International Conference and Exhibition on Molecular Medicine and Diagnostics August 24-26, 2015 West Drayton, London, UK

Carlos Fernando Prada Quiroga is Biologist, MSc in genetics and evolution of Universidade Federal de Sao Carlos in Brazil. He has completed his PhD in genetics at the age of 32 years from Universidad Autónoma de Barcelona, Spain. He has had teaching experience in countries like 2 Brazil, Spain and Colombia. He is Associate Professor and director of Bacteriology and Clinical Laboratory research group. He has published more than 10 papers in reputed journals. His research focuses on the evolutionary origin of genomic rearrangements and its relationship to different human pathologies, using mainly bioinformatics tools.

Mitochondrial genome plays a variety of important roles, including the generation of ATP through respiration and oxidative phosphorylation (OXPHOS), production of reactive oxygen species (ROS), and initiation and execution of apoptosis. Therefore, variation in these genes can directly influence metabolic performance. The mitochondrial genome presents a gene structure relatively stable through the evolution of species with a relatively low rate of gene rearrangements compared to the nuclear genome. The recent increase in the number of sequenced mitochondrial genomes of hundreds of species, it has become clear that some groups of species the gene order is more conserved than in other groups. For example, mammals and birds have relatively stable gene orders, while in amphibians, reptiles and fish rearrangements are more common. Recent studies have found that the mammalian mitochondrial genome has a higher rate of rearrangements than expected. 244 mitochondrial events were identified: Inversions, deletions and non-homologous regions (non-HR: less than 10% nucleotide identity). Inversions were found at a frequency of 84.0 % (205/244). These inversions, classified as microinversions (<1Kb) are recurrent in tRNAs genes; deletion are detected on D-loop region and tRNAs; and non-HR located on control region. Based on the breakpoints (BP) of mitochondrial inversions, there were postulated fourteen fragile regions in the human mitochondrial genome. Over 250 pathogenic human mtDNA mutations have been characterized to show the cause a wide variety of diseases with heterogeneity of phenotypes and a variable age of onset. Most of deletions and duplications BP reported in the human mitochondrial genome match with the fragile regions previously postulated. These findings could indicate the important role of tRNA in the origin of rearrangements in the human mitochondrial genome and in mammals in general.

Laura Hill is a Senior Scientist in the Design and Development department at QIAGEN Manchester, UK. She has significant experience of companion diagnostic development, specialising in primer and multiplex assay design, feasibility studies and novel technologies. She previously worked at Life Technologies on the development and manufacture of primer mixes for DNA testing kits. She has a degree in Genetics from the University of Liverpool.

The Modaplex system is a fully automated platform that combines a real time PCR module with a capillary electrophoresis detection module. The combination of these two modules allows for a large number of targets to be detected in a single reaction. Multiplexing systems that allow such a broad range of targets to be detected are desirable in companion diagnostic assays; to reduce intrusive procedures to patients, by requiring less sample material, and to reduce laboratory processing time. To investigate how the Modaplex system can make use of these benefits, we have undertaken a feasibility study to combine the detection of 42 mutation targets and 2 control targets in a single reaction. Gene panels comprising of 13 KRAS mutation targets and 29 EGFR mutation targets are the focus of this multiplexing challenge. EGFR and KRAS are key molecules in the MAPK cellular signaling pathway. Since mutations in these genes alter cellular functions, determining the mutation status is a key requirement for personalised cancer therapies. The primer designs for the 44-plex assay have initially been tested using synthetic oligonucleotides and will be further challenged with the use of FFPE extracted clinical samples. This feasibility study will demonstrate the multiplexing capabilities of the Modaplex system to meet the growing market need for quicker, less intrusive mutation detection.

