Commenced in January 2007
Frequency: Monthly
Edition: International
Paper Count: 129

Search results for: phenotype

129 The Prediction of Evolutionary Process of Coloured Vision in Mammals: A System Biology Approach

Authors: Shivani Sharma, Prashant Saxena, Inamul Hasan Madar

Abstract:

Since the time of Darwin, it has been considered that genetic change is the direct indicator of variation in phenotype. But a few studies in system biology in the past years have proposed that epigenetic developmental processes also affect the phenotype thus shifting the focus from a linear genotype-phenotype map to a non-linear G-P map. In this paper, we attempt at explaining the evolution of colour vision in mammals by taking LWS/ Long-wave sensitive gene under consideration.

Keywords: evolution, phenotypes, epigenetics, LWS gene, G-P map

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128 Exposure to Natural Outdoor Environment and Positive Health Impacts: A Synthesis of Empirical Research

Authors: Joris Zufferey, Roderick John Lawrence

Abstract:

This paper provides an overview of the state of the art about the positive health impacts of exposure to natural outdoor environments. It presents the results of a “review of reviews” in terms of empirical evidence and identifies some key questions. Finally, the authors stress the need to develop more interdisciplinary and systemic contributions. This synthesis of empirical research has been done as part of the EU- FP7 PHENOTYPE research project.

Keywords: Exposure, environment, phenotype, salutogenic effects

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127 Species Distribution and Incidence of Inducible Clindamycin Resistance in Coagulase-Negative Staphylococci Isolated from Blood Cultures of Patients with True Bacteremia in Turkey

Authors: Fatma Koksal Cakirlar, Murat Gunaydin, Nevri̇ye Gonullu, Nuri Kiraz

Abstract:

During the last few decades, the increasing prevalence of methicillin resistant-CoNS isolates has become a common problem worldwide. Macrolide-lincosamide-streptogramin B (MLSB) antibiotics are effectively used for the treatment of CoNS infections. However, resistance to MLSB antibiotics is prevalent among staphylococci. The aim of this study is to determine species distribution and the incidence of inducible clindamycin resistance in CoNS isolates caused nosocomial bacteremia in our hospital. Between January 2014 and October 2015, a total of 484 coagulase-negative CoNS isolates were isolated from blood samples of patients with true bacteremia who were hospitalized in intensive care units and in other departments of Istanbul University Cerrahpasa Medical Hospital. Blood cultures were analyzed with the BACTEC 9120 system (Becton Dickinson, USA). The identification and antimicrobial resistance of isolates were determined by Phoenix automated system (BD Diagnostic Systems, Sparks, MD). Inducible clindamycin resistance was detected using D-test. The species distribution was as follows: Staphylococcus epidermidis 211 (43%), S. hominis 154 (32%), S. haemolyticus 69 (14%), S. capitis 28 (6%), S. saprophyticus 11 (2%), S. warnerii 7 (1%), S. schleiferi 5 (1%) and S. lugdunensis 1 (0.2%). Resistance to methicillin was detected in 74.6% of CoNS isolates. Methicillin resistance was highest in S.hemoliticus isolates (89%). Resistance rates of CoNS strains to the antibacterial agents, respectively, were as follows: ampicillin 77%, gentamicin 20%, erythromycin 71%, clindamycin 22%, trimethoprim-sulfamethoxazole 45%, ciprofloxacin 52%, tetracycline 34%, rifampicin 20%, daptomycin 0.2% and linezolid 0.2%. None of the strains were resistant to vancomycin and teicoplanin. Fifteen (3%) CoNS isolates were D-test positive, inducible MLSB resistance type (iMLSB-phenotype), 94 (19%) were constitutively resistant (cMLSB -phenotype), and 237 (46,76%) isolates were found D-test negative, indicating truly clindamycin-susceptible MS phenotype (M-phenotype resistance). The incidence of iMLSB-phenotypes was higher in S. epidermidis isolates (4,7%) compared to other CoNS isolates.

Keywords: bacteremia, inducible MLSB resistance phenotype, methicillin-resistant, staphylococci

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126 Mouse Knockouts for Elucidating the Role of Cysteine-Rich Angiogenic Inducer 61 in Tendon Development and Maintenance

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

Abstract:

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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125 Phenotype Prediction of DNA Sequence Data: A Machine and Statistical Learning Approach

Authors: Darlington Mapiye, Mpho Mokoatle, James Mashiyane, Stephanie Muller, Gciniwe Dlamini

Abstract:

Great advances in high-throughput sequencing technologies have resulted in availability of huge amounts of sequencing data in public and private repositories, enabling a holistic understanding of complex biological phenomena. Sequence data are used for a wide range of applications such as gene annotations, expression studies, personalized treatment and precision medicine. However, this rapid growth in sequence data poses a great challenge which calls for novel data processing and analytic methods, as well as huge computing resources. In this work, a machine and statistical learning approach for DNA sequence classification based on k-mer representation of sequence data is proposed. The approach is tested using whole genome sequences of Mycobacterium tuberculosis (MTB) isolates to (i) reduce the size of genomic sequence data, (ii) identify an optimum size of k-mers and utilize it to build classification models, (iii) predict the phenotype from whole genome sequence data of a given bacterial isolate, and (iv) demonstrate computing challenges associated with the analysis of whole genome sequence data in producing interpretable and explainable insights. The classification models were trained on 104 whole genome sequences of MTB isoloates. Cluster analysis showed that k-mers maybe used to discriminate phenotypes and the discrimination becomes more concise as the size of k-mers increase. The best performing classification model had a k-mer size of 10 (longest k-mer) an accuracy, recall, precision, specificity, and Matthews Correlation coeffient of 72.0 %, 80.5 %, 80.5 %, 63.6 %, and 0.4 respectively. This study provides a comprehensive approach for resampling whole genome sequencing data, objectively selecting a k-mer size, and performing classification for phenotype prediction. The analysis also highlights the importance of increasing the k-mer size to produce more biological explainable results, which brings to the fore the interplay that exists amongst accuracy, computing resources and explainability of classification results. However, the analysis provides a new way to elucidate genetic information from genomic data, and identify phenotype relationships which are important especially in explaining complex biological mechanisms

Keywords: AWD-LSTM, bootstrapping, k-mers, next generation sequencing

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124 Phenotype Prediction of DNA Sequence Data: A Machine and Statistical Learning Approach

Authors: Mpho Mokoatle, Darlington Mapiye, James Mashiyane, Stephanie Muller, Gciniwe Dlamini

Abstract:

Great advances in high-throughput sequencing technologies have resulted in availability of huge amounts of sequencing data in public and private repositories, enabling a holistic understanding of complex biological phenomena. Sequence data are used for a wide range of applications such as gene annotations, expression studies, personalized treatment and precision medicine. However, this rapid growth in sequence data poses a great challenge which calls for novel data processing and analytic methods, as well as huge computing resources. In this work, a machine and statistical learning approach for DNA sequence classification based on $k$-mer representation of sequence data is proposed. The approach is tested using whole genome sequences of Mycobacterium tuberculosis (MTB) isolates to (i) reduce the size of genomic sequence data, (ii) identify an optimum size of k-mers and utilize it to build classification models, (iii) predict the phenotype from whole genome sequence data of a given bacterial isolate, and (iv) demonstrate computing challenges associated with the analysis of whole genome sequence data in producing interpretable and explainable insights. The classification models were trained on 104 whole genome sequences of MTB isoloates. Cluster analysis showed that k-mers maybe used to discriminate phenotypes and the discrimination becomes more concise as the size of k-mers increase. The best performing classification model had a k-mer size of 10 (longest k-mer) an accuracy, recall, precision, specificity, and Matthews Correlation coeffient of 72.0%, 80.5%, 80.5%, 63.6%, and 0.4 respectively. This study provides a comprehensive approach for resampling whole genome sequencing data, objectively selecting a k-mer size, and performing classification for phenotype prediction. The analysis also highlights the importance of increasing the k-mer size to produce more biological explainable results, which brings to the fore the interplay that exists amongst accuracy, computing resources and explainability of classification results. However, the analysis provides a new way to elucidate genetic information from genomic data, and identify phenotype relationships which are important especially in explaining complex biological mechanisms.

