considering the need to develop newer anticancer agents, new phenazinamine derivatives were designed by subjecting the nucleus to molecular modeling, viz., GQSAR analysis and docking studies. Simultaneously, these designed derivatives were subjected to in silico prediction of biological activity through PASS studies and then in silico toxicity risk assessment studies. In PASS studies, it was found that all the derivatives exhibited a good spectrum of biological activities confirming its anticancer potential. The toxicity risk assessment studies revealed that all the derivatives obey Lipinski’s rule. Amongst these series, compounds 4c, 5b and 6c were found to possess logP and drug-likeness values comparable with the standard Imatinib (used for anticancer activity studies) and also with the standard drug methotrexate (used for antimitotic activity studies). One of the most notable mutations is the threonine to isoleucine mutation at codon 315 (T315I), which is known to be resistant to all currently available TKI. Enzyme assay planned for confirmation of target selective activity.

Keywords: drug design, tyrosine kinases, anticancer, Phenazinamine

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Authors: Irene Alevizos, Jessica Lewis, Marina Harper, John Boyce

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Acinetobacter baumannii causes a range of severe nosocomial-acquired infections, and many strains are multi-drug resistant. A. baumannii possesses survival mechanisms allowing it to thrive in competitive polymicrobial environments, including a Type VI Secretion System (T6SS) that injects effector proteins into other bacteria to give a competitive advantage. The effects of T6SS firing are broad and depend entirely on the effector that is delivered. Effects can include toxicity against prokaryotic or eukaryotic cells and the acquisition of essential nutrients. The T6SS of some species can deliver ‘specialised effectors’ that are fused directly to T6SS components, such as PAAR proteins. PAAR proteins are predicted to form the piercing tip of the T6SS and are essential for T6SS function. Although no specialised effectors have been identified in A. baumannii, many strains encode multiple PAAR proteins. Analysis of PAAR proteins across the species identified 12 families of PAAR proteins with distinct C-terminal extensions. A. baumannii AB307-0294 encodes two PAAR proteins, one of which has a C-terminal extension. Mutation of one or both of the PAAR-encoding genes in this strain showed that expression of either PAAR protein was sufficient for T6SS function. We employed a heterologous expression approach and determined that PAAR proteins from different A. baumannii strains, as well as the closely related A. baylyi species, could complement the A. baumannii ∆paar mutant and restore T6SS function. Furthermore, we showed that PAAR fusions could be used to deliver artificially cloned protein fragments by generating Histidine- and Streptavidin- tagged PAAR specialised effectors, which restored T6SS activity. This provides evidence that the fusion of protein fragments onto PAAR proteins in A. baumannii is compatible with a functional T6SS. Successful delivery by this mechanism extends the scope of what the T6SS can deliver, including user designed proteins.

Keywords: A. baumannii, effectors, PAAR, T6SS

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Authors: Rong Li, Brian D. Wirth, Bing Liu

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Graphite is the matrix, and structural material in the high temperature gas-cooled reactor exhibits an irradiation response. It is of significant importance to analyze the defect production and evaluate the role of graphite under irradiation. A vast experimental literature exists for graphite on the dimensional change, mechanical properties, and thermal behavior. However, simulations have not been applied to the atomistic perspective. Remarkably few molecular dynamics simulations have been performed to study the irradiation response in graphite. In this paper, irradiation-induced damage cascades in graphite were investigated with molecular dynamics simulation. Statistical results of the graphite defects were obtained by sampling a wide energy range (1–30 KeV) and 10 different runs for every cascade simulation with different random number generator seeds to the velocity scaling thermostat function. The chemical bonding in carbon was described using the adaptive intermolecular reactive empirical bond-order potential (AIREBO) potential coupled with the standard Ziegler–Biersack–Littmack (ZBL) potential to describe close-range pair interactions. This study focused on analyzing the number of defects, the final cascade morphology and the distribution of defect clusters in space, the length-scale cascade properties such as the cascade length and the range of primary knock-on atom (PKA), and graphite mechanical properties’ variation. It can be concluded that the number of surviving Frenkel pairs increased remarkably with the increasing initial PKA energy but did not exhibit a thermal spike at slightly lower energies in this paper. The PKA range and cascade length approximately linearly with energy which indicated that increasing the PKA initial energy will come at expensive computation cost such as 30KeV in this study. The cascade morphology and the distribution of defect clusters in space mainly related to the PKA energy meanwhile the temperature effect was relatively negligible. The simulations are in agreement with known experimental results and the Kinchin-Pease model, which can help to understand the graphite damage cascades and lifetime span under irradiation and provide a direction to the designs of these kinds of structural materials in the future reactors.

