international society for enzymology annual meetingise.biol.uoa.gr/files/ise presentation...

International Society for Enzymology Annual Meeting

2

22th ISE International Conference All Aspects of Laboratory Medicine

June 30 – July 3, 2018 Naxos Island, Greece

ISE 2018

Under the auspices of the:

National and Kapodistrian University of Athens


3

Acknowledgements:

• Commission of the European Community and General Secretary for

Research & Technology, Greece, through the INsPiRE project (EU-FP7-

REGPOT-2011-1, proposal 284460).

• Special Account for Research Grants, National and Kapodistrian University

of Athens.


4

Organizing Committee: Diamandis E.P, Scorilas A

Local:

Scorilas A., Avgeris M., Kontos C.K,

Adamopoulos P.G, Diamantopoulos M., Panoutsopoulou K.,

Papachristopoulou G., Tsiakanikas P.

International Scientific Committee:

Adeli K. (Toronto, Canada)

Acan L. N. (Ankara, Turkey)

Boyages S.C. (Sydney, Australia)

Brandslund I. (Vejle, Denmark)

Diamandis E.P. (Toronto, Canada)

Garbis S. (Southampton, UK)

Jarolim P. (Boston, USA)

Oellerich M. (Gottingen, Germany)

Plebani M. (Padova, Italy)

Prassas I. (Toronto, Canada)

Salvatore F. (Naples, Italy)

Scorilas A. (Athens, Greece)

Fahnestock M. (Hamilton, Canada)

Yousef G.M. (Toronto, Canada)


5

Dear Colleagues

Following the successful International Society for Enzymology Annual Conference

2017 in the island of Santorini, Greece, we are now announcing the next ISE Annual

Conference 2018. The conference will take place in the nice island of Naxos, Greece,

between the dates Saturday June 30 - Tuesday July 3, 2018. Naxos is one of the most

beautiful and interesting places in the world. The conference will be held at the Finikas

Luxury Hotel Naxos on the Pyrgaki beach.

As usual, the guiding principles of this Annual Conference are: Top science related

to all aspects of laboratory medicine, in a wonderful location, with a limited number of

participants. The ISE2018 conference will cover a wide range of themes including, but not

limited to: New biomarkers for cancer and other diseases, informatics, automation,

genomics, proteomics, epigenomics, transcriptomics, other omics, enzymes in health and

disease, theranostics, micro RNAs, long non-coding RNAs, quality assurance, patient safety

and harmonization. Presentations on specific diseases are also welcome.

As last year, the meeting will be restricted to a maximum of 50 registrants, on a

first-come-first served basis. All participants will be responsible for their expenses and

will be invited to present on a topic of their choice as time permits, but this is not

necessary. The meeting expenses will be covered from the registration fees. Spouses and

other family members are also welcome.

Please take time to visit the old city of Naxos and its surroundings as well as some

of the island's wonderful beaches and villages. You may also combine your trip with a stop

in Athens.

We are looking forward to seeing you, your colleagues and your families in Naxos

for a wonderful scientific meeting and an overall enjoyable experience.

Information: http://ise.biol.uoa.gr

With best wishes

Sincerely,

The Organizers:

Dr. Eleftherios P. Diamandis

Dr. Andreas Scorilas

https://www.google.gr/search?q=naxos+Island&client=firefox-b&source=lnms&tbm=isch&sa=X&ved=0ahUKEwjV3_a5qMnVAhXJyRQKHUUtCG4Q_AUICigB&biw=1920&bih=924�

http://www.finikashotel.gr/�


6

Day

one

–Fr

iday

, 16th

June

201

7

15:00 -17:00 Registration

17:00 - 18:00 Opening Ceremonies Eleftherios P. Diamandis & Andreas Scorilas

History of Greek Music and of the Island Santorini (E.P Diamandis)

18:00 – 18:30 Modern Greece between East and West

Sotiris Mitralexis

18:30 – 19:00 Questions & Discussion

19:00 – 20:00 Opening Mixer (Dinner on your own)

Scientific Programme

Invited Speakers’ Abstracts

Poster Abstracts

CONTENTS


7

15:00 -17:30 Registration

17:30 - 18:00 Opening Ceremonies Eleftherios P. Diamandis & Andreas Scorilas

18:00 - 19:00 History of Greek Music and of the Island Naxos (E. P. Diamandis & I. Prassas)

19:00 - 19:30 Opening Mixer (Dinner on your own)

Saturday, 30th June 2018


8

Day

two

–Sat

urda

y, 1

7th Ju

ne 2

017

Day

thre

e –S

unda

y, 1

8th Ju

ne 2

017

Day

thre

e –S

unda

y, 1

8th Ju

ne 2

017

Day

two

–Sat

urda

y, 1

7th Ju

ne 2

017

09:30 - 09:45 Irreproducibility in science

Eleftherios P. Diamandis

09:45 - 10:00 Managing accreditation performance in

Europe

Bernard Gouget

10:00 - 10:15 Updates of Biomarkers in Prostate Cancer

Qing Meng


10:45 -11:30 BREAK (e-poster viewing)

11:30 - 11:45 The human gut microbiome: its role in

some pathological conditions

Francesco Salvatore

11:45 -12:00 Enzymes, life molecules

Enrique de la Morena

12:00 - 12:30 Questions & Discussion

12:30 -15:00 Lunch

15:00 -15:15 The five rights of Laboratory medicine

Mario Plebani

15:30 -15:45 Our last 7-8- years research in diabetes

Ivan Brandslund

15:45 -16:00 Immune based diagnostics for cancer

detection

Karen Anderson

16:00 - 16:15 Small-coding RNAs as novel tumor

biomarkers in prostate and bladder cancer

Margaritis Avgeris

16:15 -16:45 Questions & Discussion

11:45 -12:00 KLK6 proteolysis is implicated in the regulation of extracellular alpha-synuclein species and may represent a novel therapeutic approach.. Georgia Sotiropoulou

12:00 -12:15 Enhanced proteolytic activities in Acral Peeling Skin Syndrome: A role of transglutaminase 5 in epidermal homeostasis”. Dimitra Kiritsi


12:30 -15:00 Lunch

15:00 -15:15 New targeted multimodal therapeutic approaches for type 2 diabetes mellitus: the new kids on the block. Steven C. Boyages

15:15 -15:30 Therapeutic modulation of BDNF signaling in autism Margaret Fahnestock

15:30 -15:45 Discovery of novel tumor biomarkers in prostate cancer using high sensitive proteomic methodologies Spiros D. Garbis


Session I: Coordinators: Andreas Scorilas & Litsa Talieri 09:30 - 09:50. Personalized cancer biomarkers Eleftherios P. Diamandis

09:50 - 10:10. From robotization of clinical laboratories to artificial Intelligence for evaluation of test results Ivan Brandslund 10:10 - 10:30. Studies with some mycobacterial culture filtrates for immunotherapeutical activity Naciye Leyla Acan 10:30 – 11:00 Questions & Discussion


……………………………………………………………………………………

Session II: Coordinators: Ivan Brandslund & Naciye Leyla Acan 11:40 - 12:00. The Human Genome and its Role in Predictive Medicine Francesco Salvatore 12:00 -12:20. Advances in the use of GcfDNA in transplantation Michael Oellerich

Sunday, 1st July 2018


9


13:00 -15:30 Lunch

…………………………………………………………………………………

Session III: Coordinators: Michael Oellerich & Francesco Salvatore 15:30-15:50.Characterization and Free Thyroid Hormones Response of Chemically Modified Polyethylene Terephthalate Blood Collection Tubes Raffick Bowen 15:50 -16:10. Greater transparency in publishing, and why all scientific research matters. Sabina Alam 16:10 -16:40 Questions & Discussion

Sunday, 1st July 2018


10

Day

two

–Sat

urda

y, 1

7th Ju

ne 2

017

09:30 - 09:45 Irreproducibility in science

Eleftherios P. Diamandis

09:45 - 10:00 Managing accreditation performance in

Europe

Bernard Gouget

10:00 - 10:15 Updates of Biomarkers in Prostate Cancer

Qing Meng



11:30 - 11:45 The human gut microbiome: its role in

some pathological conditions

Francesco Salvatore

11:45 -12:00 Enzymes, life molecules

Enrique de la Morena


12:30 -15:00 Lunch

15:00 -15:15 The five rights of Laboratory medicine

Mario Plebani

15:30 -15:45 Our last 7-8- years research in diabetes

Ivan Brandslund

15:45 -16:00 Immune based diagnostics for cancer

detection

Karen Anderson

16:00 - 16:15 Small-coding RNAs as novel tumor

biomarkers in prostate and bladder cancer

Margaritis Avgeris


11:45 -12:00 KLK6 proteolysis is implicated in the regulation of extracellular alpha-synuclein species and may represent a novel therapeutic approach.. Georgia Sotiropoulou

12:00 -12:15 Enhanced proteolytic activities in Acral Peeling Skin Syndrome: A role of transglutaminase 5 in epidermal homeostasis”. Dimitra Kiritsi


12:30 -15:00 Lunch

15:00 -15:15 New targeted multimodal therapeutic approaches for type 2 diabetes mellitus: the new kids on the block. Steven C. Boyages

15:15 -15:30 Therapeutic modulation of BDNF signaling in autism Margaret Fahnestock

15:30 -15:45 Discovery of novel tumor biomarkers in prostate cancer using high sensitive proteomic methodologies Spiros D. Garbis


Session IV: Coordinators: Eleftherios P. Diamandis & Christos Kontos 09:30-09:50. Humility and Hubris in Sciences Nick Bouras 9:50-10:10. High-sensitive assays for Cardiac troponins (I and T) measurement. Mario Plebani 10:10-10:30. Knowledge translation in laboratory medicine George Yousef 10:30 - 11:00 Questions & Discussion


……………………………………………………………………………………...