Keywords: AWD-LSTM, bootstrapping, k-mers, next generation sequencing

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123 The MSC-MCF-7 Duet Playing Tumor Vasculogenesis and Angiogenesis Onto the Chick Embryo Chorioallantoic Membrane

Authors: Serban Comsa, Amalia-Raluca Ceausu, Roxana Popescu, Simona Sarb, Anca-Maria Cimpean, Marius Raica

Abstract:

Background/Aim: The human mesenchymal stem cells (hMSC) represent a versatile cell population, able to modulate the tumor microenvironment Our aim was to recreate an open scene for the in vivo interaction between hMSC and the MCF-7 breast cancer cells (MCF-7), in order to enlighten the intimate involvement of hMSC in tumor vasculogenesis and angiogenesis. Materials and Methods: hMSC and MCF-7 were seeded onto the chick embryo chorioallantoic membrane (CAM) and incubated for seven days. Consecutively, the morphology and the immunohistochemical profile of CAM were assessed. Results: Following this complex interaction, MCF-7 acquired a more aggressive phenotype, hMSC switched to a vascular precursor phenotype whileCAM underwent a major reset to an earlier stage, with hotspots of angiogenesis, vasculogenesis and hematopoiesis. Conclusion: The hallmark of this study was the establishment of a veritable in vivo experimental model of MSC involvement in tumor vasculogenesis and angiogenesis, allowing further analysis in the field.

Keywords: angiogenesis, chorioallantoic membrane, MCF-7, mesenchymal stem cells, vasculogenesis

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122 Evaluation of Mito-Uncoupler Induced Hyper Metabolic and Aggressive Phenotype in Glioma Cells

Authors: Yogesh Rai, Saurabh Singh, Sanjay Pandey, Dhananjay K. Sah, B. G. Roy, B. S. Dwarakanath, Anant N. Bhatt

Abstract:

One of the most common signatures of highly malignant gliomas is their capacity to metabolize more glucose to lactic acid than normal brain tissues, even under normoxic conditions (Warburg effect), indicating that aerobic glycolysis is constitutively upregulated through stable genetic or epigenetic changes. However, oxidative phosphorylation (OxPhos) is also required to maintain the mitochondrial membrane potential for tumor cell survival. In the process of tumorigenesis, tumor cells during fastest growth rate exhibit both high glycolytic and high OxPhos. Therefore, metabolically reprogrammed cancer cells with combination of both aerobic glycolysis and altered OxPhos develop a robust metabolic phenotype, which confers a selective growth advantage. In our study, we grew the high glycolytic BMG-1 (glioma) cells with continuous exposure of mitochondrial uncoupler 2, 4, dinitro phenol (DNP) for 10 passages to obtain a phenotype of high glycolysis with enhanced altered OxPhos. We found that OxPhos modified BMG (OPMBMG) cells has similar growth rate and cell cycle distribution but high mitochondrial mass and functional enzymatic activity than parental cells. In in-vitro studies, OPMBMG cells showed enhanced invasion, proliferation and migration properties. Moreover, it also showed enhanced angiogenesis in matrigel plug assay. Xenografted tumors from OPMBMG cells showed reduced latent period, faster growth rate and nearly five folds reduction in the tumor take in nude mice compared to BMG-1 cells, suggesting that robust metabolic phenotype facilitates tumor formation and growth. OPMBMG cells which were found radio-resistant, showed enhanced radio-sensitization by 2-DG as compared to the parental BMG-1 cells. This study suggests that metabolic reprogramming in cancer cells enhances the potential of migration, invasion and proliferation. It also strengthens the cancer cells to escape the death processes, conferring resistance to therapeutic modalities. Our data also suggest that combining metabolic inhibitors like 2-DG with conventional therapeutic modalities can sensitize such metabolically aggressive cancer cells more than the therapies alone.

Keywords: 2-DG, BMG, DNP, OPM-BMG

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121 Human Leukocyte Antigen Class 1 Phenotype Distribution and Analysis in Persons from Central Uganda with Active Tuberculosis and Latent Mycobacterium tuberculosis Infection

Authors: Helen K. Buteme, Rebecca Axelsson-Robertson, Moses L. Joloba, Henry W. Boom, Gunilla Kallenius, Markus Maeurer

Abstract:

Background: The Ugandan population is heavily affected by infectious diseases and Human leukocyte antigen (HLA) diversity plays a crucial role in the host-pathogen interaction and affects the rates of disease acquisition and outcome. The identification of HLA class 1 alleles and determining which alleles are associated with tuberculosis (TB) outcomes would help in screening individuals in TB endemic areas for susceptibility to TB and to predict resistance or progression to TB which would inevitably lead to better clinical management of TB. Aims: To be able to determine the HLA class 1 phenotype distribution in a Ugandan TB cohort and to establish the relationship between these phenotypes and active and latent TB. Methods: Blood samples were drawn from 32 HIV negative individuals with active TB and 45 HIV negative individuals with latent MTB infection. DNA was extracted from the blood samples and the DNA samples HLA typed by the polymerase chain reaction-sequence specific primer method. The allelic frequencies were determined by direct count. Results: HLA-A*02, A*01, A*74, A*30, B*15, B*58, C*07, C*03 and C*04 were the dominant phenotypes in this Ugandan cohort. There were differences in the distribution of HLA types between the individuals with active TB and the individuals with LTBI with only HLA-A*03 allele showing a statistically significant difference (p=0.0136). However, after FDR computation the corresponding q-value is above the expected proportion of false discoveries (q-value 0.2176). Key findings: We identified a number of HLA class I alleles in a population from Central Uganda which will enable us to carry out a functional characterization of CD8+ T-cell mediated immune responses to MTB. Our results also suggest that there may be a positive association between the HLA-A*03 allele and TB implying that individuals with the HLA-A*03 allele are at a higher risk of developing active TB.

Keywords: HLA, phenotype, tuberculosis, Uganda

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120 Improving the Bioprocess Phenotype of Chinese Hamster Ovary Cells Using CRISPR/Cas9 and Sponge Decoy Mediated MiRNA Knockdowns

Authors: Kevin Kellner, Nga Lao, Orla Coleman, Paula Meleady, Niall Barron

Abstract:

Chinese Hamster Ovary (CHO) cells are the prominent cell line used in biopharmaceutical production. To improve yields and find beneficial bioprocess phenotypes genetic engineering plays an essential role in recent research. The miR-23 cluster, specifically miR-24 and miR-27, was first identified as differentially expressed during hypothermic conditions suggesting a role in proliferation and productivity in CHO cells. In this study, we used sponge decoy technology to stably deplete the miRNA expression of the cluster. Furthermore, we implemented the CRISPR/Cas9 system to knockdown miRNA expression. Sponge constructs were designed for an imperfect binding of the miRNA target, protecting from RISC mediated cleavage. GuideRNAs for the CRISPR/Cas9 system were designed to target the seed region of the miRNA. The expression of mature miRNA and precursor were confirmed using RT-qPCR. For both approaches stable expressing mixed populations were generated and characterised in batch cultures. It was shown, that CRISPR/Cas9 can be implemented in CHO cells with achieving high knockdown efficacy of every single member of the cluster. Targeting of one miRNA member showed that its genomic paralog is successfully targeted as well. The stable depletion of miR-24 using CRISPR/Cas9 showed increased growth and specific productivity in a CHO-K1 mAb expressing cell line. This phenotype was further characterized using quantitative label-free LC-MS/MS showing 186 proteins differently expressed with 19 involved in proliferation and 26 involved in protein folding/translation. Targeting miR-27 in the same cell line showed increased viability in late stages of the culture compared to the control. To evaluate the phenotype in an industry relevant cell line; the miR-23 cluster, miR-24 and miR-27 were stably depleted in a Fc fusion CHO-S cell line which showed increased batch titers up to 1.5-fold. In this work, we highlighted that the stable depletion of the miR-23 cluster and its members can improve the bioprocess phenotype concerning growth and productivity in two different cell lines. Furthermore, we showed that using CRISPR/Cas9 is comparable to the traditional sponge decoy technology.