Keywords: graphite damage cascade, molecular dynamics, cascade morphology, cascade distribution

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Authors: Josephine Hai, Jie Jiang, Karen M. Lyons

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Of the musculoskeletal tissues, tendon is least understood in terms of biological development. The current study examines Cysteine-rich angiogenic inducer 61, or CCN1, a member of the CCN family of secreted matricellular proteins that regulate cell behavior via intercellular signaling. Though CCN1 is notable in limiting fibrosis by inducing senescence in fibroblasts, little is known about its role in normal fibrous tissue, where it may be essential to the development of ECM-rich structures like tendons. We found that CCN1 knockout mice (using limb-specific Prx1-Cre) exhibited clubfoot and waddling gaits, a unique phenotype not described in any other mutant to date. Histological analysis showed that the Achilles and patellar tendons, where we previously found high CCN1 expression in adult reporter mice, were thicker and denser in the Prx1-Cre knockouts than in their wildtype littermates. We then hypothesized that CCN1 is required directly in tendon progenitor cells for normal tendon development and generated tendon-specific CCN1 knockout mice using Scx-Cre. We observed similar Achilles/patellar tendon morphology among the Scx-Cre and Prx1-Cre mutants, indicating that the phenotype is a direct result of CCN1’s loss in tendon. To further study phenotype onset and progression, we will histologically characterize these tendons across different developmental time-points. We will also perform RNA-seq and qPCR to analyze tenocyte gene expression and expect fibrotic marker upregulation in the Scx-Cre mutants if CCN1 is required to maintain a normal tendon phenotype. Thus, our study aims to elucidate the molecular mechanisms underlying tendon formation and maintenance. Understanding tendons at the most basic level invites novel approaches to tendon repair.

Keywords: development, matricellular, musculoskeletal, tendon

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Authors: Kinnsley Travis, Camerron M. Crowder

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Mapk8ip3 (Mitogen-Activated Protein Kinase 8 Interacting Protein 3) is a gene that codes for the JIP3 protein, which is a part of the JIP scaffolding protein family. This protein is involved in axonal vesicle transport, elongation and regeneration. Variants in the Mapk8ip3 gene are associated with a rare-genetic condition that results in a neurodevelopmental disorder that can cause a range of phenotypes including global developmental delay and intellectual disability. Currently, there are 18 known individuals diagnosed to have sequenced confirmed Mapk8ip3 genetic disorders. This project focuses on examining the impact of a subset of missense patient variants on the Jip3 protein function by overexpressing the mRNA of these variants in a zebrafish knockout model for Jip3. Plasmids containing cDNA with individual missense variants were reverse transcribed, purified, and injected into single-cell zebrafish embryos (Wild Type, Jip3 -/+, and Jip3 -/-). At 6-days post mRNA microinjection, morphological, behavioral, and microscopic phenotypes were examined in zebrafish larvae. Morphologically, we compared the size and shape of the zebrafish during their development over a 5-day period. Total locomotive activity was assessed using the Microtracker assay and patterns of movement over time were examined using the DanioVision assay. Lastly, we used confocal microscopy to examine sensory axons for swelling and shortened length, which are phenotypes observed in the loss-of-function knockout Jip3 zebrafish model. Using these assays during embryonic development, we determined the impact of various missense variants on Jip3 protein function, compared to knockout and wild-type zebrafish embryo models. Variants in the gene Mapk8ip3 cause rare-neurodevelopmental disorders due to an essential role in axonal vesicle transport, elongation and regeneration. A subset of missense variants was examined by overexpressing the mRNA of these variants in a Jip3 knock-out zebrafish. Morphological, behavioral, and microscopic phenotypes were examined in zebrafish larvae. Using these assays, the spectrum of disorders can be phenotypically determined and the impact of variant location can be compared to knockout and wild-type zebrafish embryo models.