Session V: Coordinators: Mario Plebani & George Yousef 11:40 -12:00. Obesity: Understanding its pathophysiology and what we can do to address it Steven Boyages

Monday, 2nd July 2018


11

12:00 -12:20. Brain-derived neurotrophic factor: why diet and exercise are good for your brain. Margaret Fahnestock 12:20 - 13:00 Questions & Discussion

13:00 -15:30 Lunch

…………………………………………………………………………………..

Session VI: Coordinators: Spyros Garbis, Margaret Fahnestock, Margaritis Avgeris 15:30 -15:50. Identification of biomarkers of response and toxicity to pembrolizumab, using proteomics Ioannis Prassas 15:50 -16:10. Molecular cloning of novel alternative splice variants of the human tissue kallikrein (KLK1) and kallikrein-related peptidases (KLKs), using Next-Generation Sequencing (NGS) technology Christos Kontos 16:10 -16:20. Identification of novel splice variants of the human carcinoembryonic antigen-related cell adhesion molecule 19 gene (CEACAM19). Zafeiro Zisi 16:20 -16:50 Questions & Discussion

Monday, 2nd July 2018


12

Session VII: Coordinators: Steven Boyages & Ioannis Prassas

09:30 - 09:50. Increased circulating resistin correlates with longer disease-free survival in early-onset breast cancer:Quantitative proteomics results from the POSH multi-center cohort Spyros Garbis 09:50 - 10:10. Network-based analysis for drug repurposing and biomarker discovery: applications in NAFLD, CKD, and CVD.

Leonidas Alexopoulos 10:10 - 10:30. lncRNAs as novel biomarkers in bladder cancer management. Margaritis Avgeris 10:30 - 11:00 Questions & Discussion

11:00 -11:30 CLOSING REMARKS

11:30-13:30 ISE Board meeting

Tuesday, 3rd July 2018


13

ABSTRACTS


14

Personalized cancer biomarkers ---------------------------------------- Eleftherios P. Diamandis1,2,3* ----------------------------------------

1Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada,

2Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada,

3Department of Clinical Biochemistry, University Health Network, Toronto, Ontario, Canada *email: [email protected]

---------------------------------------- The cancer biomarker discovery pipeline is progressing slowly. The difficulties of finding novel and effective biomarkers for diagnosis and management of cancer patients are well-known. We speculate that it is unlikely to discover new serological biomarkers characterized by high sensitivity and specificity. This projection is supported by recent findings that cancers are genetically highly heterogeneous. Here, we propose a new way of improving the landscape ofcancer biomarker research. There are currently hundreds, if not thousands, of described biomarkers which perform at high specificity (>90%), but at relatively low sensitivity (<30%). We call these “rare tumour markers.” Borrowing from the principles of precision medicine, we advocate that among these low sensitivity markers, some may be useful for specific patients. We suggest screening new patients for hundreds to thousands of cancer biomarkers, to identify a few that are informative, and then use them clinically. This is similar to what is currently done with genomics, to identify personalized therapies. We further suggest this approach may explain as to why some biomarkers are elevated in only a small group of patients. It is likely that these differences in expression are linked to specific genomic alterations which could then be found with genomic sequencing.

----------------------------------------


15

From robotization of clinical laboratories to artificial Intelligence for evaluation of test results

---------------------------------------- Ivan Brandslund*

---------------------------------------- Department of Biochemistry and Immunology, Lillebaelt Hospital, Vejle, Denmark.

Institute of Regional Health Research, University of Southern Denmark, Odense, Denmark. *email: [email protected]

---------------------------------------- In our Lab we conceived the idea of transporting blood samples already labeled with patient IDfrom the wards to the Laboratory in a dedicated pressurerised tube transport system, The Tempus 600. The samples are received in a continuous flow first in first out robot reception system and transferred to the GLP/Sysmex conveyor belt systems for analysis. From installing this system in 2015 we have gained a reduction in turnaround times from sampling to test result from average 2½ hours to less than 60 min. Further the system has reduced the total marginal average cost of the 100 most frequent analytical tests to 0.5 Euro. This means that our hospitals 60.000 emergency reception patients can now be offered the full analytical repertoire of 100 tests within 60 min. of admission for a cost of less than 70 Euro. The results can be processed in a big data miner program from the SAS Institute and results presented in a user friendly and a commented way to the clinician while the doctor is still examining the patient. The data will be presented as 1. Critical test results to be addressed 2. A patient risk profile 3. The most probable diagnostic possibilities. The system is developed in a coorporation with the SAS Institute, Stockholm.

----------------------------------------


16

Studies with some mycobacterial culture filtrates for immunotherapeutical activity ----------------------------------------

Zeliha Ertürk1, Esra Büber1, Haluk Özen2, Ömür Çelikbıçak3, Bekir Salih3, N. Leyla Açan1* ----------------------------------------

1Department of Medical Biochemistry, Faculty of Medicine, Hacettepe University, Ankara, Turkey

2Department of Urology, Faculty of Medicine, Hacettepe University, Ankara, Turkey 3 Department of Physical Chemistry, Faculty of Science, Ankara, Turkey

*email: [email protected] ----------------------------------------

Intravesical application of Mycobacterium bovis (BCG) is used as a gold standard for the therapy of non-muscle invasive bladder cancer. Since undesirable side effects may occur during this therapy, new approaches with less toxic mycobacterial strains are being sought. Three mycobacterial strains (M. brumae, M. chitae and M. murale), cell walls of which showed high cytotoxic activities on bladder cancer cell line were selected for this study. It is aimed to separate and investigate the culture filtrates of these strains. The separation included ammonium sulphate precipitation, acetone extraction, Sephadex G25 column chromatography and separation of the fractions by HPLC on C18 column. Each fraction was analysed by matrix assisted laser desorption/ionization mass spectroscopy. Tumour necrosis factor-alpha stimulating activities of the fractions on human monocyte cell lines were also investigated. Some of the fractions showed high cytokine stimulation activities. These findings might be useful in development of a novel drug for the therapy of non-muscle invasive bladder cancer.

---------------------------------------- Acknowledgements: This project was supported in part by the Scientific and Technical Research Institute of Turkey, project no. 105S361.


17

The Human Genome and its Role in Predictive Medicine ----------------------------------------

Francesco Salvatore1,2* ----------------------------------------

1CEINGE-Biotecnologie Avanzate, Naples, Italy 2Department of Molecular and Medical Biotechnologies, University of Naples Federico II,

Naples, Italy *email: [email protected]

---------------------------------------- Each human genome (constituted by 3.3 billion bp) is unique due to a s e rie s of nucle otide

sequence variants. This is the mechanism by which DNA variants can change the physiology of each stretch of sequence, whether a protein gene or an RNA molecule. These sequence variants are also the basis for predictive medicine since their study may reveal the effect they can have on a given phenotype. In some cases, a single variant may produce one of the so-called monogenic diseases (which however are often modulated by epistatic genes). In almost all other cases in which the variant produces a putative mutation, the carrier may be considered to have a disease predisposition, which means he/she is at risk of a given disease. Notably, the disease occurs only when the other genes in the carrier are compatible with that disease. Thanks to the advent of next-generation sequencing, increasingly more gene variant sequences are being identified. Indeed, this technology produces an increase of analytical sensitivity, which makes the approach to disease prediction more efficient. This technology is now moving towards “third-generation sequencing” that can sequence a single-strand of DNA measuring up to 150,000 base pairs, or even more. Therefore, the effects that this enormous number of sequences have on the phenotype of an individual must be interpreted in order to make an early estimate of disease prediction risk and thus to plan preventive measures in each individual examined. The functional analysis of the variants in protein genes are performed in silico on a series of softwares and pipelines, which are giving increasing support to their possible deleterious effects. However, only in vitro and in vivo experimental approaches can improve these results. I will present a few examples regarding cardiopathies (both in different types of RNA splicing and regarding cell electrophysiological patch-clamp measurements), and in DNA repair mechanisms in breast cancer. Notably, a variant may be designated “pathogenetic”, “likely pathogenetic”, “of uncertain significance”, “likely benign” or “benign”. As a sequencing approach, in our laboratory we have constructed about 25 gene panels, several of which are made of more than 100 genes for a total of over 1,000 genes. The gene panels are designed to distinguish between similar confounding diseases, or in some cases constructed on the basis of costs. The latter panels range from more frequently mutated genes that have a high diagnostic sensitivity (i.e., cardiopathies), to second-tier panels, namely panels containing less frequently mutated genes. Lastly, whole exome sequencing (WES), clinical exomes and even whole genome sequencing (WGS) are starting to be used as alternatives to gene panels. The knowledge and understanding of variant functions in a gene is giving increasingly more accurate information about personal prediction based on genome sequencing, so that many scientists suggest that each individual should ask for his/her entire DNA sequence. In conclusion, this will give a complete picture of predicting personalized information based on the genome, that increasingly furnishes the possibility of utilizing that information to effectively monitoring at a very early stage many types of diseases.