The Chemoprevention of seasonal malaria with AQ + SP was adopted in areas with seasonal malaria transmission. The emergence and spread of antimalarial drug resistance poses a serious public health problem. Prophylactic or therapeutic failures LEAD re-emergence of malaria accompanied by an increase in transmission, morbidity and mortality especially among children. The spread of resistance to SP but also to AQ resulting in mutations located on pfdhfr, pfdhps, pfcrt and pfmdr1 genes could affect the effectiveness of this strategy.
A child born of immune mothers are protected during the first months of his life, by passive transfer of maternal immunity. The child is exposed to infection and begins to develop his own immunity, a process that will take several years before becomes effective. Hence the need to check whether SMC will have an impact on immunity in children. As well as objectives of this thesis we i) examine the prevalence of molecular markers of resistance of Plasmodium falciparum to SP and AQ and ii) evaluate the impact of seasonal malaria chemoprevention on the acquisition of antibodies directed against malaria. To do this, blood samples on filter paper were made during cross-sectional surveys conducted after the intervention in three health districts in central Senegal in 2008, 2009 and 2010. We determined the mutation associated with resistance to SP (pfdhfr and pfdhps) and AQ (pfcrt and pfmdr1) by PCR and secondly measuring the production of antibodies against antigens of MSP1 and AMA1 of P. falciparum.
The results show about the prevalence of molecular markers of resistance that the triple mutation pfdhfr were high between 2008 and 2010. The quintuple mutation pfdhfr / pfdhps were not observed either in control areas or in SMC area after three years of implementation. However, the absolute prevalence of resistance markers was lower in areas with SMC. Mutations pfdhfr 164L and 540E pfdhps were not found.
Regarding immunity, it was found that children living in control areas produced more antibodies (MSP1 and AMA1) than those in areas with strategy. So it seems that the SMC retards the development of immunity in these children. In view of our results, it is likely that the SMC has no impact on the prevalence of molecular markers of resistance to SP and AQ but appears to delay the acquisition of anti malarial immunity in children undergoing strategy.

Ashraf A Khalil holds a position of professor at City of Scientific Research (SRTA-City), Egypt. He got postdoctoral trainings at Dept of Biotechnology, Dept of Developmental Biology, Dept of Protein Chemistry and Dept of Neurosurgery. All were at Lund University, Sweden. In 2007, he came back Egypt and built his own team and supervising many biotechnological-oriented studies, including disease biomarkers, natural products, bioactive proteins and proteomics. Along his career he published more than 35 articles in peer-reviewed journals. He is a member of the editorial board of many peer-reviewed journals and is a fellow of European Organization for Molecular Biology (EMBO) and American Association for Cancer Research (AACR). Prof Khalil is the founder of Euro-Mediterranean Association of Life Sciences (EMALS). EMALS initiated a series of conferences called Euro-Mediterranean Conferences of Natural Products (BioNat) which were held four times successfully, in which Prof Khalil was the key leader.

PPARγ, a ligand-stimulated transcription factor with differentiation promoting activity is overexpressed in a variety of cancers. Perturbation of PPAR-γ signaling is now believed to be a strategy for treatment of several cancers, including breast cancer. A set of genes regulated by PPAR-γ ligands is expected to mediate the antiproliferative and prodifferentiation effects in cancer cells. Because 14-3-3 family of proteins shows a debatable activity and varying expression levels in different tumors, in the studies presented here we explored the transcriptional regulatory role of Pioglitazone on the seven 14-3-3 isoforms presenting in MCF-7 breast cancer cells. This study demonstrated that the potent PPAR-γ agonist, Pioglitazone exerted a regulatory role on expression of 14-3-3 genes where it upregulated 14-3-3 gamma, epsilon, zeta and tau by 3.8, 5.2, 2.7 and 739 folds, respectively. However, it had a negative regulatory effect on 14-3-3 beta, sigma and Eta by 16.94, 4.58 and 2.12 folds, respectively compared with control cells. These results correlated with growth arrest and a great increase in BRCA1 gene expression by 1076 folds. In summary, these findings are the first time showing that PPAR-γ regulates 14-3-3 genes and raises question whether PPAR-γ ligands mediate their anticancer effects via regulation of 14-3-3 proteins.

Minhee Ku is attending doctoral course at Brain Korea 21 Plus Project for Medical Science, Yonsei university. During this period she’s studying metastatic potential of cancer based on enzymatic activity.