Keywords: rare disease, neurodevelopmental disorders, mrna overexpression, zebrafish research

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Authors: Sungsoo Na, Chanho Park

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Lung cancer is one of the most common severe diseases driving to the death of a human. Lung cancer can be divided into two cases of small-cell lung cancer (SCLC) and non-SCLC (NSCLC), and about 80% of lung cancers belong to the case of NSCLC. From several studies, the correlation between epidermal growth factor receptor (EGFR) and NSCLCs has been investigated. Therefore, EGFR inhibitor drugs such as gefitinib and erlotinib have been used as lung cancer treatments. However, the treatments result showed low response (10~20%) in clinical trials due to EGFR mutations that cause the drug resistance. Patients with resistance to EGFR inhibitor drugs usually are positive to KRAS mutation. Therefore, assessment of EGFR and KRAS mutation is essential for target therapies of NSCLC patient. In order to overcome the limitation of conventional therapies, overall EGFR and KRAS mutations have to be monitored. In this work, the only detection of EGFR will be presented. A variety of techniques has been presented for the detection of EGFR mutations. The standard detection method of EGFR mutation in ctDNA relies on real-time polymerase chain reaction (PCR). Real-time PCR method provides high sensitive detection performance. However, as the amplification step increases cost effect and complexity increase as well. Other types of technology such as BEAMing, next generation sequencing (NGS), an electrochemical sensor and silicon nanowire field-effect transistor have been presented. However, those technologies have limitations of low sensitivity, high cost and complexity of data analyzation. In this report, we propose a label-free and high-sensitive detection method of lung cancer using quartz crystal microbalance based platform. The proposed platform is able to sense lung cancer mutant DNA with a limit of detection of 1nM.

Keywords: cancer DNA, resonance frequency, quartz crystal microbalance, lung cancer

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Authors: Aswini M. S.

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Tomato (Solanum lycopersicum L.) is one of the most significant vegetable crops in terms of its economic benefits. Both fresh and processed tomatoes are consumed. Tomatoes have a limited genetic base, which makes breeding extremely challenging. Plant breeding has become much simpler and more effective with genome editing tools of CRISPR and CRISPR-associated 9 protein (CRISPR/Cas9), which address the problems with traditional breeding, chemical/physical mutagenesis, and transgenics. With the use of CRISPR/Cas9, a number of tomato traits have been functionally distinguished and edited. These traits include plant architecture as well as flower characters (leaf, flower, male sterility, and parthenocarpy), fruit ripening, quality and nutrition (lycopene, carotenoid, GABA, TSS, and shelf-life), disease resistance (late blight, TYLCV, and powdery mildew), tolerance to abiotic stress (heat, drought, and salinity) and resistance to herbicides. This study explores the potential of CRISPR/Cas9 genome editing for enhancing yield in tomato plants. The study utilized the CRISPR/Cas9 genome editing technology to functionally edit various traits in tomatoes. The de novo domestication of elite features from wild cousins to cultivated tomatoes and vice versa has been demonstrated by the introgression of CRISPR/Cas9. The CycB (Lycopene beta someri) gene-mediated Cas9 editing increased the lycopene content in tomato. Also, Cas9-mediated editing of the AGL6 (Agamous-like 6) gene resulted in parthenocarpic fruit development under heat-stress conditions. The advent of CRISPR/Cas has rendered it possible to use digital resources for single guide RNA design and multiplexing, cloning (such as Golden Gate cloning, GoldenBraid, etc.), creating robust CRISPR/Cas constructs, and implementing effective transformation protocols like the Agrobacterium and DNA free protoplast method for Cas9-gRNAs ribonucleoproteins (RNPs) complex. Additionally, homologous recombination (HR)-based gene knock-in (HKI) via geminivirus replicon and base/prime editing (Target-AID technology) remains possible. Hence, CRISPR/Cas facilitates fast and efficient breeding in the improvement of tomatoes.