----------------------------------------


18

Advances in the use of graft-derived cfDNA in transplantation ----------------------------------------

Michael Oellerich* ----------------------------------------

Institute for Clinical Pharmacology, University Medical Center Goettingen, Goettingen, Germany


Because traditional methods to assess transplant organ damage are imprecise, new biomarkers for noninvasive monitoring of graft integrity are needed to personalize immunosuppression. In a recent prospective study of 157 lung transplant recipients, allograft injury was detected by elevated GcfDNA a median of 2.8 months before the clinical diagnosis of ABMR (antibody-mediated rejection) using spirometry or histopathology and these GcfDNA elevations were associated with concurrent increases in DSA levels (1). In another multicenter study of 102 kidney transplant (KTx) recipients, GcfDNA percentages discriminated between patients whose biopsy specimens showed a biopsy-proven acute rejection (BPAR) and biopsy negative controls (diagnostic accuracy: 87%). Positive and negative predictive values for ABMR at a cut-off of 1% GcfDNA were 44% and 69%, respectively (2). In a prospective observational trial, our group evaluated both GcfDNA % and absolute quantification of GcfDNA copies (GcfDNA cp/mL) in plasma collected at pre-specified visits in 76 KTx recipients followed for at least one year post transplantation (3). In patients (N=14) with samples (n=21) drawn during BPAR periods, median GcfDNA cp/mL was 5-fold and median GcfDNA % 2.6-fold higher than the medians observed in samples (n=267) from 62 clinically stable patients without rejection. Diagnostic accuracy was 88% for GcfDNA cp/mL, and 79% for GcfDNA %. Diagnostic sensitivity was 89% for GcfDNA cp/mL, and 84% for GcfDNA %. Diagnostic specificity was 78% for GcfDNA cp/mL, and 74% for GcfDNA %. The threshold at maximum Youden Index was 37 for GcfDNA cp/mL, and 0.43 for GcfDNA %. The correlation between GcfDNA cp/mL and plasma creatinine was 0.37. In a selected patient subgroup (N=24) without clinically suspected rejection and a change of tacrolimus concentrations > 60% in samples (n=75) collected at ≥ 3 consecutive visits, there was a negative correlation (r= - 0.46) between tacrolimus and GcfDNA cp/mL. This finding suggests that GcfDNA may detect silent graft damage due to under-immunosuppression that could increase the risk of de novo DSA formation and subsequent graft loss. Results showed that absolute GcfDNA quantification allowed for better discrimination than GcfDNA % of KTx recipients with acute rejection and graft injuries, presumably due to lack of influence from recipient DNA changes (e.g. infection, exercise).

----------------------------------------

1. Agbor-Enoh S et al. (2018) J Heart Lung Transplant; in press. 2. Bloom RD et al. (2017) J Am Soc Nephrol 28: 2221-2232. 3. Oellerich M et al. (2018) Clin Chem Annual Meeting Abstracts; submitted


19

Characterization and Free Thyroid Hormones Response of Chemically Modified Polyethylene Terephthalate Blood Collection Tubes

---------------------------------------- R. A. R. Bowen1*, E. Jalali Dil2, S. C. Kim3, A. Saffar2, A. Ajji2, R. N. Zare1,

A. Sattayapiwat1, V. Esguerra1

---------------------------------------- 1Stanford University, Stanford, CA, USA

2Ecole Polytechnique de Montreal, Montreal, QC, Canada 3University of California, San Francisco, San Francisco, CA, USA

*e-mail: [email protected] ---------------------------------------

Background: Blood collection tubes (BCTs) are not inert specimen carriers, as shown by some studies reporting significant differences in serum hormone concentrations collected in polyethylene terephthalate (PET) BCTs due to surfactants. We developed chemically modified BCTs (chemoPETs) that have an interior surface similar to glass BCT and lacks surfactant(s). Objectives: To characterize and compare unmodified PET and chemoPET film surfaces to determine if the latter is significantly hydrophilic and to determine if the there are significant biases in serum free triiodothyronine (FT3) and free thyroxine (FT4) concentrations collected in chemoPET and other PET BCTs compared to glass tubes. Materials and Methods: PET and chemoPET film surfaces were characterized by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), and scanning electron microscopy (SEM). The PET film contact angles and surface energy were determined with liquid probes. Thromboelastography (TEG) was used to evaluate and compare the thrombogenicity of the inner walls of various types of BCTs. For serum specimens, one glass (control BCT) and five plastic (Vacuette™, SST™, RST™, plain red-top, and chemoPET) BCT types were examined. FT3 and FT4 concentrations in the different BCTs were measured on an Immulite 1000 instrument and compared to glass tubes. Results: XPS results indicate that the carbon/oxygen ratio on the PET film surfaces decreased from 2.9 for an unmodified PET to 2.1 for chemoPET surfaces. ToF-SIMS shows a higher peak intensity of oxygen and hydroxyl groups on the chemoPET compared to unmodified PET film surface. SEM demonstrated that the chemoPET film surface was smooth without any holes. Water contact angle measurements show a decrease from 70° for an unmodified PET to 44° for chemoPET film surfaces, and the surface energy and polarity increased from 37.9 to 51.9 (mJ/m2) and 25% to 71%, respectively.TEG tracings from various BCTs demonstrated differences in the reaction and coagulation times but not in clot strength. It was determined that the interference of chemoPET tubes be negligible compared to that of commercially available PET BCTs. Conclusion: BCTs with no surfactants coated to its interior tube wall surface may be developed to eliminate its potential interference with hormone immunoassays.

---------------------------------------


20

Greater transparency in publishing, and why all scientific research matters ----------------------------------------

Sabina Alam* ----------------------------------------

Faculty of 1000, Middlesex House, 34-42 Cleveland St, London, UK *email: [email protected] ----------------------------------------

The landscape of scholarly publishing and peer review has been evolving, with manymodels trying to address the issues around delays, editorial bias, issues aroundaccess, lack of transparency and irreproducible research. Although open accesspublishing allows global access, and different peer review models at variousjournals are being tested to determine if there are any ‘best’ solutions, none haveso far adequately addressed the problems outlined above.The post-publication peer review model pioneered by F1000Research, and now adoptedby Wellcome Open Research, Gates Open Research and other funder or institutionpublishing platforms operates a model of publishing that aims to address all of these issues. By removing focus from impact factors, the publishing model insteadfocuses on removing bias by giving space for all types of research outputs to bepublished, including null findings or replication studies. The model encourages thepublication of study protocols and supports registered reports, and also allows fordata notes and method articles to be published as separate papers. To encouragetransparency and reproducibility, the publishing model mandates the sharing of datathat support all findings presented in a paper.To support speed, the articles undergo post-publication invited open peer review,which allows articles to be shared in a ‘preprint’ form first and then peer-reviewed transparently. In this way, articles can become ‘living’, so that even indexedarticles can continue to be updated and peer-reviewed again when needed.

----------------------------------------


21

Humility and Hubris in Sciences ----------------------------------------

Nick Bouras* ----------------------------------------

King’s College London, Institute of Psychiatry, Psychology and Neuroscience *email: [email protected] ----------------------------------------

There has been an increasing awareness of the importance of leadership and decision making, including scientists and academics, over recent times. By whom and how decisions are made can have serious implications across all levels of society. Several people have been successful in their life and have been inflicted by excessive pride and self-confidence. There are times when the manifestations of such behaviours demonstrate noticeable signs of narcissism and on extreme cases, hubris. Hubris is an old concept originated from the Greek mythology. The risk of hubris affects politicians, leaders in business, scientists, academia, the military, entertainers, athletes and doctors (among many others). Power, especially absolute and unchecked power, is intoxicating and is manifested behaviourally in a variety of ways, ranging from amplified cognitive functions to lack of inhibition, poor judgment, extreme narcissism, deviant behaviour, and even cruelty. Hubristic behaviour of overconfidence, extreme pride together with an unwillingness to disregard advice makes powerful people in leadership positions to over-reach themselves with negative consequences for themselves and others. Responsible leaders, including acclaimed scientists should exercise greater humility to the complexity and inherent uncertainty of their activities and strive to seek out and challenge hubristic behaviours.