Biological optical imaging in the NIR window includes strategies for diagnostic imaging of solid tumors, separation from autofluorescence enabling higher signal-to background ration and increased penetration into tissue than visible light. The use of NIR fluorescent cyanine dyes (Cy7) has desirable NIR optical properties and we developed a near-infrared (NIR) in vivo imaging probe for simultaneous metastatic tumor targeting and fluorescence imaging. The metastatic cell phenotype was considered as essential information in multiple tumor types. Among the many extracellular proteases, Membrane-type matrix metalloproteinase 1 (MT1-MMP) is considered key promoters in extracellular matrix (ECM)-degrading membrane protrusions of cancer cells. Therefore, the sensitive imaging of proteolytic enzyme activity and presence is important for prediction of the metastatic ability cancer cells. In this study, a fluorogenic sensing probe was fabricated by conjugation of Cy7 modified MT1-MMP specific cleavable peptides with gold nanorods (GNRs). GNR provide a versatile method for NIR photoabsorption and nanoparticle surface energy transfer (NSET). We have demonstrated metastatic tumor selectivity of NIR optical imaging probes based on proteolytic activity of MT1-MMP in vitro and in vivo. We conclude the potential benefits of NIR optical imaging probe for studying metastatic properties by tumor detection, biomarker visualization, and molecular activity. This work was supported by the Brain Korea 21 PLUS Project for Medical Science, Yonsei University

Jun-Beom Park was graduated from Seoul National University, Republic of Korea with DDS degree. He has completed his PhD from Seoul National University. He has postdoctoral experience at University of Michigan. He is Clinical Assistant Professor at The Catholic University of Korea, Republic of Korea. His research focuses on the mesenchymal stem cells.

Background: Recently, cell-based approaches have been applied in tissue engineering. Stem cells are reported to produce growth factors including nerve growth factor, vascular endothelial growth factor and hepatocyte growth factor. Stem cells are usually applied after they are expanded and/or differentiated. In this approach, there is risk of immune reaction and possibility of rejection if host’s own cells are not used, even though stem cells are reported to have immune modulatory function. Thus, the co-delivery of stem cells in immune protective device was suggested. The aim of this study is to fabricate the microcapsules loaded with stem cells and to test the stability and viability of the stem cells. Materials and methods: Microcapsulation of stem cells was performed using an electrostatic bead generator. The stability of the microcapsules was tested under mechanical stress. The viability of stem cells was determined by the Live/Dead® viability test kit. Results: The capsule showed relatively uniform size with diameter of 350-500µm. Light microscopy of encapsulated stem cells revealed that most cells were located toward the periphery with only a few cells located at the center. The shape of most microcapsules under mechanical stress was maintained up to the final evaluation point of two hours. The viability of stem cells was maintained up to two weeks of the culture as ascertained by Live/Dead® test. Conclusions: This preliminary study gives the possibility of microencapsulation technology to deliver stem cells. Further study is needed to evaluate the feasibility of this approach.

Hye Min Choi has completed her M.D. and Ph.D. at Korea University School of Medicine. She has completed her residentship in the department of Internal medicine from Korea University hospital, and worked as a basic science researcher in the department of Internal medicine of Michigan University in US between 2008~2009. After finishing the fellowship in the division of Nephrology of Korea University, now she works as an assistant professor in Seonam University Medical School Myongji Hospital in Korea.

Background: It is believed that patients with neurogenic bladder (NB) have a significantly higher risk of developing chronic kidney disease (CKD) than the general population. However, data are limited except a few studies that examined the incidence of renal failure in spina bifida or myelomeningocele in pediatric patients. In addition, serum creatinine is not a reliable marker for renal function in NB patients because they present muscle wasting due to disuse or denervation. We examined the prevalence of CKD in NB patients from spinal cord injury using biomarker cystatin-C, and the risk factors for progression to CKD. Methods: This was a cross sectional study in Korea workers’ compensation & welfare Hospital, which is a specialized center for patients from industrial accident. Patients with NB were under regular examination including regular laboratory test and urologic study such as urodynamic study, Sonography and voiding cystourethrography. Patients who visited urology department for routine check-up underwent additional measurement of serum cystatin-C for 3 months. Results: Serum cystatin-C was checked in 314 patients (mean age 58.1±8.8 yr, mean time period after injury 18.9±9.3yr). Detrusor hyperreflexia and areflexia accounted for 66.0% and 22.2%, respectively. The overall prevalence of CKD, defined as estimated glomerular filtration rate (eGFR) <60ml/min/1.73m2 was 22.4% and 8.0% by cystatin-C and creatinine-based CKD-EPI equation, respectively, and was greater than in age-matched general population (Korean National Health and Nutritional Examination Surveys). Initial eGFR, co-morbid diabetes, proteinuria, bladder volume and the presence of recurrent urinary tract infection (UTI) were the independent risk factors for CKD development in the multivariable analysis while different bladder emptying modalities was not significantly associated. Conclusions: The prevalence of CKD is higher in NB patients than in the general population and cystatin-C was more sensitive than creatinine for detecting CKD in NB patients. Co-morbid diabetes, proteinuria, bladder volume and the presence of recurrent UTI seem to be the important risk factors for development of CKD in NB patients.