Keywords: Chinese Hamster ovary cells, CRISPR/Cas9, microRNAs, sponge decoy technology

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119 Prevalence of Cyp2d6 and Its Implications for Personalized Medicine in Saudi Arabs

Authors: Hamsa T. Tayeb, Mohammad A. Arafah, Dana M. Bakheet, Duaa M. Khalaf, Agnieszka Tarnoska, Nduna Dzimiri

Abstract:

Background: CYP2D6 is a member of the cytochrome P450 mixed-function oxidase system. The enzyme is responsible for the metabolism and elimination of approximately 25% of clinically used drugs, especially in breast cancer and psychiatric therapy. Different phenotypes have been described displaying alleles that lead to a complete loss of enzyme activity, reduced function (poor metabolizers – PM), hyperfunctionality (ultrarapid metabolizers–UM) and therefore drug intoxication or loss of drug effect. The prevalence of these variants may vary among different ethnic groups. Furthermore, the xTAG system has been developed to categorized all patients into different groups based on their CYP2D6 substrate metabolization. Aim of the study: To determine the prevalence of the different CYP2D6 variants in our population, and to evaluate their clinical relevance in personalized medicine. Methodology: We used the Luminex xMAP genotyping system to sequence 305 Saudi individuals visiting the Blood Bank of our Institution and determine which polymorphisms of CYP2D6 gene are prevalent in our region. Results: xTAG genotyping showed that 36.72% (112 out of 305 individuals) carried the CYP2D6_*2. Out of the 112 individuals with the *2 SNP, 6.23% had multiple copies of *2 SNP (19 individuals out of 305 individuals), resulting in an UM phenotype. About 33.44% carried the CYP2D6_*41, which leads to decreased activity of the CYP2D6 enzyme. 19.67% had the wild-type alleles and thus had normal enzyme function. Furthermore, 15.74% carried the CYP2D6_*4, which is the most common nonfunctional form of the CYP2D6 enzyme worldwide. 6.56% carried the CYP2D6_*17, resulting in decreased enzyme activity. Approximately 5.73% carried the CYP2D6_*10, consequently decreasing the enzyme activity, resulting in a PM phenotype. 2.30% carried the CYP2D6_*29, leading to decreased metabolic activity of the enzyme, and 2.30% carried the CYP2D6_*35, resulting in an UM phenotype, 1.64% had a whole-gene deletion CYP2D6_*5, thus resulting in the loss of CYP2D6 enzyme production, 0.66% carried the CYP2D6_*6 variant. One individual carried the CYP2D6_*3(B), producing an inactive form of the enzyme, which leads to decrease of enzyme activity, resulting in a PM phenotype. Finally, one individual carried the CYP2D6_*9, which decreases the enzyme activity. Conclusions: Our study demonstrates that different CYP2D6 variants are highly prevalent in ethnic Saudi Arabs. This finding sets a basis for informed genotyping for these variants in personalized medicine. The study also suggests that xTAG is an appropriate procedure for genotyping the CYP2D6 variants in personalized medicine.

Keywords: CYP2D6, hormonal breast cancer, pharmacogenetics, polymorphism, psychiatric treatment, Saudi population

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118 Angiogenic and Immunomodulatory Properties and Phenotype of Mesenchymal Stromal Cells Can Be Regulated by Cytokine Treatment

Authors: Ekaterina Zubkova, Irina Beloglazova, Iurii Stafeev, Konsyantin Dergilev, Yelena Parfyonova, Mikhail Menshikov

Abstract:

Mesenchymal stromal cells from adipose tissue (MSC) currently are widely used in regenerative medicine to restore the function of damaged tissues, but that is significantly hampered by their heterogeneity. One of the modern approaches to overcoming this obstacle is the polarization of cell subpopulations into a specific phenotype under the influence of cytokines and other factors that activate receptors and signal transmission to cells. We polarized MSC with factors affecting the inflammatory signaling and functional properties of cells, followed by verification of their expression profile and ability to affect the polarization of macrophages. RT-PCR evaluation showed that cells treated with LPS, interleukin-17, tumor necrosis factor α (TNF α), primarily express pro-inflammatory factors and cytokines, and after treatment with polyninosin polycytidic acid and interleukin-4 (IL4) anti-inflammatory factors and some proinflammatory factors. MSC polarized with pro-inflammatory cytokines showed a more robust pro-angiogenic effect in fibrin gel bead 3D angiogenesis assay. Further, we evaluated the possibility of paracrine effects of MSCs on the polarization of intact macrophages. Polarization efficiency was assesed by expression of M1/M2 phenotype markers CD80 and CD206. We showed that conditioned media from MSC preincubated in the presence of IL-4 cause an increase in CD206 expression similar to that observed in M2 macrophages. Conditioned media from MSC polarized in the presence of LPS or TNF-α increased the expression of CD80 antigen in macrophages, similar to that observed in M1 macrophages. In other cases, a pronounced paracrine effect of MSC on the polarization of macrophages was not detected. Thus, our study showed that the polarization of MSC along the pro-inflammatory or anti-inflammatory pathway allows us to obtain cell subpopulations that have a multidirectional modulating effect on the polarization of macrophages. (RFBR grants 20-015-00405 and 18-015-00398.)

Keywords: angiogenesis, cytokines, mesenchymal, polarization, inflammation

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117 Inhibition of Variant Surface Glycoproteins Translation to Define the Essential Features of the Variant Surface Glycoprotein in Trypanosoma brucei

Authors: Isobel Hambleton, Mark Carrington

Abstract:

Trypanosoma brucei, the causal agent of a range of diseases in humans and livestock, evades the mammalian immune system through a population survival strategy based on the expression of a series of antigenically distinct variant surface glycoproteins (VSGs). RNAi mediated knockdown of the active VSG gene triggers a precytokinesis cell cycle arrest. To determine whether this phenotype is the result of reduced VSG transcript or depleted VSG protein, we used morpholino antisense oligonucleotides to block translation of VSG mRNA. The same precytokinesis cell cycle arrest was observed, suggesting that VSG protein abundance is monitored closely throughout the cell cycle. An inducible expression system has been developed to test various GPI-anchored proteins for their ability to rescue this cell cycle arrest. This system has been used to demonstrate that wild-type VSG expressed from a T7 promoter rescues this phenotype. This indicates that VSG expression from one of the specialised bloodstream expression sites (BES) is not essential for cell division. The same approach has been used to define the minimum essential features of a VSG necessary for function.

Keywords: bloodstream expression site, morpholino, precytokinesis cell cycle arrest, variant surface glycoprotein

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116 Characteristics of Phytophthora infestans: The Causal Fungus of Potato Late Blight Disease

Authors: A. E. Elkorany, Eman Elsrgawy

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Eighty six isolates of Phytophthora infestans dating back to 2006 were recovered from potato tubers that were on sale in Alexandria markets, Egypt. The isolates were characterized for mating type and colony morphology. Both A1 and A2 mating types were detected in the isolate collection, however, the A2 constituted 5.8% of the total isolates made while the A1 mating type isolates constituted 91.9%. The self-fertile phenotype was also detected but at a lower percentage of 2.3% of the total isolates. This indicated that Mexico, the probable origin of the disease, is no longer the only place where A2 mating type ever exists. The lumpy phenotype was the only trait observed linked to the A2 mating type isolates on rye A agar medium. The self-fertile isolates, however, exhibited colonies of a waxy appearance with little aerial hyphae and the culture were backed full with oospores. The A1 mating colonies were of smooth white abundant aerial hyphae. The metalaxyl resistant isolates were also detected among the analyzed isolates and constituted 4.6% of the total (86) isolates investigated. The appearance of the A2 mating type outside Mexico and the variation revealed in the population of Phytophthora infestans investigated supported the hypothesis of a second worldwide migration of the fungus from its origin which could constitute a threat to potato cultivation around the world.