---------------------------------------- Eleftherios P Diamandis and Nick Bouras: Hubris and Sciences. F1000Research 2018, 7:133


22

High-sensitive assays for Cardiac troponins (I and T) measurement. ---------------------------------------

Mario Plebani* ---------------------------------------

Department of Laboratory Medicine, University-Hospital of Padova, Padova – Italy *email: [email protected]. ---------------------------------------

The international guidelines “on the Redefinition of AMI”, issued in the year 2000 by the Joint European Society of Cardiology/American College of Cardiology Committee, recommended that an increase in cardiac troponin I (cTnI) or cTnT levels outside the 99th percentile upper reference limit (99th URL) should be considered clinically relevant, and indicated that this cut off value should be measured with an imprecision of 10 CV%. Subsequently the guidelines for the Universal Definition of Myocardial Infarction, edited by the joint ESC/ACCF/AHA/WHF task force in 2007 and 2012, confirmed that cTnI and cTnT are the preferred biomarkers for the differential diagnosis of acute coronary syndrome (ACS), and also that the 99th URL value should be measured with an imprecision of 10 CV%. However, measurement of the 99th URL of cTnI and cTnT levels is a challenging analytical issue due to low biomarker concentrations present in healthy subjects. Only after 2006 some manufacturers set-up the first new generation of cTnI and cTnT immunoassays with improved analytical sensitivity in accordance with the quality specifications indicated in international guidelines and consensus documents. Importantly, highly sensitive methods should also be able to measure troponin levels in the majority of healthy adults subjects (50%), as suggested in some consensus documents. In order to measure the extremely low circulating levels of the biomarker in all healthy subjects, a highly sensitive method for cTnI or cTnT should have a low detection limit greatly lower than 5 ng/L. The main advamntages of high-sensitive assays for measuring cTn (T and/or I) should be summarized as follows: a) higher analytical and diagnostic sensitivity; b) eraly diagnosis of ACS/AMI, thus avoiding the need of an “early” biomarker; c) adoption of biochemical algorithms based on cTn measurement (from 0-3 to 0-2 and 0-1 hours and even undetectable cTn at o time), d) better prognostic value to be used for disease stratification; e) better utilization for personalized diagnostic and therapeutic purposes. However, there are still some open questions about: a) definition of the 99° percentile as the criteria and number fpr reference subjects enrollment are under discussion; b) clinical usefulness of cut off age- and gender-related; c) value of the “delta” (difference between two serial results; d)biological variation and reference change value (RCV) of cTnI/T.

---------------------------------------


23

Knowledge translation in laboratory medicine ---------------------------------------

George M Yousef* ---------------------------------------

Department of Laboratory Medicine and Pathobiology, University of Toronto, Canada *email: [email protected]

--------------------------------------- Knowledge translation is defined as the dynamic interactive process that includes synthesis, dissemination, exchanges, and ethically sound application of knowledge to improve health and provide more effective health services and products, and to strengthen the health care system. In translating discovery into bedside, a number of issues need to be addressed. At the technical level, challenges include the interpretation of advanced technology results like next generation sequencing (for instance the type of SNIP or mutation whether somatic or gremlin). Another important issue is the presence of evidences that support clinical relevance. Ethical considerations especially for incidental findings and the genetic variants of unknown significant should be also considered. Also there is an urgent need for standardization and to ensure reproducibility among different platforms and technologies. Another important issue that should be addressed is the knowledge and skills of health care providers. In general, there is a lack of genomic knowledge among physicians and laboratory professionals. There is also a need for clear guideline that enables counseling patients about genetic risk of diseases with confidence. Added to this is the lack of uniform access to genetic services, and the time needed for proper interpretation and counseling. Moving forward, to enhance the synthesis of knowledge, there should be continuos updating then contextualization and integration of individualized research finding. Processing and exchange of this overwhelming and exponentially increasing amount of molecular results requires a new era of collaboration between knowledge producer and decision makers.

---------------------------------------


24

Obesity: Understanding its pathophysiology and what we can do to address it ---------------------------------------

Steven C. Boyages* ---------------------------------------

Department of Endocrinology, Westmead Hospital, Sydney, Australia The University of Sydney, Sydney, Australia

*email: [email protected] ---------------------------------------

Excess weight, especially obesity, is a major risk factor for cardiovascular disease, type 2 diabetes, some musculoskeletal conditions and some cancers. As the level of excess weight increases, so does the risk of developing these conditions. In addition, being overweight can hamper the ability to control or manage chronic conditions. The prevalence of obesity is rising rapidly in developed as well as developing countries.Worldwide obesity has nearly tripled since 1975.In 2016, more than 1.9 billion adults, 18 years and older, were overweight. Of these over 650 million were obese.39% of adults aged 18 years and over were overweight in 2016, and 13% were obese.Most of the world's population live in countries where overweight and obesity kills more people than underweight.Obesity is treatable and its consequences preventable. This paper will discuss individual management strategies of nutrition, activity, medical and surgical therapies for the treatment of the disorder. Finally, public health measures to prevent obesity will be discussed.

---------------------------------------


25

Brain-derived neurotrophic factor: why diet and exercise are good for your brain ---------------------------------------

Margaret Fahnestock* ---------------------------------------

Department of Psychiatry and Behavioural Neurosciences, McMaster University, Hamilton, ON, Canada


Brain-derived neurotrophic factor (BDNF) is an activity-dependent protein that is essential for synaptic plasticity and learning and memory. Cortical and hippocampal BDNF levels are reduced in aging and Alzheimer’s disease and correlate with memory scores. We and others have shown that raising brain BDNF levels improves cognition. We have used rodent and dog models to demonstrate that environmental enrichment (including exercise) interacts with diet to yield higher BDNF levels and better cognition than either intervention alone. Most recently, we demonstrated in healthy young human subjects that exercise alone selectively increases performance in a high-interference memory task that is linked to hippocampal function. Those who exhibited greater fitness improvements also showed higher BDNF levels and better memory performance when exercise was combined with cognitive training, suggesting that synergistic effects may depend upon BDNF.

---------------------------------------


26

Identification of Biomarkers of Response and Toxicity to Pembrolizumab, Using Proteomics

--------------------------------------- Ioannis Prassas*

--------------------------------------- Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Canada


Immune checkpoint blockade (ICB) is a breakthrough form of cancer immunotherapy, but the lack of sensitive and specific predictive biomarkers of response and toxicity have limited its efficacy. Predictive biomarkers are non-invasive molecular tools that can provide valuable information about a patient’s response to a treatment. Cancer immunotherapy has several significant limitations, including extremely high cost, potential severe toxicity from immune-related adverse effects (IRAEs), and a limited success rate (10-40%). ICB therapy employs antibody-targeting of specific inhibitory receptors and ligands that are overexpressed by cancer cells, such as programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte associated antigen 4 (CTLA-4). IRAEs induced by ICB can range from mild dermatitis to serious colitis, as well as endocrine inflammatory events. Predictive biomarkers of ICB are not currently widespread in clinical use, despite the growing need for a personalized approach to cancer treatment. It has been challenging to find novel cancer biomarkers that are sufficiently sensitive and specific, in part due to the genetic heterogeneity of tumors. Effective immunotherapy causes tumor cell death, which releases tumor-associated antigens (TAAs) into circulation. This results in abnormal presentation of these antigens to immune cells, leading to B-cell autoantibody production against them. Our research hypothesis is that responders to pembrolizumab (anti-PD-1) will develop high titers of serum autoantibodies, indicative of a strong humoral immune response to TAAs released during immunotherapy. Non-responders will have low levels of these autoantibodies, due to a weaker or non-existent anti-tumor and humoral immune response. Secondly, patients who develop serious toxicity from immunotherapy will also develop several autoantibodies indicative of an autoimmune-like response. Our goal is to discover sensitive and specific autoantibody biomarkers early in treatment, which have the potential to predict if a patient will respond favorably to therapy, as well as identify patients who are at risk of developing toxicity to treatment. Towards this goal, we will perform a proteome-wide search for serum autoantibodies against proteinaceous antigens to find potential serum biomarkers of response and toxicity to pembrolizumab. The screening process will consist of patient serum samples from responders, non-responders and patients with toxicity to pembrolizumab treatment, collected from the “INSPIRE” clinical trial at Princess Margaret Hospital.

---------------------------------------


27

Molecular cloning of novel alternative splice variants of the human tissue kallikrein (KLK1) and kallikrein-related peptidases 2, 5 – 12 (KLK2, KLK5 – KLK12), using Next-

Generation Sequencing (NGS) technology ----------------------------------------

Christos K. Kontos, Panagiotis G. Adamopoulos, Andreas Scorilas* ----------------------------------------

Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, Athens, Greece


Alternative splicing of cancer-related genes is a common cellular mechanism accounting for cancer cell transcriptome complexity and affecting cell cycle control, proliferation, apoptosis, angiogenesis, invasion, and metastasis. In this study, we describe the discovery and molecular cloning of novel alternative splice variants of the human tissue kallikrein (KLK1) and kallikrein-related peptidases 2, 5 – 12 (KLK2, KLK5 – KLK12), using 3′ rapid amplification of cDNA ends (3′ RACE) and Next-Generation Sequencing (NGS) technology, as well as their expression analysis in many established cell lines, originating from several distinct cancerous and normal tissues. Extensive bioinformatic analysis revealed novel splice variants of these five members of the KLK family, comprising entirely new exons, previously unknown boundaries of the already annotated exons (extensions and truncations) as well as alternative splicing events between these exons. Nested RT-PCR in a panel of human cell lines originating from seventeen cancerous and two normal tissues with the use of variant-specific pairs of primers was carried out for expression analysis of these novel splice variants, and Sanger sequencing of the respective amplicons confirmed our NGS results. Since several splice variants of KLK family members possess clinical value, novel alternatively spliced transcripts appear as new candidate biomarkers for diagnostic and/or prognostic purposes and as targets for therapeutic strategies.