After getting the PhD degree of Pathology and Pathophysiology in Shantou University Medical College (SUMC), China, Dr. Ting Long serves as an associate professor in Department of Physiology at SUMC. He dedicates in diabetes associated male sexual dysfunction studies investigating the mechanism of diabetic erectile dysfunction and sexual behavior disorder. There were several TNF-α related publication recently which suggests that TNF-α is both a significant molecular biomarker and highly potential target in diabetes associated male sexual disorder.

Background: Sexual dysfunction, including decreased libido, sexual behavior disorder and erectile dysfunction (ED), is common in male patients with diabetes mellitus (DM). We previously demonstrated that increased peripheral tumor necrosis factor alpha (TNF-α) expression, associated with inflammation in DM, contributes to ED in the rat corpus cavernosum. However, the role of TNF-α in the central pathophysiology of DM-associated male sexual dysfunction is unknown. In this study, we examined the effects of TNF-α inhibition, i.e. etanercept (ETN) via chronic intra-cerebroventricular (ICV) infusion on nNOS expression in the hypothalamic paraventricular nucleus (PVN) and sexual behavior disorder in male diabetic rats.
Results: Male diabetic rats with ICV aCSF treatment displayed significantly severe sexual disorder accompanied with blunted nNOS expression and activity in the PVN in addition to local upregulated TNF-α and TNFR-1 expression, and increased ROS generation compared with non-diabetic controls. The sexual behavioral parameters were significantly improved in the treated group with ETN. ICV ETN significantly inhibited TNF-α and TNFR-1 expression and reduced ROS generation in the PVN in diabetic rats. In addition, ICV ETN appeared to induce marked increased in nNOS expression in the PVN of diabetic animals compared with ICV aCSF-treated diabetic rats.
Conclusion: Increased TNF-α and TNFR1 expression in the hypothalamic PVN associated with DM contributes to male sexual disorder by centrally inhibiting nNOS expression and activity in the PVN via promoting local ROS generation. Central TNF-α blockade may have beneficial effects on the male sexual disorder in diabetes through improvement of NO pathway within the PVN.

Lei Shang has completed his PhD at the age of 28 years from Central South University. He is the research worker of Hunan Cancer Hospital in the department of Translational Medicine Research, a premier Cancer Research Institute in Central South Region of China. He has published more than 10 papers in reputed journals.

Necroptosis is an important neuronal death mode in retinal ischemia, but the mechanism still needs clarify. RIP3 is characterized as an N-terminal Serine/Threonine kinase, which participates in cell death signaling. Previous studies indicated RIP3 may participate in neuronal necroptosis, and the activation of caspase-8 could cleave RIP3 to inactive form. In the present study, we explored the effects of RIP3 in retinal necroptosis following elevated hydrostatic pressure (EHP) and discussed the possible role of caspase-8 on regulation of RIP3 activity. Necrosis levels detection were repeated with pretreatment of Nec-1 of 24h to confirm the existence of necroptosis. The expression of RIP3, downstream molecules in the pathway of RIP3-induced necroptosis and necrosis levels of RGC-5 cells were detected by immunoblotting, immunofluorescence and flow cytometry at 6h, 12h or 24h after EHP. Then, RNAi to rip3 was used for further confirming RIP3’s effects on retinal necroptosis. Finally, caspase-8 inhibitor and activity peptide were applied to try to unveil the regulated mechanism of RIP3 activity. The results showed that, RIP3 expression was up-regulated and RIP3 enhance-labeled cells were coexisted with PI-positive cells after injury. PI-positive cells were reduced and ratios of necrosis were decreased after injury when treating with Nec-1and rip3 RNAi. The ROS and PYGL levels in pathway of RIP3-induced necroptosis had been found to be decreased after rip3 knockdown. Caspase-8 inhibitor and activity peptide usage affected ratio of necrosis and levels of ROS or PYGL. Our results indicated RIP3 participated in RGC-5 necroptosis following EHP and caspase-8 may interference RIP3-induced necroptosis. This work was supported by the National Natural Science Foundation of China (No.81070729), and National Key Technologies Research and Development Program of China(No. 2012BAK14B03) .