Keywords: CRISPR-Cas, biotic and abiotic stress, flower and fruit traits, genome editing, polygenic trait, tomato and trait introgression

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Authors: Abanoub Mikhael, Darryl Hardie, Derek Smith, Helena Petrosova, Robert Ernst, David Goodlett

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Lipopolysaccharide (LPS) is a hallmark virulence factor of Gram-negative bacteria. It is a complex, structurally het- erogeneous mixture due to variations in number, type, and position of its simplest units: fatty acids and monosaccharides. Thus, LPS structural characterization by traditional mass spectrometry (MS) methods is challenging. Here, we describe the benefits of field asymmetric ion mobility spectrometry (FAIMS) for analysis of intact R-type lipopolysaccharide complex mixture (lipooligo- saccharide; LOS). Structural characterization was performed using Escherichia coli J5 (Rc mutant) LOS, a TLR4 agonist widely used in glycoconjugate vaccine research. FAIMS gas phase fractionation improved the (S/N) ratio and number of detected LOS species. Additionally, FAIMS allowed the separation of overlapping isobars facilitating their tandem MS characterization and un- equivocal structural assignments. In addition to FAIMS gas phase fractionation benefits, extra sorting of the structurally related LOS molecules was further accomplished using Kendrick mass defect (KMD) plots. Notably, a custom KMD base unit of [Na-H] created a highly organized KMD plot that allowed identification of interesting and novel structural differences across the different LOS ion families, i.e., ions with different acylation degrees, oligosaccharides composition, and chemical modifications. Defining the composition of a single LOS ion by tandem MS along with the organized KMD plot structural network was sufficient to deduce the composition of 181 LOS species out of 321 species present in the mixture. The combination of FAIMS and KMD plots allowed in-depth characterization of the complex LOS mixture and uncovered a wealth of novel information about its structural variations.

Keywords: lipopolysaccharide, ion mobility MS, Kendrick mass defect, Tandem mass spectrometry

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Authors: G. Barański, P. Kacejko, M. Wendeker

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The impact of the ignition advance control algorithms of the ASz-62IR-16X aircraft piston engine on a combustion process has been presented in this paper. This aircraft engine is a nine-cylinder 1000 hp engine with a special electronic control ignition system. This engine has two spark plugs per cylinder with an ignition advance angle dependent on load and the rotational speed of the crankshaft. Accordingly, in most cases, these angles are not optimal for power generated. The scope of this paper is focused on developing algorithms to control the ignition advance angle in an electronic ignition control system of an engine. For this type of engine, i.e. radial engine, an ignition advance angle should be controlled independently for each cylinder because of the design of such an engine and its crankshaft system. The ignition advance angle is controlled in an open-loop way, which means that the control signal (i.e. ignition advance angle) is determined according to the previously developed maps, i.e. recorded tables of the correlation between the ignition advance angle and engine speed and load. Load can be measured by engine crankshaft speed or intake manifold pressure. Due to a limited memory of a controller, the impact of other independent variables (such as cylinder head temperature or knock) on the ignition advance angle is given as a series of one-dimensional arrays known as corrective characteristics. The value of the ignition advance angle specified combines the value calculated from the primary characteristics and several correction factors calculated from correction characteristics. Individual cylinder control can proceed in line with certain indicators determined from pressure registered in a combustion chamber. Control is assumed to be based on the following indicators: maximum pressure, maximum pressure angle, indicated mean effective pressure. Additionally, a knocking combustion indicator was defined. Individual control can be applied to a single set of spark plugs only, which results from two fundamental ideas behind designing a control system. Independent operation of two ignition control systems – if two control systems operate simultaneously. It is assumed that the entire individual control should be performed for a front spark plug only and a rear spark plug shall be controlled with a fixed (or specific) offset relative to the front one or from a reference map. The developed algorithms will be verified by simulation and engine test sand experiments. This work has been financed by the Polish National Centre for Research and Development, INNOLOT, under Grant Agreement No. INNOLOT/I/1/NCBR/2013.

Keywords: algorithm, combustion process, radial engine, spark plug

Procedia PDF Downloads 266

Authors: Yuanyuan Zhang, Yujuan Zheng, Giuseppina Barutello, Sumako Kameishi, Kungchun Chiu, Katharina Hennig, Martial Balland, Federica Cavallo, Lars Holmgren