---------------------------------------- Acknowledgements: This work was supported by Special Account for Research Grants of the National and Kapodistrian University of Athens.


28

Discovery of novel splice variants of the human carcinoembryonic antigen-related cell adhesion molecule 19 gene (CEACAM19), using Next-Generation Sequencing

---------------------------------------- Zafeiro Zisi, Panagiotis G. Adamopoulos, Christos K. Kontos and Andreas Scorilas*

---------------------------------------- Department of Biochemistry and Molecular Biology, Faculty of Biology, National and

Kapodistrian University of Athens, Athens, Greece *email: [email protected] ---------------------------------------

Cancer is one of the leading causes of death worldwide with devastating effects on the patients. Biomarkers constitute excellent tools for accurate and efficient identification and monitoring of the disease. Alternative splicing is a complexity-inducing mechanism commonly involved in carcinogenesis. The differential expression of cancer related genes leads to changes in essential processes including cell cycle control, cell proliferation and apoptosis which eventually result in disease development. The gene encoding the carcinoembryonic antigen-related cell adhesion molecule 19 (CEACAM19) is located in chromosome 19 and is physiologically expressed in various cell types. Recent studies of different cancer types have shown that CEACAM19 is overexpressed in malignant tumors and its detection is associated with poor patient outcomes and reduced survival. In this study, we identified sixteen novel transcripts of the human CEACAM19 gene using 3′ rapid amplification of cDNA ends (3′ RACE) and Next-Generation Sequencing (NGS) technology and also conducted expression analysis of the gene in a wide panel of established human cell lines. Analysis of the NGS data with bioinformatic tools uncovered new CEACAM19 splice variants consisting of new exons, extensions of already known exons, a novel intron retention and also novel alternative splicing events between already annotated exons. We investigated the expression of the gene’s variants using nested PCR with variant-specific pairs of primers and validated our results using Sanger sequencing. The wide expression of CEACAM19 in a large number of tissues, along with findings of being overexpressed in ovarian and breast tumours indicate its potential as a biomarker for prognostic and diagnostic purposes.

---------------------------------------- Acknowledgements: Part of this work was financially supported by the interdisciplinary program of postgraduate studies “Clinical Biochemistry – Molecular Diagnostics”, National and Kapodistrian University of Athens.


29

Increased circulating resistin correlates with longer disease-free survival in early-onset breast cancer: Quantitative proteomics results from the POSH multi-center cohort

---------------------------------------- Spiros D. Garbis*

---------------------------------------- Faculty of Medicine, University of Southampton, UK


Early-onset breast cancer (EOBC) affects about one in 300 women aged 40 years or younger and is associated with worse outcomes than later onset breast cancer. This study explored novel serum proteins as surrogate markers of prognosis in patients with EOBC. Serum samples from EOBC patients (stages 1-3) were analysed using agnostic high-precision quantitative proteomics. Patients received anthracycline-based chemotherapy. The discovery cohort (n = 399) either had more than 5-year disease-free survival (DFS) (good outcome group, n = 203) or DFS of less than 2 years (poor outcome group, n = 196). Expressed proteins were assessed for differential expression between the two groups. Bioinformatics pathway and network analysis in combination with literature research were used to determine clinically relevant proteins. ELISA analysis against an independent sample set from the Prospective study of Outcomes in Sporadic versus Hereditary breast cancer (POSH) cohort (n = 181) was used to validate expression levels of the selected target. Linear and generalized linear modelling was applied to determine the effect of target markers, body mass index (BMI), lymph node involvement (LN), oestrogen receptor (ER), progesterone receptor and human epidermal growth factor receptor 2 status on patients' outcome. A total of 5346 unique proteins were analysed (peptide FDR p ≤ 0.05). Of these, 812 were differentially expressed in the good vs poor outcome groups and showed significant enrichment for the insulin signalling (p = 0.01) and the glycolysis/gluconeogenesis (p = 0.01) pathways. These proteins further correlated with interaction networks involving glucose and fatty acid metabolism. A consistent nodal protein to these metabolic networks was resistin (upregulated in the good outcome group, p = 0.009). ELISA validation demonstrated resistin to be upregulated in the good outcome group (p = 0.04), irrespective of BMI and ER status. LN involvement was the only covariate with a significant association with resistin measurements (p = 0.004). An ancillary in-silico observation was the induction of the inflammatory response, leucocyte infiltration, lymphocyte migration and recruitment of phagocytes (p < 0.0001, z-score > 2). Survival analysis showed that resistin overexpression was associated with improved DFS. Higher circulating resistin correlated with node-negative patients and longer DFS independent of BMI and ER status in women with EOBC. Overexpression of serum resistin in EOBC may be a surrogate indicator of improved prognosis.

----------------------------------------

Zeidan et al, Breast Cancer Res, 2018 doi: 10.1186/s13058-018-0938-6.


30

Network-based analysis for drug repurposing and biomarker discovery: applications in NAFLD, CKD, and CVD.

---------------------------------------- Leonidas Alexopoulos*

---------------------------------------- Department of Mechanical Engineering, National Technical University of Athens, Athens

Greece *email: [email protected]

---------------------------------------- A major challenge for bringing safe and effective new treatment to patients is the deep understanding of a disease. Here, we describe multi-omics data acquisition technologies and systems biology algorithms for tackling major questions in the drug discovery pipeline: (i) identification of disease mechanisms, (ii) matching disease mechanisms to drug mode of action (MoA), and (iii) prediction of compound toxicity and efficacy. Our network-based approach is able to predict the drugs' main target and uncover off-target effects. Subsequently, machine learning algorithms can select MoAs with reduced toxicity, increased efficacy and tailor drugs to specific disease mechanisms. So far, we have applied our approach to liver cancer, osteoarthritis, multiple sclerosis, non-alcoholic fat liver disease, chronic kidney disease and more recently in melanoma. Our pathway analysis algorithms and high throughput multiplex platform pave the road for new solutions for early drug discovery.

----------------------------------------


31

lncRNAs as novel biomarkers in bladder cancer management ----------------------------------------

Margaritis Avgeris and Andreas Scorilas*



Bladder cancer (BlCa) heterogeneity in the cellular and molecular levels drives the highly variable patients’ treatment outcome. The lack of accurate disease prognosis forces the generic (non-personalized) active treatment of the patients, as well as the lifelong surveillance and monitoring strategies, classifying BlCa as the most expensive per-patient-to-treat cancer for the healthcare systems. In the present study, we have analysed the clinical value of GAS5 and H19 lncRNAs in improving patients’ prognosis and risk-stratification.GAS5 and H19 levels were quantified in a screening cohort of 176 BlCa patients (363 fresh-frozen tissue specimens of bladder tumors and matched adjacent normal bladder specimens), as well as 43 normal bladder specimens from BPH patients, by specific qPCR assays. The Hedegaard et al, 2016 (n=476) and the TCGA provisional (n=413) were used as validation cohorts for NMIBC and MIBC, respectively. Survival analysis was performed by Kaplan-Meier and Cox regression analysis using disease recurrence and progression as clinical endpoint events for NMIBC (TaT1), or death for MIBC (T2-T4) patients. Internal validation was performed by bootstrap analysis, and decision curve analysis was used to evaluate the clinical net benefit on disease outcome. GAS5 and H19 lncRNAs levels were significantly deregulated in bladder tumors and associated with unfavorable disease features, such as invasive disease stages, high-grade tumors, as well as high-EORTC risk group and recurrence at FFC of NMIBC (TaT1) patients. GAS5 and H19 loss was strongly correlated with higher risk for early-relapse and progression of the NMIBC patients, independently of tumor’s stage, grade and EORTC-risk score. The analysis of Hedegaard et al. and TCGA validation cohorts clearly confirmed the association of GAS5 loss with NMIBC worse prognosis. Finally, multivariate models incorporating GAS5 and H19 loss with disease established markers resulted to improved stratification specificity and higher clinical net benefit for NMIBC prognosis. GAS5 and H19 loss is associated with short-term relapse and progression of NMIBC and results to improved positive prediction of NMIBC patients’ poor outcome, supporting personalized treatment decisions.