Xiaolin Lu has completed her MM at the Capital Institute of Pediatrics in 2007, and became a young researcher at Beijing Municipal Key Laboratory of Child Development and Nutriomics, Capital Institute of Pediatrics.The main work of her research is about the interaction of genetics and epigenetics in the mechanism of birth defects. Since 2013, she has published about 2 articles on genetic and epigenetic alternations in birth defects as the first author and joint first author.

SHH signaling pathway played an important role in the formation of dorsal ventral neural plate. It has been shown in mice that genes of SHH signaling pathway mutations increased the incidence of NTDs, such as spina bifida and brain anomalies. Moreover, the single nucleotide polymorphisms (SNPs) of several key genes belonging to SHH pathway have been verified to increase the risk of NTDs in our previous studies. GLI2 is a key mediator of the Sonic hedgehog (Shh) signaling pathway and plays an important role in neural tube development during vertebrate embryogenesis; however, the role of gli2 in human folate-related neural tube defects remains unclear. In this study, we compared the methylation status and polymorphisms of gli2 between spina bifida patients and a control group to explore the underlying mechanisms related to folate deficiency in spina bifida. No single nucleotide polymorphism was distributed significantly differently between the two groups, although gli2 methylation levels were significantly increased in spina bifida samples, accompanied by aberrant GLI2 expression. Moreover, a significant negative correlation was found between the folate level of brain tissue and the gli2 methylation status (r=−0.41, P=0.014), while gli2 hypermethylation increased the risk of spina bifida with an odds ratio of 12.45 (95% confidence interval: 2.71–57.22, P=0.001). We also used a cell model to illustrate the effect of gli2 expression and the accessibility of chromatin affected by methylation. High gli2 and gli1 mRNA expression was detected in 5-Aza-treated cells, while gli2 hypermethylation resulted in chromatin inaccessibility and a reduced association with nuclear proteins containing transcriptional factors. More meaningful to the pathway, the effect gene of the Shh pathway, gli1, was found to have less expression along with decreased expression of gli2 in the cell model. Aberrant high methylation resulted in the low expression of gli2 in spina bifida, which was affected by the change in chromatin status and the capacity of transcription factor binding.

Zhen Wang has completed her MM at the Capital Institute of Pediatrics in 2012, and became a young researcher at Beijing Municipal Key Laboratory of Child Development and Nutriomics, Capital Institute of Pediatrics. The main work of her research is about the interaction of genetics and epigenetics in the mechanism of birth defects. Since 2012, she has published about 4 articles on genetic and epigenetic alternations in birth defects as the first author or joint first author.

SHH signaling pathway played an important role in the formation of dorsal ventral neural plate, Neural tube defects (NTDs) were caused through the overactive of SHH signaling pathway. It has been shown in mice that genes of SHH signaling pathway mutations increased the incidence of NTDs, such as spina bifida and brain anomalies. However, the relationship between the single nucleotide polymorphisms (SNPs) of SHH signaling pathway and neural tube defects NTDs remains unclear. A case-control study was designed; NTDs and control samples were obtained from the Lvliang area of Shanxi Province in northern China. Polymorphisms were genotyped for 187 NTD and 212 control samples and genotyping was conducted with a MassArray high-throughput DNA analyzer. The G allele of rs357564 in PTCH1 increased the risk of NTDs especially spina bifida. The A allele of rs12132032 in PKA signifcantly increased the incidence of NTDs especially anencephaly. The AG allele of rs10786691 in SUFU was related with NTDs and encephalocele. The C allele of rs3824 in SMO increased the risk of spina bifda. High methylation levels around rs357564 were detected in the control group with the G phenotype of the site in PTCH1. Data shown here implying that the gene polymorphisms of SHH signaling pathway maybe a potential risk factor for NTDs in our study population, and the SNP may have an impact on surrounding methylation status.