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Angiogenesis describes that new blood vessels migrate from pre-existing ones to form 3D lumenized structure and remodeling. During directional migration toward the gradient of pro-angiogenic factors, the endothelial cells, especially the tip cells need filopodia to sense the environment and exert the pulling force. Of particular interest are the integrin proteins, which play an essential role in focal adhesion in the connection between migrating cells and extracellular matrix (ECM). Understanding how these biomechanical complexes orchestrate intrinsic and extrinsic forces is important for our understanding of the underlying mechanisms driving angiogenesis. We have previously identified Angiomotin (Amot), a member of Amot scaffold protein family, as a promoter for endothelial cell migration in vitro and zebrafish models. Hence, we established inducible endothelial-specific Amot knock-out mice to study normal retinal angiogenesis as well as tumor angiogenesis. We found that the migration ratio of the blood vessel network to the edge was significantly decreased in Amotec- retinas at postnatal day 6 (P6). While almost all the Amot defect tip cells lost migration advantages at P7. In consistence with the dramatic morphology defect of tip cells, there was a non-autonomous defect in astrocytes, as well as the disorganized fibronectin expression pattern correspondingly in migration front. Furthermore, the growth of transplanted LLC tumor was inhibited in Amot knockout mice due to fewer vasculature involved. By using MMTV-PyMT transgenic mouse model, there was a significantly longer period before tumors arised when Amot was specifically knocked out in blood vessels. In vitro evidence showed that Amot binded to beta-actin, Integrin beta 1 (ITGB1), Fibronectin, FAK, Vinculin, major focal adhesion molecules, and ITGB1 and stress fibers were distinctly induced by Amot transfection. Via traction force microscopy, the total energy (force indicater) was found significantly decreased in Amot knockdown cells. Taken together, we propose that Amot is a novel partner of the ITGB1/Fibronectin protein complex at focal adhesion and required for exerting force transition between endothelial cell and extracellular matrix.

Keywords: angiogenesis, angiomotin, endothelial cell migration, focal adhesion, integrin beta 1

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Authors: Mehrdad Sheikhvatan, Mohammad Ali Boroumand, Mehrdad Behmanesh, Shayan Ziaee

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Contradictory results have been obtained regarding the role of integrin, beta 3 (ITGB3) gene polymorphisms in occurrence of acute myocardial infarction (MI) in patients with coronary artery disease (CAD). Hence, we aimed to assess the association between 1565C/T polymorphism of ITGB3 gene and increased risk for acute MI in patients who suffered premature CAD in Iranian population. Our prospective study included 1000 patients (492 men and 508 women aged 21 to 55 years) referred to Tehran Heart center during a period of four years from 2008 to 2011 with the final diagnosis of premature CAD and classified into two groups with history of MI (n = 461) and without of MI (n = 539). The polymorphism variants were determined by PCR-RFLP technique by entering 10% of randomized samples and then genotyping of the polymorphism was also conducted by High Resolution Melting (HRM) method. Among study samples, 640 were followed with a median follow-up time 45.74 months for determining association of long-term major adverse cardiac events (MACE) and genotypes of polymorphisms. There was no significant difference in the frequency of 1565C/T polymorphism between the MI and non-MI groups. The frequency of wild genotype was 69.2% and 72.2%, the frequency of homozygous genotype was 21.3% and 18.4%, and the frequency of mutant genotype was 9.5% and 9.5%, respectively (p=0.505). Results were also similar when adjusted for covariates in a multivariate logistic regression model. No significant difference was also found in total-MACE free survival rate between the patients with different genotypes of 1565C/T polymorphism in both MI and non-MI group. The carriage of the 1565C/T polymorphism of ITGB3 gene seems unlikely to be a significant risk factor for the development of MI in Iranian patients with premature CAD. The presence of this ITGB3 gene polymorphism may not also predict long-term cardiac events.

Keywords: coronary artery disease, myocardial infarction, gene, integrin, beta 3, polymorphism