----------------------------------------


32

Identification of new transcripts of the human KLK1 and KLK2 genes ----------------------------------------

Panagiotis G. Adamopoulos, Christos K. Kontos and Andreas Scorilas* ----------------------------------------

Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, Athens, Greece


Tissue kallikrein, kallikrein-related peptidases (KLKs), and plasma kallikrein form the largest group of serine proteases in the human genome, sharing many structural and functional properties. Several KLK transcripts have been found aberrantly expressed in numerous human malignancies, confirming their prognostic or/and diagnostic values. Alternative splicing of cancer-related genes is a common cellular mechanism accounting for cancer cell transcriptome complexity and affecting cell cycle control, proliferation, apoptosis, angiogenesis, invasion, and metastasis. However, the process of alternative splicing can now be studied in-depth due to the development of Next-Generation Sequencing (NGS). In the present study, we used NGS to discover novel transcripts of the KLK1 and KLK2 genes, after nested touchdown PCR. Bioinformatics analysis and PCR experiments revealed a total of eleven novel KLK transcripts (two KLK1 and nine KLK2 transcripts). Nested RT-PCR in a panel of human cell lines originating from seventeen cancerous and two normal tissues with the use of variant-specific pairs of primers was carried out for expression analysis of these novel splice variants, and Sanger sequencing of the respective amplicons confirmed our NGS results. Since KLKs are implicated in human malignancies, qualifying as potential biomarkers, the quantification of the presented novel transcripts in human samples may have clinical applications in different types of cancer.

----------------------------------------


33

Biomarkers for Alzheimer’s disease ---------------------------------------- Eleftherios P. Diamandis1,2,3* ----------------------------------------

1Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada,

2Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada,


---------------------------------------- Alzheimer’s disease (AD) is the most common type of dementia, with progressive onset of clinical symptoms. The main pathological hallmarks are brain deposits of extracellular amyloid beta plaques and intracellular neurofibrillary tangles (NFT). Cerebrospinal fluid reflects pathological changes in the brain; amyloid beta 1-42 is a marker of amyloid plaques, while total and phosphorylated tau are markers of NFT formation. Additional biomarkers associated with disease pathogenesis are needed, for better prognosis, more specific diagnosis, prediction of disease severity and progression and for improved patient classification in clinical trials. The aim of the present study was to evaluate numerous brain-specific proteins as potential biomarkers of progression of AD. Overall, 30 candidate proteins were quantified in CSF samples from patients with mild cognitive impairment (MCI) and mild, moderate and severe AD dementia (n=101) using mass spectrometry-based selected reaction monitoring assays. The best discrimination between MCI and more advanced AD stages (moderate and severe dementia) was observed for protein neuronal pentraxin receptor-1 (NPTXR) (area under the curve, AUC=0.799). A statistically different abundance of this protein was observed between the two groups, with severe AD patients having progressively lower levels (p<0.05). We conclude that NPTXR protein in CSF is a novel potential biomarker of AD progression and could have important utility in assessing treatment success in clinical trials.

----------------------------------------


34

Advances in circulating tumor DNA ---------------------------------------- Eleftherios P. Diamandis1,2,3* ----------------------------------------

1Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada

2Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada


---------------------------------------- Early detection has been in the forefront of cancer research for decades as it often enables gentler treatments and better patient outcomes. Unfortunately, the current methods for early diagnosis of cancer lack sensitivity and specificity, especially in population screening scenarios. Consequently, very few cancer screening programs are recommended by professional bodies. Thus, for most major cancers, including breast, prostate, lung and pancreatic cancer, screening is still a controversial issue. Circulating tumor DNA (ctDNA) is an exciting new cancer biomarker with potential applicability to most, if not all, cancer types. Numerous investigations have shown that genetic alterations in ctDNA could be an early sign of cancer. Tests harnessing this knowledge have attracted tremendous attention for various applications, including patient management, early diagnosis and population screening. Recently, a major biotechnology company, Grail, was founded, with the goal of revolutionizing early cancer detection by using molecular techniques applied to ctDNA. Here, we summarize the latest progress in Grail’s early diagnosis efforts. We compare these results with our recently published predictions, derived by considering plasma ctDNA abundance and tumor volume. Despite progress, it seems clear that the sensitivity and specificity of ctDNA as a screening test for cancer are currently too low for clinical use.

----------------------------------------


35

MicroRNA-92a-3p overexpression in peripheral blood mononuclear cells is an independent predictor of prolonged overall survival of patients with chronic lymphocytic

leukemia ----------------------------------------

Marios A. Diamantopoulos1, Christos K. Kontos1, Sotirios G. Papageorgiou2, Vasiliki Pappa2, Andreas Scorilas1* ----------------------------------------

1Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, Athens, Greece

2 Second Department of Internal Medicine, University General Hospital “Attikon”, Athens, Greece


Many small non-coding RNAs, including microRNAs (miRNAs) are aberrantly expressed in cancer and leukemia. MicroRNA-92a-3p (miR-92a-3p) is a member of the miR-17/92 cluster and the cancer-related miR19 family. miR-92a-3p regulates important transcription factors such as hypoxia-inducible factor 1 (HIF1) and inhibits SOCS1 to enhance the anti-apoptotic STAT3/IL6 signaling route. In this study, we examined the putative prognostic value of miR-92a-3p in chronic lymphocytic leukemia (CLL). Total RNA, which was isolated from peripheral blood mononuclear cells (PBMCs), collected from 88 CLL patients and 36 non-leukemic blood donors. After polyadenylation of 2 μg total RNA by poly(A) polymerase and subsequent reverse transcription with an oligo-dT adapter primer, we quantified miR-92-3p levels using an in-house–developed reverse-transcription real-time quantitative PCR method, based on the SYBR Green chemistry using SNORD43 (RNU43) as endogenous control. Extensive biostatistical analysis revealed that miR-92a-3p levels were significantly lower in PBMCs of CLL patients. Kaplan–Meier overall survival (OS) analysis and bootstrap univariate Cox regression showed that high miR-92a-3p expression predicts significantly prolonged OS for CLL patients. Interestingly, miR-92a-3p overexpression retains its favorable prognostic role. In conclusion, this study showed that high miR-92a-3p expression may represent a novel molecular biomarker of favorable prognosis in CLL, meriting further investigation in a large cohort of CLL patients. Ideally, miR-92a-3p expression status could be combined with surrogate prognostic biomarkers in a multi-parametric prognostic index with regard to this hematological malignancy, as the high prognostic significance of miR-92a-3p overexpression is independent of clinical staging and other established biomarkers (IGHV mutational status and CD38 expression) that are used to predict CLL patients’ outcome. Furthermore, it would be interesting to conduct mechanistic studies of miR92a-3p in CLL patients of intermediate risk or stratified according to established prognostic factors.

----------------------------------------


36

Enzymatic kinetics analysis of phytase using Isothermal Titration Calorimetry: Using ITC for complex/multiple stage reactions.

---------------------------------------- Theofilos Kempapidis1, Niall Bradshaw1, Aaron Cowieson2,

Duncan Cameron1, Robert Falconer1* ----------------------------------------

1 The University of Sheffield, Sheffield, U.K. 2 DSM Nutritional Products, Kaiseraugst, Switzerland

*e-mail: [email protected] ---------------------------------------

Isothermal titration calorimetry (ITC) is a technique that has been commonly used in for the investigation of binding interactions between molecules and for the analysis of enzymatic kinetics. Despite its power as an analytical tool ITC has primarily been used for the analysis of simple/one stage reactions due to the complexity of the data generated from multiple-step reactions. The reaction between phytase and phytic acid is known to be a complex reaction due to the stoichiometry of the phytic acid molecule. In this work, ITC was used to study the action of phytase when cleaving phosphate groups from phytic acid sodium salt hydrate. The thermogram obtained from this enzymatic reaction is complex, with at least three clear peaks produced. This was expected since phytases have a preferential mechanism of degrading phytic acid. To deconvolute the data obtained by ITC, a ‘real-time’ mass spec analysis of the same reaction was performed. By quantifying the peaks produced in the mass spectra by the lower inositol phosphates throughout the reaction, the pattern of phytic acid dephosphorylation was revealed. The phytase was found to rapidly cleave the first two phosphate groups of phytic acid, after which the rate of dephosphorylation slows to eventually produce a final product of the singly phosphorylated inositol monophosphate. By combining the ITC data with the MS data, the thermogram obtained by ITC can be deconvoluted and each step of the reaction can be identified since both datasets are against time. Thus, the kinetics of phytase can be calculated for the different steps of the reaction.

----------------------------------------

Ariza, A. et al., 2013. Degradation of Phytate by the 6-Phytase from Hafnia alvei: A Combined Structural and Solution Study. PLoS ONE, 8(5). Acknowledgements: This work was supported by DSM Nutritional Products.