Keywords: Phytophthora infestans, potato, Egypt, fungus

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115 AAV-Mediated Human Α-Synuclein Expression in a Rat Model of Parkinson's Disease –Further Characterization of PD Phenotype, Fine Motor Functional Effects as Well as Neurochemical and Neuropathological Changes over Time

Authors: R. Pussinen, V. Jankovic, U. Herzberg, M. Cerrada-Gimenez, T. Huhtala, A. Nurmi, T. Ahtoniemi

Abstract:

Targeted over-expression of human α-synuclein using viral-vector mediated gene delivery into the substantia nigra of rats and non-human primates has been reported to lead to dopaminergic cell loss and the formation of α-synuclein aggregates reminiscent of Lewy bodies. We have previously shown how AAV-mediated expression of α-synuclein is seen in the chronic phenotype of the rats over 16 week follow-up period. In the context of these findings, we attempted to further characterize this long term PD related functional and motor deficits as well as neurochemical and neuropathological changes in AAV-mediated α-synuclein transfection model in rats during chronic follow-up period. Different titers of recombinant AAV expressing human α-synuclein (A53T) were stereotaxically injected unilaterally into substantia nigra of Wistar rats. Rats were allowed to recover for 3 weeks prior to initial baseline behavioral testing with rotational asymmetry test, stepping test and cylinder test. A similar behavioral test battery was applied again at weeks 5, 9,12 and 15. In addition to traditionally used rat PD model tests, MotoRater test system, a high speed kinematic gait performance monitoring was applied during the follow-up period. Evaluation focused on animal gait between groups. Tremor analysis was performed on weeks 9, 12 and 15. In addition to behavioral end-points, neurochemical evaluation of dopamine and its metabolites were evaluated in striatum. Furthermore, integrity of the dopamine active transport (DAT) system was evaluated by using 123I- β-CIT and SPECT/CT imaging on weeks 3, 8 and 12 after AAV- α-synuclein transfection. Histopathology was examined from end-point samples at 3 or 12 weeks after AAV- α-synuclein transfection to evaluate dopaminergic cell viability and microglial (Iba-1) activation status in substantia nigra by using stereological analysis techniques. This study focused on the characterization and validation of previously published AAV- α-synuclein transfection model in rats but with the addition of novel end-points. We present the long term phenotype of AAV- α-synuclein transfected rats with traditionally used behavioral tests but also by using novel fine motor analysis techniques and tremor analysis which provide new insight to unilateral effects of AAV α-synuclein transfection. We also present data about neurochemical and neuropathological end-points for the dopaminergic system in the model and how well they correlate with behavioral phenotype.

Keywords: adeno-associated virus, alphasynuclein, animal model, Parkinson’s disease

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114 Genetics of Pharmacokinetic Drug-Drug Interactions of Most Commonly Used Drug Combinations in the UK: Uncovering Unrecognised Associations

Authors: Mustafa Malki, Ewan R. Pearson

Abstract:

Tools utilized by health care practitioners to flag potential adverse drug reactions secondary to drug-drug interactions ignore individual genetic variation, which has the potential to markedly alter the severity of these interactions. To our best knowledge, there have been limited published studies on the impact of genetic variation on drug-drug interactions. Therefore, our aim in this project is the discovery of previously unrecognized, clinically important drug-drug-gene interactions (DDGIs) within the list of most commonly used drug combinations in the UK. The UKBB database was utilized to identify the top most frequently prescribed drug combinations in the UK with at least one route of interaction (over than 200 combinations were identified). We have recognised 37 common and unique interacting genes considering all of our drug combinations. Out of around 600 potential genetic variants found in these 37 genes, 100 variants have met the selection criteria (common variant with minor allele frequency ≥ 5%, independence, and has passed HWE test). The association between these variants and the use of each of our top drug combinations has been tested with a case-control analysis under the log-additive model. As the data is cross-sectional, drug intolerance has been identified from the genotype distribution as presented by the lower percentage of patients carrying the risky allele and on the drug combination compared to those free of these risk factors and vice versa with drug tolerance. In GoDARTs database, the same list of common drug combinations identified by the UKBB was utilized here with the same list of candidate genetic variants but with the addition of 14 new SNPs so that we have a total of 114 variants which have met the selection criteria in GoDARTs. From the list of the top 200 drug combinations, we have selected 28 combinations where the two drugs in each combination are known to be used chronically. For each of our 28 combinations, three drug response phenotypes have been identified (drug stop/switch, dose decrease, or dose increase of any of the two drugs during their interaction). The association between each of the three phenotypes belonging to each of our 28 drug combinations has been tested against our 114 candidate genetic variants. The results show replication of four findings between both databases : (1) Omeprazole +Amitriptyline +rs2246709 (A > G) variant in CYP3A4 gene (p-values and ORs with the UKBB and GoDARTs respectively = 0.048,0.037,0.92,and 0.52 (dose increase phenotype)) (2) Simvastatin + Ranitidine + rs9332197 (T > C) variant in CYP2C9 gene (0.024,0.032,0.81, and 5.75 (drug stop/switch phenotype)) (3) Atorvastatin + Doxazosin + rs9282564 (T > C) variant in ABCB1 gene (0.0015,0.0095,1.58,and 3.14 (drug stop/switch phenotype)) (4) Simvastatin + Nifedipine + rs2257401 (C > G) variant in CYP3A7 gene (0.025,0.019,0.77,and 0.30 (drug stop/switch phenotype)). In addition, some other non-replicated, but interesting, significant findings were detected. Our work also provides a great source of information for researchers interested in DD, DG, or DDG interactions studies as it has highlighted the top common drug combinations in the UK with recognizing 114 significant genetic variants related to drugs' pharmacokinetic.

Keywords: adverse drug reactions, common drug combinations, drug-drug-gene interactions, pharmacogenomics

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113 Novel Molecular Mechanisms Involved in Macrophage Phenotypic Polarization

Authors: Mansi Srivastava, Uzma Saqib, Adnan Naim, Anjali Roy, Dongfang Liu, Deepak Bhatnagar, Ravinder Ravinder, Mirza S. Baig

Abstract:

Macrophages polarize to proinflammatory M1 or anti-inflammatory M2 states with distinct physiological functions. This transition within the M1 to M2 phenotypes decides the nature, duration, and severity of an inflammatory response. However, inspite of a substantial understanding of the fate of these phenotypes, the underlying molecular mechanisms are not well understood. We have investigated the role of Neuronal nitric oxide synthase (NOS1) mediated regulation of Activator protein 1 (AP-1) transcription factor in macrophages as a critical effector of macrophage phenotypic change. Activator protein 1 (AP-1) is a group of dimeric transcription factors composed of jun, Fos, and ATF family proteins. We determined that NOS1-derived nitric oxide (NO) facilitate Fos and jun interaction which induces IL12 & IL23 expression. Pharmacological inhibition of NOS1 inhibits Fos and jun interaction but increases ATF2 and Fos dimerization. Switching of Fos and jun dimer to ATF2 and jun dimerization switches phenotype from IL–12high IL-23high IL-10low to IL–12low IL-23lowIL-10high phenotype, respectively. Together, these findings highlight a key role of the TLR4-NOS1-AP1 signaling axis in regulating macrophage polarization.