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Authors: Leslie Nobler

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African fabrics based specifically on the Dutch Wax Print, or Ankara, popularized during Africa's colonial era, have had an enormous impact on western fashion (especially in the US and UK), in the last half-decade. The trend has had an effect on the world of visual arts as well, which circuitously, also heavily impacts fashion design. In fashion, and notably in celebrity apparel choices, this is in part due to ‘identity’ and taking pride in one's African roots; in the visual arts, artists such as Yinka Shonibare and Njideka Akunyili Crosby are making statements about identity politics, colonialism up through post-colonialism, and racism. The ‘global village’ brought on by the internet has driven this proliferation, as have improvements in the printing technology with which the Ankara print is made, combining wax-resist with roller printing. The newest patterns can now be designed authentically in western African and easily sent electronically to Europe for printing. Examples of Ankara's new reach across the Atlantic abound. They have taken several paths, which the paper will detail. Briefly, the first is its greater utilization in the fashion world, from authentic textile shops in African American neighborhoods to copied (knocked-off) low-end reproductions in discount chains. Secondly, we are seeing far more uses of these textiles/patterns in important works of fine arts from major museums, in Philadelphia to Palm Beach to the Mass MOCA (in the US), all the way to the Israel Museum in Jerusalem, and everywhere in between. And lastly, but quite significantly, we see this trend throughout social media thanks to Instagram, Pinterest and celebrity photos –even at the recent royal wedding. What shall sustain this major new design direction is that Ankara changes with and adapts to the times. Some of it is now printed in West Africa, often in the Nigeria area. And some may be designed in Europe or even at knock-off apparel studios in NY or Asia. But it stays utterly relevant because the motifs are based on objects and scenes in everyday life. In my design studio and university design classes, this idea is first and foremost, from our big spiritual eye motifs to drawings of our art supplies to the ‘politically-loaded’ chain patterns. This first-hand creativity experience becomes part of the research of this paper, along with historic and contemporary sources of inquiry, both through a literature/image search and anecdotal experience into what is behind this exciting and surprising trend.

Keywords: African wax print, Ankara, identity (politics), textile design, surface design

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Authors: Mouna Baassi, Mohamed Moussaoui, Sanchaita Rajkhowa, Hatim Soufi, Said Belaaouad

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The objective of this paper is to address the challenging task of targeting Human Immunodeficiency Virus type 1 Reverse Transcriptase (HIV-1 RT) in the treatment of AIDS. Reverse Transcriptase inhibitors (RTIs) have limitations due to the development of Reverse Transcriptase mutations that lead to treatment resistance. In this study, a combination of statistical analysis and bioinformatics tools was adopted to develop a mathematical model that relates the structure of compounds to their inhibitory activities against HIV-1 Reverse Transcriptase. Our approach was based on a series of compounds recognized for their HIV-1 RT enzymatic inhibitory activities. These compounds were designed via software, with their descriptors computed using multiple tools. The most statistically promising model was chosen, and its domain of application was ascertained. Furthermore, compounds exhibiting comparable biological activity to existing drugs were identified as potential inhibitors of HIV-1 RT. The compounds underwent evaluation based on their chemical absorption, distribution, metabolism, excretion, toxicity properties, and adherence to Lipinski's rule. Molecular docking techniques were employed to examine the interaction between the Reverse Transcriptase (Wild Type and Mutant Type) and the ligands, including a known drug available in the market. Molecular dynamics simulations were also conducted to assess the stability of the RT-ligand complexes. Our results reveal some of the new compounds as promising candidates for effectively inhibiting HIV-1 Reverse Transcriptase, matching the potency of the established drug. This necessitates further experimental validation. This study, beyond its immediate results, provides a methodological foundation for future endeavors aiming to discover and design new inhibitors targeting HIV-1 Reverse Transcriptase.

Keywords: QSAR, ADMET properties, molecular docking, molecular dynamics simulation, reverse transcriptase inhibitors, HIV type 1

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Authors: Gemma Reynolds, Deborah Bailey Rodriguez, Maria Paula Valdivieso Rueda

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In recent years, the mental health of Higher Education (HE) students has been a growing concern. This has been further exacerbated by the stresses associated with the Covid-19 pandemic, placing students at even greater risk of developing mental health issues. Support available to students in HE tends to follow an established and traditional route. The demands for counselling services have grown, not only with the increase in student numbers but with the number of students seeking support for mental health issues. One way of improving well-being and mental health in HE students is through the use of brief interventions, such as expressive writing (EW). This intervention involves encouraging individuals to write continuously for at least 15-20 minutes for three to five sessions (often on consecutive days) about their deepest thoughts and feelings to explore significant personal experiences in a meaningful way. Given the brevity, simplicity and cost-effectiveness of EW, this intervention has considerable potential as an intervention for HE populations. The current study, therefore, employed a mixed-methods design to explore the effectiveness of EW in reducing anxiety, general stress, academic stress and depression in HE students while improving well-being. HE students at MDX were randomly assigned to one of three conditions: (1) The UniExp-EW group were required to write about their emotions and thoughts about any stressors they have faced that are directly relevant to their university experience (2) The NonUniExp-EW group were required to write about their emotions and thoughts about any stressors that are NOT directly relevant to their university experience, and (3) The Control group were required to write about how they spent their weekend, with no reference to thoughts or emotions, and without thinking about university. Participants were required to carry out the EW intervention for 15minutes per day for four consecutive days. Baseline mental health and wellbeing measures were taken before the intervention via a battery of standardised questionnaires. Following completion of the intervention on day four, participants were required to complete the questionnaires a second time and again one week later. Participants were also invited to attend focus groups to discuss their experience of the intervention. This will allow an in-depth investigation into students’ perceptions of EW as an effective intervention to determine whether they would choose to use this intervention in the future. The quantitative findings will be discussed at the conference as well as a discussion of the important implications of the findings. The study is fundamental because if EW is an effective intervention for improving mental health and well-being in HE students, its brevity and simplicity means it can be easily implemented and can be freely-available to students. Improving the mental health and well-being of HE students can have knock-on implications for improving academic skills and career development.