37

Analysis of small non-coding RNA sequencing data: challenges and solutions through bioinformatics tools

---------------------------------------- Fotini E. Koukouzeli, Panagiotis G. Adamopoulos, Paraskevi C. Skourou,

Andreas Scorilas* ----------------------------------------

Department of Biochemistry and Molecular Biology, National and Kapodistrian University of Athens, Athens, Greece

--------------------------------------- Over the last decade, high-throughput RNA sequencing (RNA-seq) has been fueling innovative and unexpected discoveries, leading us away from the linear view of the “Central Dogma of Biology” and towards a framework in which non-coding RNAs (ncRNAs) are as important as proteins. RNA-seq has greatly facilitated the study of such molecules, making it possible to find new categories and further advance our understanding of long-established classes of ncRNAs. Here, we describe the challenges of analysing small ncRNA sequencing data from Next-Generation Sequencing (NGS) platforms and present algorithms used both for expression analysis as well as for predicting putative novel small ncRNAs, such as microRNAs (miRNAs) and tRFs (tRNA-derived fragments). MiRNAs represent a class of single-strand RNAs of ~ 22 nucleotides in length, which post-transcriptionally regulate the expression of the vast majority of human genes, thus playing an important role in a wide range of biological processes. Regarding the class of mature transfer RNAs (tRNAs), they were viewed as the sole products of the respective genomic locus used in translation. Recent advances in deep-sequencing technologies have been reshaping this understanding, revealing that tRNA loci produce regulatory fragments, known as tRFs. The role of tRFs in human health is only now starting to emerge, but several functions have already been attributed, marking their potential biomedical significance in neurodegeneration, cancer and immune modulation. Perhaps the most challenging problem associated with RNA-seq experiments is the analysis of the large resulting datasets. The miRDeep2 algorithm identifies known and potential novel human miRNAs, while tools such as tDRmapper and MINTmap provide a detailed profiling and quantification schemes, facilitating the research of tRNA-derived RNA biology. We compared the prerequisites of using these algorithms, their possible applications, as well as the generated results, giving emphasis on the need of experimental validation of each algorithm.

---------------------------------------- Acknowledgements: Part of this work was financially supported by the interdisciplinary program of postgraduate studies “Clinical Biochemistry – Molecular Diagnostics”, National and Kapodistrian University of Athens.


38

Molecular cloning of novel alternative splice variants of the human protein arginine methyltransferase 1 (PRMT1) gene with the use of Next-Generation Sequencing

---------------------------------------- Adamantios V. Mavrogiannis, Panagiotis G. Adamopoulos, Christos K. Kontos

Diamandis Sideris and Andreas Scorilas* ----------------------------------------

Department of Biochemistry and Molecular Biology, National and Kapodistrian University of Athens, Athens, Greece

--------------------------------------- Next-generation sequencing (NGS) technologies are highly expected to help researchers disclose the complexity of alternative splicing and comprehend its association with carcinogenesis. Alternative splicing alterations are firmly associated with multiple human malignancies, in terms of functional roles in the transformation, motility or metastasis of cancer cells. One perfect example illustrating the connection between alternative splicing and cancer is the human protein arginine methyltransferase 1 gene (PRMT1), which was previously cloned from members of our research group and is involved in a variety of processes, including gene transcription, DNA repair, and signal transduction. Two transcript variants of PRMT1 (variants v.1 and v.2) are downregulated in breast cancer. In addition, PRMT1 v.2 promotes the survival and invasiveness of breast cancer cells, while it could serve as a biomarker of unfavourable prognosis in colon cancer patients. The aim of this study was the molecular cloning of novel alternative splice variants of PRMT1 with the use of 3′ RACE and the use of NGS in order to discover and identify novel alternative transcripts and 3′ UTRs. Extensive bioinformatics and computational analysis revealed a significant number of 19 novel alternative splicing events between annotated exons of PRMT1 as well as one novel exon, resulting in the discovery of novel PRMT1 transcripts. In order to validate the full novel transcript sequences, RT-PCR experiments were carried out with the use of variant-specific primers. As a result, 58 novel PRMT1 transcripts were identified, from which 34 are predicted as protein-coding mRNAs, whereas the rest 24 as candidates for nonsense-mediated mRNA decay.

----------------------------------------

Acknowledgements: Part of this work was financially supported by the interdisciplinary program of postgraduate studies “Clinical Biochemistry – Molecular Diagnostics”, National and Kapodistrian University of Athens.


39

miR-125b loss is associated with adverse survival and treatment outcome in ovarian cancer patients

---------------------------------------- Konstantina Panoutsopoulou, Margaritis Avgeris, Andreas Scorilas*



Ovarian Cancer (OvCa) is the fifth most common cancer and sixth leading cause of cancer-related death in women in developed countries. miR-125b is frequently dysregulated in various human cancers and has been associated with metastasis and poor prognosis of the patients. In the present study, we aimed to investigate the expression profile of miR-125b in ovarian tumors and to explore its clinical value in OvCa patients’ prognosis. Total RNA was extracted from 75 ovarian tumors and 10 benign ovarian lesions using TRI reagent. Following total RNA polyadenylation in 3’-end, first-strand cDNA synthesis was performed using an oligo (dT) primer. A SYBR-Green fluorescent-based quantitative real-time PCR (qPCR) assay was developed to quantify miR-125b levels in ovarian tissues, using snoRNAs RNU44 and RNU48 as endogenous reference control genes and SKOV3 ovarian cancer cells as our assay calibrator. Extensive statistical analysis has been conducted using the IBM SPSS Statistics 20 software. Significant downregulation of miR-125b has been observed in ovarian tumors compared to benign tissue samples (p<0.001). Focusing on disease clinicopathological features, miR-125b loss was associated with unfavourable disease markers and poor response to chemotherapy. More precisely, reduced miR-125b levels detected in grade 3 and undifferentiated tumors compared to grade 1 and 2 tumors (p=0.073), as well as in progressed or stable disease patients following chemotherapy in relation to those presenting complete or partial remission (p=0.044). Importantly, the levels of miR-125b are negatively correlated with CA-125 pre-operation (rs=-0.213, p=0.055), post-operation (rs=-0.384, p=0.021) and during chemotherapy (rs=-0.312, p=0.073) levels, as Spearman correlation analysis demonstrated. Finally, patients with reduced miR-125b levels highlighted to have significantly higher risk for disease short-term relapse (p=0.041) and death (p=0.054) compared to those overexpressing miR-125b, despite active treatment. Our data suggest that miR-125b could play a pivotal role in the prognosis, treatment response and monitoring in OvCa.

----------------------------------------

Acknowledgements: The research presented was carried out within the framework of a Stavros Niarchos Foundation grant to the National and Kapodistrian University of Athens.

mailto:[email protected]�


40

Isolation and biological study of secondary metabolites of microbial origin as cytotoxic agents

----------------------------------------

Konstantina-Vaia Pantiora1,2, Aikaterini Georgousaki2, Christos K. Kontos1, Nikolaos Tsafantakis2, Olga Genilloud3, Nikolas Fokialakis2*, Andreas Scorilas1*

---------------------------------------- 1 Department of Biochemistry and Molecular Biology, Faculty of Biology,

National and Kapodistrian University of Athens, Athens, Greece 2 Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy,

National and Kapodistrian University of Athens, Athens, Greece 3 Fundación MEDINA, Granada, Spain

*e-mail: [email protected], [email protected]

---------------------------------------

In this study we investigated the cytotoxic effect of secondary metabolites of microbial origin in ovarian human cancer cell lines. For this purpose the compounds selected to be investigated are trichostatin A (C17H22N2O3), the trichostatin analog g-{N-(6R,2E,4E)-7-[40-(dimethylamino)phenyl]-4,6-dimethyl-7-oxohepta-2,4-dienoyl}gluta-mine (C22H29N3O5) and thrichostatic acid (C17H21NO3), that were isolated from the actinomycete Streptomyces hygroscopicus and fulvic acid (C14H12O8), penialidin F (C13H12O6) and vinaxanthone (C28H16O14), that were isolated from the fungus Cercospora sp. Both classical chromatographic methods (HPLC), as well as more innovative techniques (FCPC), were used. The full set of spectroscopic data (LC-HRMS and NMR) was recorded for all isolated compounds in order to unambiguously elucidate their structure. Ovarian cancer cell lines Skov-3 and MDAH-2774 were treated with the aforementioned compounds using the MTT assay test at a density of 0,7 × 105 cells/ml and 0,5 × 105 cells/ml respectively. Trichostatin A as a histone deacetylase inhibitor (HDACi) was more effective than the other compounds tested and inhibits cell growth in SKOV-3 cell line with an IC50 value of 10000nM. Cell viability was measured using Neubauer hemocytometer with Trypan blue. Total RNA from non-treated and TSA-treated cells was extracted using Trizol protocol. Moreover, c-DNA was synthesized in order to study BCL2, BCL2L12, BCL2L1, MCL1 and BAX genes expression by RT-quantitative PCR to assess the effect of trichostatin A.

----------------------------------------

Acknowledgements: Part of this work was financially supported by the EU project MICROMETABOLITE (GA No 612276 ) and part from the interdisciplinary program of postgraduate studies “Clinical Biochemistry – Molecular Diagnostics”, National and Kapodistrian University of Athens.