Keywords: inflammation, macrophage, lipopolysaccharide (LPS), proinflammatory cytokines, activator protein 1 (AP-1), neuronal nitric oxide synthase (NOS1)

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112 Molecular Defects Underlying Genital Ambiguity in Egyptian Patients: A Systematic Review

Authors: Y. Z. Gad

Abstract:

Disorders of Sex Development (DSD) are defined as congenital conditions in which development of chromosomal, gonadal or anatomical sex is atypical. The DSD are relatively prevalent in Egypt. In spite of that, the relative rarity of the individual disease types or their molecular pathologies frequently resulted in reporting on single or few cases. This augmented the challenging nature of phenotype-genotype correlation in this disease group and its utilization in the management of such medical emergency. Through critical assessment of the published DSD reports, the current review aims at analyzing the clinical characteristics of the various DSD forms in relation to the underlying molecular pathologies. A systematic literature search was done in Pubmed, using relevant keywords (Egypt versus DSD, genital ambiguity or ambiguous genitalia, the old terms of 'intersex, hermaphroditism and pseudohermaphroditism', and a list of the DSD entities and their related genes). The search yielded 24 reports of molecular data in Egyptian patients presenting with ambiguous genitalia. However, only 21 publications fulfilled the criteria of inclusion of detailed clinical descriptions and definitive molecular diagnoses of individual patients. Curation of the data yielded a total of 53 cases that were ascertained from 40 families. Fifty-one patients present with ambiguous genitalia only while 2 had multiple congenital anomalies. Parental consanguinity was noted in 60% of cases. Sex of rearing at initial presentation was female in 75% and 60% in 46,XY and 46,XX DSD cases, respectively. The external genital phenotype in 2/3 of the 46,XY DSD cases showed moderate undermasculinization [Quigley scores 3 & 4] and 1/3 had severe presentations [scores 5 & 6]. For 46,XX subjects, 1 had severe virilization of the external genitalia while 8 had moderate phenotype. Hormonal data were inconclusive or contradictory to final diagnosis in a forth of cases. Collectively, 31 families [31/40, 77.5%] with 46,XY DSD had molecular defects in the genes, 5 alpha reductase 2 (SRD5A2) [12/31], 17 beta-hydroxysteroid dehydrogenase 3 [8/31], androgen receptor [7/31], Steroidogenic factor 1 [2/31], luteinizing hormone receptor [1/31], and fibroblast growth factor receptor 1 [1/31]. In a multiethnic study, 9 families afflicted with 46,XX DSD due to 11 beta hydroxylase (CYP11B1) deficiency were documented. Two recurrent mutations, G34R and N160D, in SRD5A2 were present, respectively, in 42 and 17% of cases. Similarly, 4 recurrent mutations resulted in 89% of the CYP11B1 presentations. In conclusion, this analysis highlights the importance of autosomal recessive inheritance and inbreeding among DSD presentations, the importance of founder effect in at least 2 disorders, the difficulties in relating the genotype with the indeterminate genital phenotype, the under-reporting of some DSD subtypes, and the notion that the reported mutational profiles among Egyptian DSD cases are relatively different from those reported in other ethnic groups.

Keywords: disorders of sex development, genital ambiguity, mutation, molecular diagnosis, Egypt

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111 Antigen-Presenting Cell Characteristics of Human γδ T Lymphocytes in Chronic Myeloid Leukemia

Authors: Piamsiri Sawaisorn, Tienrat Tangchaikeeree, Waraporn Chan-On, Chaniya Leepiyasakulchai, Rachanee Udomsangpetch, Suradej Hongeng, Kulachart Jangpatarapongsa

Abstract:

Human Vγ9Vδ2 T lymphocytes are regarded as promising effector cells for cancer immunotherapy since they have the ability to eliminate several tumor cells through non-peptide antigen recognition and non-major histocompatibility complex (MHC) restriction. An issue of recent interest is the capability to activate γδ T cells by use of a group of drugs, such as pamidronate, that cause accumulation of phosphoantigen which is recognized by γδ T cell receptors. Moreover, their antigen presenting cell-like phenotype and function have been confirmed in many clinical trials. In this study, Vγ9Vδ2 T cells derived from normal peripheral blood mononuclear cells were activated with pamidronate and the expanded Vγ9Vδ2 T cells can recognize and kill chronic myeloid leukemia (CML) cells treated with pamidronate through their cytotoxic activity. To support the strong role played by Vγ9Vδ2 T cells against cancer, we provide the evidence that Vγ9Vδ2 T cells activated with CML cell lysate antigen can efficiently express antigen presenting cell (APC) phenotype and function. In conclusion, pamidronate can be used in intentional activation of human Vγ9Vδ2 T cells and can increase the susceptibility of CML cells to cytotoxicity of Vγ9Vδ2 T cells. The activated Vγ9Vδ2 T cells by cancer cells lysate can show their APC characteristics, and so greatly increase the interest in exploring their therapeutic potential in hematologic malignancy.

Keywords: γδ T lymphocytes, antigen-presenting cells, chronic myeloid leukemia, cancer, immunotherapy

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110 Study into the Interactions of Primary Limbal Epithelial Stem Cells and HTCEPI Using Tissue Engineered Cornea

Authors: Masoud Sakhinia, Sajjad Ahmad

Abstract:

Introduction: Though knowledge of the compositional makeup and structure of the limbal niche has progressed exponentially during the past decade, much is yet to be understood. Identifying the precise profile and role of the stromal makeup which spans the ocular surface may inform researchers of the most optimum conditions needed to effectively expand LESCs in vitro, whilst preserving their differentiation status and phenotype. Limbal fibroblasts, as opposed to corneal fibroblasts are thought to form an important component of the microenvironment where LESCs reside. Methods: The corneal stroma was tissue engineered in vitro using both limbal and corneal fibroblasts embedded within a tissue engineered 3D collagen matrix. The effect of these two different fibroblasts on LESCs and hTCEpi corneal epithelial cell line were then subsequently determined using phase contrast microscopy, histolological analysis and PCR for specific stem cell markers. The study aimed to develop an in vitro model which could be used to determine whether limbal, as opposed to corneal fibroblasts, maintained the stem cell phenotype of LESCs and hTCEpi cell line. Results: Tissue culture analysis was inconclusive and required further quantitative analysis for remarks on cell proliferation within the varying stroma. Histological analysis of the tissue-engineered cornea showed a comparable structure to that of the human cornea, though with limited epithelial stratification. PCR results for epithelial cell markers of cells cultured on limbal fibroblasts showed reduced expression of CK3, a negative marker for LESC’s, whilst also exhibiting a relatively low expression level of P63, a marker for undifferentiated LESCs. Conclusion: We have shown the potential for the construction of a tissue engineered human cornea using a 3D collagen matrix and described some preliminary results in the analysis of the effects of varying stroma consisting of limbal and corneal fibroblasts, respectively, on the proliferation of stem cell phenotype of primary LESCs and hTCEpi corneal epithelial cells. Although no definitive marker exists to conclusively illustrate the presence of LESCs, the combination of positive and negative stem cell markers in our study were inconclusive. Though it is less traslational to the human corneal model, the use of conditioned medium from that of limbal and corneal fibroblasts may provide a more simple avenue. Moreover, combinations of extracellular matrices could be used as a surrogate in these culture models.