Jeng-Long Hsieh had completed her Master degree from Rutgers University in United States and PhD degree from National Cheng Kung University Medical College in Taiwan. She is a former Dean of College of Medicine and Life Science, Chung Hwa University of Medical Technology, and now a professor in the Department of Nursing. Her research focus on two fields, cancer gene therapy and molecular factors in bone related diseases. She has published 20 papers in reputed journals and has been serving as a reviewer of repute.

De Quervain’s disease, or stenosing tenosynovitis of the first dorsal compartment of the wrist, is a common ailment. How estrogen is involved in this disease is not clear. We previously showed that inflammation was involved in the pathogenesis of de Quervain’s disease. In this study, the expression of estrogen receptor (ER)- is further examined to delineate the possible roles of estrogen in this disease. Postoperative retinaculum samples were collected from 16 patients with de Quervain’s disease. The specimens were histologically graded by collagen structure. They were immunohistochemically evaluated by quantifying the expression of ER-, interleukin (IL)-1, IL-6, cyclooxygenase (COX)-2, vascular endothelial growth factor (VEGF), and Von Willebrand’s factor (vWF). De Quervain’s disease occurs primarily in women. The female:male ratio in our study was 7:1. ER- was detected in the retinaculum and its expression increased with the grade of the disease and the age of the patient. Moreover, disease severity was related to the inflammatory cytokines IL-1 and IL-6, the inflammatory enzyme COX-2, and the angiogenic factors VEGF and vWF in the tenosynovial tissue. The severity of de Quervain’s disease is associated with increased ER- expression, tissue inflammation, and angiogenesis. ER- might be a useful target for treating de Quervain’s disease.

I has completed his PhD at 2006 from University of Edinburgh. I have published more than 8 papers in different journals. I am doing fellow at HUMS as a student at the moment and I would like to present my work in your conferences as poster.

Stress represents a wide range of physical responses and plays an important role in modulating different stages of memory including reconsolidation. Relapse to drug taking induced by exposure to stimuli or cues associated with drugs of abuse is a main challenge to the treatment of morphine addiction.We examined the effects of forced swim stress and corticosterone on reconsolidation of a drug-related memory using a conditioned place preference (CPP) procedure. The results showed that animals acquired morphine CPP after conditioning, and that this CPP was inhibited by stress given immediately after re-exposure to morphine. Corticosterone injection which leads to immediate and sudden increase in the level of glucocorticoids fails to reinstate the terminated CPP in rats. Studies on FSS-induced reinstatement show that this behavior is blocked by administration of corticotropin-releasing hormone (CRH) antagonists. Overall, these results suggest that corticosterone plays an important role in relapse to drug seeking behavior induced by stress.

Site specificity of anti-neoplastics still poses a challenge in the pharmaceutical research. Conventional chemotherapy has limitations such as site in-specificity, inability of the drug to penetrate inside the tumor, adverse effects there by reducing the clinical application. So, in this study oxaliplatin solid lipid nanoparticles (OP-SLN) were prepared by micro emulsion method using various ratios of lipid and then covalently conjugated to TRAIL monoclonal antibody (TR-OP-SLN) for targeting colorectal cancer cells by receptor mediated internalization of drug. The prepared Immuno nanoparticles were characterized for Fourier Transform Infrared spectroscopy (FT-IR), Differential scanning calorimetry (DSC), X-ray diffraction (XRD), particle size, scanning electron microscopy (SEM), surface charge, fluorescence intensity and in vitro drug release. These were further characterized for in vitro cytotoxicity (in HT-29 cells) followed by cellular uptake and internalization by flow Cytometry along with protein assay. The optimized OP-SLN3 has shown an appreciable particle size (121±1.22nm), entrapment efficiency (78±0.09%), drug loading (32±1.01%) along with spherical surface morphology. TR-OP-SLN has shown a drug release of 81±0.01% and 27±0.12% at pH 4.5 and 7.4 respectively further confirming its ability to release the drug inside the tumor. Fluorescence study confirmed the presence of the antibody on the surface of the nanoparticles by change in their intensity. A 1.5 fold increase in cytotoxicity of immuno nanoparticles (7.5Ug/ml or more) was observed. Cellular uptake studies have shown 89% of immuno nanoparticles uptake by the cells and Immuno nanoparticle are majorly internalized in the perinuclear region. In conclusion, we demonstrate a preparation and characterization of oxaliplatin immuno nanoparticles for receptor mediated targeting of the drug.