Keywords: mental health, wellbeing, higher education students, expressive writing

Procedia PDF Downloads 49

Authors: Maria Ostroukhova, Zhanneta Zalutskaya, Elena Ermilova

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The alternative oxidase (AOX) mediates cyanide-resistant respiration, which bypasses proton-pumping complexes III and IV of the cytochrome pathway to directly transfer electrons from reduced ubiquinone to molecular oxygen. In Chlamydomonas reinhardtii, AOX is a monomeric protein that is encoded by two genes of discrete subfamilies, AOX1 and AOX2. Although AOX has been proposed to play essential roles in stress tolerance of organisms, the role of subfamily AOX2 is largely unknown. In C. reinhardtii, AOX2 was initially identified as one of constitutively low expressed genes. Like other photosynthetic organisms C. reinhardtii cells frequently experience periods of hypoxia. To examine AOX2 transcriptional regulation and role of AOX2 in hypoxia adaptation, real-time PCR analysis and artificial microRNA method were employed. Two experimental approaches have been used to induce the anoxic conditions: dark-anaerobic and light-anaerobic conditions. C. reinhardtii cells exposed to the oxygen deprivation have shown increased AOX2 mRNA levels. By contrast, AOX1 was not an anoxia-responsive gene. In C. reinhardtii, a subset of genes is regulated by transcription factor CRR1 in anaerobic conditions. Notable, the AOX2 promoter region contains the potential motif for CRR1 binding. Therefore, the role of CRR1 in the control of AOX2 transcription was tested. The CRR1-underexpressing strains, that were generated and characterized in this work, exhibited low levels of AOX2 transcripts under anoxic conditions. However, the transformants still slightly induced AOX2 gene expression in the darkness. These confirmed our suggestions that darkness is a regulatory stimulus for AOX genes in C. reinhardtii. Thus, other factors must contribute to AOX2 promoter activity under dark-anoxic conditions. Moreover, knock-down of CRR1 caused a complete reduction of AOX2 expression under light-anoxic conditions. These results indicate that (1) CRR1 is required for AOX2 expression during hypoxia, and (2) AOX2 gene is regulated by CRR1 together with yet-unknown regulatory factor(s). In addition, the AOX2-underexpressing strains were generated. The analysis of amiRNA-AOX2 strains suggested a role of this alternative oxidase in hypoxia adaptation of the alga. In conclusion, the results reported here show that C. reinhardtii AOX2 gene is stress inducible. CRR1 transcriptional factor is involved in the regulation of the AOX2 gene expression in the absence of oxygen. Moreover, AOX2 but not AOX1 functions under oxygen deprivation. This work was supported by Russian Science Foundation (research grant № 16-14-10004).

Keywords: alternative oxidase 2, artificial microRNA approach, chlamydomonas reinhardtii, hypoxia

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Authors: S. Sreeja, Radhakrishnan R. Nair, Noble Gracious, Sreeja S. Nair, M. Radhakrishna Pillai