Keywords: cornea, Limbal Stem Cells, tissue engineering, PCR

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109 Transgenerational Impact of Intrauterine Hyperglycaemia to F2 Offspring without Pre-Diabetic Exposure on F1 Male Offspring

Authors: Jun Ren, Zhen-Hua Ming, He-Feng Huang, Jian-Zhong Sheng

Abstract:

Adverse intrauterine stimulus during critical or sensitive periods in early life, may lead to health risk not only in later life span, but also further generations. Intrauterine hyperglycaemia, as a major feature of gestational diabetes mellitus (GDM), is a typical adverse environment for both F1 fetus and F1 gamete cells development. However, there is scare information of phenotypic difference of metabolic memory between somatic cells and germ cells exposed by intrauterine hyperglycaemia. The direct transmission effect of intrauterine hyperglycaemia per se has not been assessed either. In this study, we built a GDM mice model and selected male GDM offspring without pre-diabetic phenotype as our founders, to exclude postnatal diabetic influence on gametes, thereby investigate the direct transmission effect of intrauterine hyperglycaemia exposure on F2 offspring, and we further compared the metabolic difference of affected F1-GDM male offspring and F2 offspring. A GDM mouse model of intrauterine hyperglycemia was established by intraperitoneal injection of streptozotocin after pregnancy. Pups of GDM mother were fostered by normal control mothers. All the mice were fed with standard food. Male GDM offspring without metabolic dysfunction phenotype were crossed with normal female mice to obtain F2 offspring. Body weight, glucose tolerance test, insulin tolerance test and homeostasis model of insulin resistance (HOMA-IR) index were measured in both generations at 8 week of age. Some of F1-GDM male mice showed impaired glucose tolerance (p < 0.001), none of F1-GDM male mice showed impaired insulin sensitivity. Body weight of F1-GDM mice showed no significance with control mice. Some of F2-GDM offspring exhibited impaired glucose tolerance (p < 0.001), all the F2-GDM offspring exhibited higher HOMA-IR index (p < 0.01 of normal glucose tolerance individuals vs. control, p < 0.05 of glucose intolerance individuals vs. control). All the F2-GDM offspring exhibited higher ITT curve than control (p < 0.001 of normal glucose tolerance individuals, p < 0.05 of glucose intolerance individuals, vs. control). F2-GDM offspring had higher body weight than control mice (p < 0.001 of normal glucose tolerance individuals, p < 0.001 of glucose intolerance individuals, vs. control). While glucose intolerance is the only phenotype that F1-GDM male mice may exhibit, F2 male generation of healthy F1-GDM father showed insulin resistance, increased body weight and/or impaired glucose tolerance. These findings imply that intrauterine hyperglycaemia exposure affects germ cells and somatic cells differently, thus F1 and F2 offspring demonstrated distinct metabolic dysfunction phenotypes. And intrauterine hyperglycaemia exposure per se has a strong influence on F2 generation, independent of postnatal metabolic dysfunction exposure.

Keywords: inheritance, insulin resistance, intrauterine hyperglycaemia, offspring

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108 A Clinical Cutoff to Identify Metabolically Unhealthy Obese and Normal-Weight Phenotype in Young Adults

Authors: Lívia Pinheiro Carvalho, Luciana Di Thommazo-Luporini, Rafael Luís Luporini, José Carlos Bonjorno Junior, Renata Pedrolongo Basso Vanelli, Manoel Carneiro de Oliveira Junior, Rodolfo de Paula Vieira, Renata Trimer, Renata G. Mendes, Mylène Aubertin-Leheudre, Audrey Borghi-Silva

Abstract:

Rationale: Cardiorespiratory fitness (CRF) and functional capacity in young obese and normal-weight people are associated with metabolic and cardiovascular diseases and mortality. However, it remains unclear whether their metabolically healthy (MH) or at risk (AR) phenotype influences cardiorespiratory fitness in this vulnerable population such as obese adults but also in normal-weight people. HOMA insulin resistance index (HI) and leptin-adiponectin ratio (LA) are strong markers for characterizing those phenotypes that we hypothesized to be associated with physical fitness. We also hypothesized that an easy and feasible exercise test could identify a subpopulation at risk to develop metabolic and related disorders. Methods: Thirty-nine sedentary men and women (20-45y; 18.530 kg.m-2) underwent a clinical evaluation, including the six-minute step test (ST), a well-validated and reliable test for young people. Body composition assessment was done by a tetrapolar bioimpedance in a fasting state and in the folicular phase for women. A maximal cardiopulmonary exercise testing, as well as the ST, evaluated the oxygen uptake at the peak of the test (VO2peak) by an ergospirometer Oxycon Mobile. Lipids, glucose, insulin were analysed and the ELISA method quantified the serum leptin and adiponectin from blood samples. Volunteers were divided in two groups: AR or MH according to a HI cutoff of 1.95, which was previously determined in the literature. T-test for comparison between groups, Pearson´s test to correlate main variables and ROC analysis for discriminating AR from up-and-down cycles in ST (SC) were applied (p<0.05). Results: Higher LA, fat mass (FM) and lower HDL, SC, leg lean mass (LM) and VO2peak were found in AR than in MH. Significant correlations were found between VO2peak and SC (r= 0.80) as well as between LA and FM (r=0.87), VO2peak (r=-0.73), and SC (r=-0.65). Area under de curve showed moderate accuracy (0.75) of SC <173 to discriminate AR phenotype. Conclusion: Our study found that at risk obese and normal-weight subjects showed an unhealthy metabolism as well as a poor CRF and functional daily activity capacity. Additionally, a simple and less costly functional test associated with above-mentioned aspects is able to identify ‘at risk’ subjects for primary intervention with important clinical and health implications.

Keywords: aerobic capacity, exercise, fitness, metabolism, obesity, 6MST

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107 Insight into the Visual Attentional Correlates Underpinning Autistic-Like Traits in Fragile X and Down Syndrome

Authors: Jennifer M. Glennon, Hana D'Souza, Luke Mason, Annette Karmiloff-Smith, Michael S. C. Thomas

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Genetic syndrome groups that feature high rates of autism comorbidity, like Down syndrome (DS) and fragile X syndrome (FXS), have been presented as useful models for understanding risk and protective factors involved in the emergence of autistic traits. Yet despite reaching clinical thresholds, these ‘syndromic’ forms of autism appear to differ in important ways from the idiopathic or ‘non-syndromic’ autism phenotype. To uncover the true nature of these comorbidities, it is necessary to extend definitions of autism to include the cognitive characteristics of the disorder and to then apply this broadened conceptualisation to the study of syndromic autism profiles. The current study employs a variety of well-established eye-tracking paradigms to assess visual attentional performance in children with DS and FXS who reach thresholds for autism on the Social Communication Questionnaire. It investigates whether autism profiles in these children are accompanied by visual orienting difficulties (‘sticky attention’), decreased social attention, and enhanced visual search performance, all of which are characteristic of the idiopathic autism phenotype. Data is collected from children with DS and FXS aged between 6 and 10 years, in addition to two control groups matched on age and intellectual ability (i.e., children with idiopathic autism and neurotypical controls). Cross-sectional developmental trajectory analyses are conducted to enable visuo-attentional profile comparisons. Significant differences in the visuo-attentional processes underpinning autism presentations in children with FXS and DS are hypothesised, supporting notions of syndrome specificity. The study provides insight into the complex heterogeneity associated with syndromic autism presentations and autism per se, with clinical implications for the utility of autism intervention programmes in DS and FXS populations.

Keywords: autism, down syndrome, fragile X syndrome, eye tracking

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106 Sexually Dimorphic Effects of Chronic Exercise and Myocytic Androgen Receptor Overexpression on Body Composition in Sprague dawley Rats

Authors: Sabrina Barsky, Ashley Monks

Abstract:

In humans, exercise improves symptoms of various pathological states, although exercise adaptations seem to differ in response to sex. Skeletal muscle anabolism is thought to be regulated by androgen receptor (AR) through poorly specified mechanisms. Interactions of AR and exercise on muscle phenotype remain inconclusive in males, and undetermined in females. We hypothesized that sex differences in exercise adaptations are regulated by the androgenic system and the type of exercise performed. Here we examined interactions between a muscle-specific AR overexpression transgene (HSA-AR) and forced aerobic exercise paradigm on muscle and adipose exercise adaptation in male and female rats. We used dual-energy X-ray absorptiometry (DXA) to examine body composition adaptations post 9-week exercise protocol. We replicated the effects of HSA-AR on body composition, with males only having increased % lean mass and reduced % fat mass (P<0.05). Aerobic exercise improved lean body phenotype significantly, with lesser indices of total and % fat mass (P<0.01) in both sexes. Sex-specific effects of exercise included decreased total body mass (P<0.01) in males and increased lean mass % (P<0.001) in females. Surprisingly, neither AR manipulation nor exercise affected bone parameters in either sex. This varied response in total mass and lean mass according to exercise presents a sexually dimorphic response to exercise. Neither sex showed an interaction between HSA-AR and forced aerobic exercise on body composition. Future work is proposed to examine the effects of exercise type (aerobic versus resistance) and the role of gonadal androgens in sexually dimorphic exercise-mediated mitochondrial adaptations. This work implicates the development of sex-specific exercise therapies.