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Background: Tacrolimus is a potent immunosuppressant clinically used for the long term treatment of antirejection of transplanted organs in liver and kidney transplant recipients though dose optimization is poorly managed. However, So far no study has been carried out on the South Indian kidney transplant patients. The objective of this study is to evaluate the potential influence of a functional polymorphism in CYP3A5*3 gene on tacrolimus physiological availability/dose ratio in South Indian renal transplant patients. Materials and Methods: Twenty five renal transplant recipients receiving tacrolimus were enrolled in this study. Their body weight, drug dosage, and therapeutic concentration of Tacrolimus were observed. All patients were on standard immunosuppressive regime of Tacrolimus-Mycophenolate mofetil along with steroids on a starting dose of Tac 0.1 mg/kg/day. CYP3A5 genotyping was performed by PCR followed with RFLP. Conformation of RFLP analysis and variation in the nucleotide sequence of CYP3A5*3 gene were determined by direct sequencing using a validated automated generic analyzer. Results: A significant association was found between tacrolimus per dose/kg/d and CYP3A5 gene (A6986G) polymorphism in the study population. The CYP3A5 *1/*1, *1/*3 and *3/*3 genotypes were detected in 5 (20 %), 5 (20 %) and 15 (60 %) of the 25 graft recipients, respectively. CYP3A5*3 genotypes were found to be a good predictor of tacrolimus Concentration/Dose ratio in kidney transplant recipients. Significantly higher L/D was observed among non-expressors 9.483 ng/mL(4.5- 14.1) as compared with the expressors 5.154 ng/mL (4.42-6.5 ) of CYP3A5. Acute rejection episodes were significantly higher for CYP3A5*1 homozygotes compared to patients with CYP3A5*1/*3 and CYP3A5*3/*3 genotypes (40 % versus 20 % and 13 %, respectively ). The dose normalized TAC concentration (ng/ml/mg/kg) was significantly lower in patients having CYP3A5*1/*3 polymorphism. Conclusion: This is the first study to extensively determine the effect of CYP3A5*3 genetic polymorphism on tacrolimus pharmacokinetics in South Indian renal transplant recipients and also shows that majority of our patients carry mutant allele A6986G in CYP3A5*3 gene. Identification of CYP3A5 polymorphism prior to transplantation could contribute to evaluate the appropriate initial dosage of tacrolimus for each patient.

Keywords: kidney transplant patients, CYP3A5 genotype, tacrolimus, RFLP

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Authors: Islam Nowisser, Noha Farag, Mohamed El Azizi

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Helicobacter pylori is a highly important human pathogen which inhabits about 50% of the population worldwide. Infection with this bacteria is very hard to treat, with high probability of recurrence. H. pylori causes severe gastric diseases, including peptic ulcer, gastritis, and gastric cancer, which has been linked to chronic inflammation. The infection has been reported to be associated with high levels of pro-inflammatory cytokines, especially IL-1β and TNF-α. The aim of the current study is to investigate the molecular mechanisms by which H. pylori activates NLRP3 inflammasome and its contribution to Il-1 β production in an innate cellular model. H. pylori PMSS1 and G27 standard strains, as well as the PMSS1 isogenic mutant strain PMSS1ΔVacA and G27ΔVacA, G27ΔCagA in addition to clinical isolates obtained from biopsy samples from the antrum and corpus mucosa of chronic gastritis patients, were used to establish infection in RAW-264.7 macrophages. The production levels of TNF-α and IL-1β was assessed using ELISA. Since expression of these cytokines is often regulated by the transcription factor complex, nuclear factor-kB (NF-kB), the activation of NF-κB in H. pylori infected cells was also evaluated by luciferase assay. Genomic DNA was extracted from bacterial cultures of H. pylori clinical isolates as well as the standard strains and their corresponding mutants, where they were evaluated for the cagA pathogenicity island and vacA expression. The correlation between these findings and expression of the cagA Pathogenicity Island and vacA in the bacteria was also investigated. The results showed IL-1β, and TNF-α production significantly increased in raw macrophages following H. pylori infection. The cagA+ and vacA+ H. pylori strains induced significant production of IL-1β compared to cagA- and vacA- strains. The activation pattern of NF-κB was correlated in the isolates to their cagA and vacA expression profiles. A similar finding could not be confirmed for TNF-α production. Our study shows the ability of H. pylori to activate NF-kB and induce significant IL-1β production as a possible mechanism for the augmented inflammatory response seen in subjects infected with cagA+ and vacA+ H. pylori strains that would lead to the progression to more severe form of the disease.

Keywords: Helicobacter pylori, IL-1β, inflammatory cytokines, nuclear factor KB, TNF-α

Procedia PDF Downloads 99