Keywords: androgen receptor, forced exercise, muscle physiology, sexual dimorphism

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105 Peptidoglycan Vaccine-On-Chip against a Lipopolysaccharide-Induced Experimental Sepsis Model

Authors: Katerina Bakela, Ioanna Zerva, Irene Athanassakis

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Lipopolysaccharide (LPS) is commonly used in murine sepsis models, which are largely associated with immunosuppression (incretion of MDSCs cells and Tregs, imbalance of inflammatory/anti-inflammatory cytokines) and collapse of the immune system. After adapting the LPS treatment to the needs of locally bred BALB/c mice, the present study explored the protective role of Micrococcus luteus peptidoglycan (PG) pre-activated vaccine-on chip in endotoxemia. The established protocol consisted of five daily intraperitoneal injections of 0.2mg/g LPS. Such protocol allowed longer survival, necessary in the prospect of the therapeutic treatment application. The so-called vaccine-on-chip consists of a 3-dimensional laser micro-texture Si-scaffold loaded with BALB/c mouse macrophages and activated in vitro with 1μg/ml PG, which exert its action upon subcutaneous implantation. The LPS treatment significantly decreased CD4+, CD8+, CD3z+, and CD19+ cells, while increasing myeloid-derived suppressor cells (MDSCs), CD25+, and Foxp3+ cells. These results were accompanied by increased arginase-1 activity in spleen cell lysates and production of IL-6, TNF-a, and IL-18 while acquiring severe sepsis phenotype as defined by the murine sepsis scoring. The in vivo application of PG pre-activated vaccine-on chip significantly decreased the percent of CD11b+, Gr1+, CD25+, Foxp3+ cells, and arginase-1 activity in the spleen of LPS-treated animals, while decreasing IL-6 and TNF-a in the serum, allowing survival to all animals tested and rescuing the severity of sepsis phenotype. In conclusion, these results reveal a promising mode of action of PG pre-activated vaccine-on chip in LPS endotoxemia, strengthening; thus, the use of treatment is septic patients.

Keywords: myeloid-derived suppressor cells, peptidoglycan, sepsis, Si-scaffolds

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104 Inactivation of Semicarbazide-Sensitive Amine Oxidase Induces the Phenotypic Switch of Smooth Muscle Cells and Aggravates the Development of Atherosclerotic Lesions

Authors: Miao Zhang, Limin Liu, Feng Zhi, Panpan Niu, Mengya Yang, Xuemei Zhu, Ying Diao, Jun Wang, Ying Zhao

Abstract:

Background and Aims: Clinical studies have demonstrated that serum semicarbazide-sensitive amine oxidase (SSAO) activities positively correlate with the progression of atherosclerosis. The aim of the present study is to investigate the effect of SSAO inactivation on the development of atherosclerosis. Methods: Female LDLr knockout (KO) mice were given the Western-type diet for 6 and 9 weeks to induce the formation of early and advanced lesions, and semicarbazide (SCZ, 0.125%) was added into the drinking water to inactivate SSAO in vivo. Results: Despite no impact on plasma total cholesterol levels, abrogation of SSAO by SCZ not only resulted in the enlargement of both early (1.5-fold, p=0.0043) and advanced (1.8-fold, p=0.0013) atherosclerotic lesions, but also led to reduced/increased lesion contents of macrophages/smooth muscle cells (SMCs) (macrophage: ~0.74-fold, p=0.0002(early)/0.0016(advanced); SMC: ~1.55-fold, p=0.0003(early) /0.0001(advanced)), respectively. Moreover, SSAO inactivation inhibited the migration of circulating monocytes into peripheral tissues and reduced the amount of circulating Ly6Chigh monocytes (0.7-fold, p=0.0001), which may account for the reduced macrophage content in lesions. In contrast, the increased number of SMCs in lesions of SCZ-treated mice is attributed to an augmented synthetic vascular SMC phenotype switch as evidenced by the increased proliferation of SMCs and accumulation of collagens in vivo. Conclusion: SSAO inactivation by SCZ promotes the phenotypic switch of SMCs and the development of atherosclerosis. The enzymatic activity of SSAO may thus represent a potential target in the prevention and/or treatment of atherosclerosis.

Keywords: atherosclerosis, phenotype switch of smooth muscle cells, SSAO/VAP-1, semicarbazide

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103 Generalized Correlation Coefficient in Genome-Wide Association Analysis of Cognitive Ability in Twins

Authors: Afsaneh Mohammadnejad, Marianne Nygaard, Jan Baumbach, Shuxia Li, Weilong Li, Jesper Lund, Jacob v. B. Hjelmborg, Lene Christensen, Qihua Tan

Abstract:

Cognitive impairment in the elderly is a key issue affecting the quality of life. Despite a strong genetic background in cognition, only a limited number of single nucleotide polymorphisms (SNPs) have been found. These explain a small proportion of the genetic component of cognitive function, thus leaving a large proportion unaccounted for. We hypothesize that one reason for this missing heritability is the misspecified modeling in data analysis concerning phenotype distribution as well as the relationship between SNP dosage and the phenotype of interest. In an attempt to overcome these issues, we introduced a model-free method based on the generalized correlation coefficient (GCC) in a genome-wide association study (GWAS) of cognitive function in twin samples and compared its performance with two popular linear regression models. The GCC-based GWAS identified two genome-wide significant (P-value < 5e-8) SNPs; rs2904650 near ZDHHC2 on chromosome 8 and rs111256489 near CD6 on chromosome 11. The kinship model also detected two genome-wide significant SNPs, rs112169253 on chromosome 4 and rs17417920 on chromosome 7, whereas no genome-wide significant SNPs were found by the linear mixed model (LME). Compared to the linear models, more meaningful biological pathways like GABA receptor activation, ion channel transport, neuroactive ligand-receptor interaction, and the renin-angiotensin system were found to be enriched by SNPs from GCC. The GCC model outperformed the linear regression models by identifying more genome-wide significant genetic variants and more meaningful biological pathways related to cognitive function. Moreover, GCC-based GWAS was robust in handling genetically related twin samples, which is an important feature in handling genetic confounding in association studies.

Keywords: cognition, generalized correlation coefficient, GWAS, twins

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102 ROCK Signaling and Radio Resistance: The Association and the Effect

Authors: P. Annapurna, Cecil Ross, Sudhir Krishna, Sweta Srivastava

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Irradiation plays a pivotal role in cervical cancer treatment, however some tumors exhibit resistance to therapy while some exhibit relapse, due to better repair and enhanced resistance mechanisms operational in their cells. The present study aims to understand the signaling mechanism operational in resistance phenotype and in the present study we report the role of Rho GTPase associated protein kinase (ROCK) signaling in cervical carcinoma radio-resistance. ROCK signaling has been implicated in several tumor progressions and is important for DNA repair. Irradiation of spheroid cultures of SiHa cervical carcinoma derived cell line at 6Gy resulted in generation of resistant cells in vitro which had better clonogenic abilities and formed larger and more colonies, in soft agar colony formation assay, as compared to the non-irradiated cells. These cells also exhibited an enhanced motility phenotype. Cell cycle profiling showed the cells to be blocked in G2M phase with enhanced pCDC2 levels indicating onset of possible DNA repair mechanism. Notably, 3 days post-irradiation, irradiated cells showed increased ROCK2 translocation to the nucleus with enhanced protein expression as compared to the non-irradiated cells. Radio-sensitization of the resistant cells was enhanced using Y27632, an inhibitor to ROCK signaling. The treatment of resistant cells with Y27632 resulted in increased cell death upon further irradiation. This observation has been confirmed using inhibitory antibodies to ROCK1/2. Result show that both ROCK1/2 have a functional contribution in radiation resistance of cervical cancer cells derived from cell lines. Interestingly enrichment of stem like cells (Hoechst negative cells) was also observed upon irradiation and these cells were markedly sensitive to Y27632 treatment. Our results thus suggest the role of ROCK signaling in radio-resistance in cervical carcinoma. Further studies with human biopsies, mice models and mechanistic of ROCK signaling in the context of radio-resistance will clarify the role of this molecule further and allow for therapeutics development.

Keywords: cervical carcinoma, radio-resistance, ROCK signaling, cancer treatment

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101 Association between a Serotonin Re-Uptake Transporter Gene Polymorphism and Mucosal Serotonin Level in Women Patients with Irritable Bowel Syndrome and Healthy Control: A Pilot Study from Northern India

Authors: Sunil Kumar, Uday C. Ghoshal

Abstract:

Background and aims: Serotonin (5-hydroxtryptamine, 5-HT) is an important factor in gut function, playing key roles in intestinal peristalsis and secretion, and in sensory signaling in the brain-gut axis. Removal from its sites of action is mediated by a specific protein called the serotonin reuptake transporter (SERT). Polymorphisms in the promoter region of the SERT gene have effects on transcriptional activity, resulting in altered 5-HT reuptake efficiency. Functional polymorphisms may underlie disturbance in gut function in individuals suffering with disorders such as irritable bowel syndrome (IBS). The aim of this study was to assess the potential association between SERT polymorphisms and the diarrhea predominant IBS (D-IBS) phenotype Subjects: A total of 36 northern Indian female patients and 55 female northern Indian healthy controls (HC) were subjected to genotyping. Methods: Leucocyte DNA of all subjects was analyzed by polymerase chain reaction based technologies for SERT polymorphisms, specifically the insertion/deletion polymorphism in the promoter (SERT-P). Statistical analysis was performed to assess association of SERT polymorphism allele with the D-IBS phenotype. Results: The frequency of distribution of SERT-P gene was comparable between female patients with IBS and HC (p = 0.086). However, frequency of SERT-P deletion/deletion genotype was significantly higher in female patients with D-IBS compared to C-IBS and A-IBS [17/19 (89.5%) vs. 4/12 (33.3%) vs. 1/5 (20%), p=0.001, respectively]. The mucosal level of serotonin was higher in D-IBS compared to C-IBS and A-IBS [Median, range (159.26, 98.78–212.1) vs. 110.4, 67.87–143.53 vs. 92.34, 78.8–166.3 pmol/mL, p=0.001, respectively]. The mucosal level of serotonin was higher in female patients with IBS with SERT-P deletion/deletion genotype compared deletion/insertion and insertion/insertion [157.65, 67.87–212.1 vs. 110.4, 78.1–143.32 vs. 100.5, 69.1–132.03 pmol/mL, p=0.001, respectively]. Patients with D-IBS with deletion/deletion genotype more often reported symptoms of abdominal pain, discomfort (p=0.025) and bloating (p=0.039). Symptoms development following lactose ingestion was strongly associated with D-IBS and SERT-P deletion/deletion genotype (p=0.004). Conclusions: Significant association was observed between D-IBS and the SERT-P deletion/deletion genotype, suggesting that the serotonin transporter is a potential candidate gene for D-IBS in women.

Keywords: serotonin, SERT, inflammatory bowel disease, genetic polymorphism

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100 Temporal Profile of T2 MRI and 1H-MRS in the MDX Mouse Model of Duchenne Muscular Dystrophy

Authors: P. J. Sweeney, T. Ahtoniemi, J. Puoliväli, T. Laitinen, K.Lehtimäki, A. Nurmi, D. Wells

Abstract:

Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscle wasting disease for which there are currently no treatment that effectively prevents the muscle necrosis and progressive muscle loss. DMD is among the most common of inherited diseases affecting around 1/3500 live male births. MDX (X-linked muscular dystrophy) mice only partially encapsulate the disease in humans and display weakness in muscles, muscle damage and edema during a period deemed the “critical period” when these mice go through cycles of muscular degeneration and regeneration. Although the MDX mutant mouse model has been extensively studied as a model for DMD, to-date an extensive temporal, non-invasive imaging profile that utilizes magnetic resonance imaging (MRI) and 1H-magnetic resonance spectroscopy (1H-MRS) has not been performed.. In addition, longitudinal imaging characterization has not coincided with attempts to exacerbate the progressive muscle damage by exercise. In this study we employed an 11.7 T small animal MRI in order to characterize the MRI and MRS profile of MDX mice longitudinally during a 12 month period during which MDX mice were subjected to exercise. Male mutant MDX mice (n=15) and male wild-type mice (n=15) were subjected to a chronic exercise regime of treadmill walking (30 min/ session) bi-weekly over the whole 12 month follow-up period. Mouse gastrocnemius and tibialis anterior muscles were profiled with baseline T2-MRI and 1H-MRS at 6 weeks of age. Imaging and spectroscopy was repeated again at 3 months, 6 months, 9 months and 12 months of age. Plasma creatine kinase (CK) level measurements were coincided with time-points for T2-MRI and 1H-MRS, but also after the “critical period” at 10 weeks of age. The results obtained from this study indicate that chronic exercise extends dystrophic phenotype of MDX mice as evidenced by T2-MRI and1H-MRS. T2-MRI revealed extent and location of the muscle damage in gastrocnemius and tibialis anterior muscles as hyperintensities (lesions and edema) in exercised MDX mice over follow-up period.. The magnitude of the muscle damage remained stable over time in exercised mice. No evident fat infiltration or cumulation to the muscle tissues was seen at any time-point in exercised MDX mice. Creatine, choline and taurine levels evaluated by 1H-MRS from the same muscles were found significantly decreased in each time-point, Extramyocellular (EMCL) and intramyocellular lipids (IMCL) did not change in exercised mice supporting the findings from anatomical T2-MRI scans for fat content. Creatine kinase levels were found to be significantly higher in exercised MDX mice during the follow-up period and importantly CK levels remained stable over the whole follow-up period. Taken together, we have described here longitudinal prophile for muscle damage and muscle metabolic changes in MDX mice subjected to chronic exercised. The extent of the muscle damage by T2-MRI was found to be stable through the follow-up period in muscles examined. In addition, metabolic profile, especially creatine, choline and taurine levels in muscles, was found to be sustained between time-points. The anatomical muscle damage evaluated by T2-MRI was supported by plasma CK levels which remained stable over the follow-up period. These findings show that non-invasive imaging and spectroscopy can be used effectively to evaluate chronic muscle pathology. These techniques can be also used to evaluate the effect of various manipulations, like here exercise, on the phenotype of the mice. Many of the findings we present here are translatable to clinical disease, such as decreased creatine, choline and taurine levels in muscles. Imaging by T2-MRI and 1H-MRS also revealed that fat content or extramyocellar and intramyocellular lipids, respectively, are not changed in MDX mice, which is in contrast to clinical manifestation of the Duchenne’s muscle dystrophy. Findings show that non-invasive imaging can be used to characterize the phenotype of a MDX model and its translatability to clinical disease, and to study events that have traditionally been not examined, like here rigorous exercise related sustained muscle damage after the “critical period”. The ability for this model to display sustained damage beyond the spontaneous “critical period“ and in turn to study drug effects on this extended phenotype will increase the value of the MDX mouse model as a tool to study therapies and treatments aimed at DMD and associated diseases.

Keywords: 1H-MRS, MRI, muscular dystrophy, mouse model

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