41

Downregulation of the tumor-suppressor microRNA-497 is associated with breast cancer aggressiveness and higher risk of recurrence

---------------------------------------- Papachristopoulou G1,2, Dokou A3, Drizou M3, Magou E3, Nasi D3, Tripodaki E3, Atlani C3,

Ardavanis A3, Scorilas A1* ----------------------------------------

1 Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, Athens, Greece

2 Department of Pathology, “Saint Savvas” Cancer Hospital of Athens, Athens, Greece 3First Department of Medical Oncology, “Saint Savvas” Cancer Hospital of Athens, Athens,

Greece ----------------------------------------

Aberrations in the expression of miRNAs have been correlated with variations in the corresponding transcript levels of their target-genes frequently constituting tumor suppressor genes or oncogenes. Data suggests that miR-497 act as a suppressor in breast cancer cells through negative regulation of BCL-2. The purpose of the present study was to evaluate the expression of miR-497 in 140 malignant and 70 benign breast tumors, in order to explore its differential diagnostic and prognostic value in breast cancer. Toward this direction, total RNA was extracted, polyadenylated and reversely transcripted from the tissue specimens analyzed herein. The end product was used in a highly sensitive real-time PCR protocol for the expression analysisof miR-497 and snoRNA RNU48, the latter of which served as a reference gene. Thereafter, the relative levels of miR-497 were estimated by applying the 2-ΔΔC

T method, whilst their diagnostic accuracy and correlation with the clinicopathological features of breast malignancies were assessed by comprehensive statistical analysis. Our results showed that miR-497 significantly upregulated (P<0.001) in benign compared to malignant breast tumors. Specifically, miR-497 levels showed the ability to accurately discriminate between cancerous and benign specimens, based on ROC curve analysis (AUC:0.848; 95% CI: 0.792-0.905; P<0.001). In addition, miR-497 expression decreased remarkably in tumors with high mitotic activity (Ki67>20%) (P=0.026), poor differentiated cells (Grade III) (P=0.007) and, advanced primary tumor staging (T3) (P<0.001). Moreover, Kaplan-Meier and Cox regression analyses showed that patients with elevated miR-497 levels had a significantly longer disease-free survival (P=0.028) and a lower risk to relapse (HR=0.44; 95% CI: 0.205 – 0.935; P=0.033). Overall these results recommended that miR-497 expression constitutes a novel, promising biomarker of favorable prognosis in breast cancer.

----------------------------------------

Acknowledgements: Part of this work has been financially supported by the Hellenic Society of Medical Oncology.


42

The evaluation of ΔNp63/miR-205/miR-203 axis in prognosis of bladder cancer patients ----------------------------------------

Maria-Alexandra Papadimitriou1, Athanasios Kontes1, Margaritis Avgeris1, Panagiotis Levis2, Theodoros Tokas2, Konstantinos Stravodimos2 and Andreas Scorilas1*

---------------------------------------- 1 Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Athens,

Panepistimiopolis, 15701, Athens, Greece 2 First Department of Urology, “Laiko” General Hospital, Medical School, University of Athens,

AgiouThoma 17, 11527, Athens Greece ----------------------------------------

Bladder cancer (BlCa) represents the second most commonly diagnosed urologic malignancy of the Western male population. Accurate disease prognosis is essential in establishing optimal and personalized therapeutic decisions. ΔNp63 is one of the two main isoforms of the TP63, a member of the p53/p63/p73 family of transcription factors. ΔΝp63 regulates the expression of miR-205 and the role of ΔΝp63/miR-205 axis in BlCa is poorly studied. Furthermore miR-203 and miR-92 have been validated to regulate the expression of ΔΝp63 and recent studies have highlighted their implication in human malignancies. The aim of the present study is the analysis of ΔNp63/miR-205, miR-203 and miR-92 expression regulation in bladder tumors and their clinical significance for disease outcome. Total RNA was extracted following pulverization of bladder tumors and matched adjacent normal urothelium from 109 BlCa patients. cDNA synthesis performed by the reverse transcription of 1μg of total RNA using oligo(dT) primer and MMLV for ΔΝp63 analysis, or 1μg of polyadenylated total RNA using a poly(T) primer and MMLV for miR-205/203/92. ΔNp63 mRNA and miR-205/203/92 levels were quantified by in-house developed and validated qPCR assays. ΔNp63 and miR-205 expression levels are significantly increased in bladder tumors compared to normal urothelium (ΔΝp63: p<0.001; miR-205: p<0.001). However, reduced ΔNp63 expression levels revealed in muscle-invasive (T2-T4) compared to TaT1 tumors (ΔΝp63: p<0.001; miR-205: p=0.020), as well as in high grade tumors compared to low grade (ΔΝp63: p<0.001; miR-205: p=0.022). The levels of miR-203 (p=0.002) were significantly overexpressed, while miR-92 (p=0.004) was downregulated in bladder tumors compared to matched normal adjacent tissues and urothelium from healthy controls. The survival analysis, using Kaplan-Meier curves and Cox models, clearly highlighted that the loss of ΔNp63/miR-205/203 expression correlates with the stronger risk for relapse and progression to muscle-invasive stage of the TaT1 patients. Our study highlights the correlation of ΔNp63/miR-205/203 expression loss with significantly worse progression of bladder cancer patients following treatment.

---------------------------------------- Acknowledgement: The study was supported by Hellenic Society of Medical Oncology (HeSMO)


43

Elevated miR-20b-5p expression levels in peripheral blood mononuclear cells predict favorable prognosis in chronic lymphocytic leukemia

---------------------------------------- Panagiotis Tsiakanikas1, Christos K. Kontos1, Sotirios G. Papageorgiou2, Vasiliki

Pappa2, Andreas Scorilas1* ----------------------------------------

1Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, Athens, Greece

2 Second Department of Internal Medicine, University General Hospital “Attikon”, Athens, Greece


Small non-coding RNAs, including microRNAs (miRNAs) are crucial molecules during the initial phase and progression of human malignancies and leukemias. MicroRNA-20b-5p (miR-20b-5p) is part of the miR-106a/363 cluster and a member of the cancer-related miR-17 family. Moreover, miR-20b-5p regulates important transcription factors, including hypoxia-inducible factor 1 (HIF1) and signal transducer and activator of transcription 3 (STAT3). In this study, we investigated the prognostic and diagnostic potential of miR-20b-5p in chronic lymphocytic leukemia (CLL) and evaluated its putative clinical application as a molecular biomarker in this hematological malignancy. Therefore, total RNA was extracted from peripheral blood mononuclear cells (PBMCs) collected from 88 CLL patients and 36 non-leukemic donors. After polyadenylation of 2 μg total RNA by poly(A) polymerase and subsequent first-strand cDNA synthesis using an oligo-dT–adapter primer, we quantified miR-20-5p expression levels using an in-house–developed real-time quantitative PCR methodology, based on the SYBR Green chemistry using SNORD43 (RNU43) as endogenous control. Kaplan-Meier analysis revealed significantly better OS for CLL patients overexpressing miR-20b-5p in comparison to them with decreased miR-20b-5p expression levels (p < 0.001). Bootstrap univariate Cox regression analysis uncovered a significant lower risk of death for CLL patients with elevated miR-20b-5p expression status (Hazard Ratio = 0.27, BCa 95% Bootstrap CI = 0.11 – 0.53, bootstrap p value < 0.001). Most importantly, the favorable prognostic significance of miR-20b-5p overexpression was shown to be independent of CD38 expression, mutational status of the immunoglobulin heavy chain variable (IGHV) region, and the clinical stage (Binet or Rai stage) or risk group (p < 0.001), as demonstrated in the multivariate Cox regression analysis. Interestingly, elevated expression of miR-20b-5p retains its favorable prognostic value in distinct subgroups of CLL patients, stratified according to established prognostic factors. Specifically, CLL patients with mutated IGHV and attenuated miR-20b-5p expression levels had significantly lower OS probabilities than those with mutated IGHV and positive miR-20b-5p expression status (p < 0.001). In conclusion, this study showed that high miR -20b-5p expression represents a novel, potential molecular biomarker of favorable prognosis in CLL independent of clinical staging and other already established biomarkers (IGHV mutational status and CD38 expression) that are used to predict CLL patients’ outcome.

---------------------------------------- Acknowledgements: The research presented was carried out within the framework of a Stavros Niarchos Foundation grant to the National and Kapodistrian University of Athens.



44

Expression analysis and clinical evaluation of miR-221, miR-222 and miR-143 in breast cancer

---------------------------------------- Dimitra Varthali1, Margaritis Avgeris1, Alexandros Ardavanis2, Andreas Scorilas1*

---------------------------------------- 1Department of Biochemistry and Molecular Biology, Faculty of Biology, National and

Kapodistrian University of Athens, Panepistimiopolis 15701 Athens, Greece 2First Department of Medical Oncology, “Saint Savvas” Cancer Hospital of Athens, Athens,

Greece ---------------------------------------

Breast cancer remains the most frequent diagnosed malignancy amongst women worldwide. The microRNAs (miRNAs) are small (~22 nt) non-protein-coding RNAs with tumor-suppressive or -promoting role in human malignancies and are implicated also in modern molecular diagnostics of breast cancer patients, prognosis and treatment response. The aim of the present study was the expression analysis of miR-221, miR-222 and miR-143 in breast tumors and the evaluation of their clinical significance for breast cancer patients. miR-221 has an oncogenic role, inhibits apoptosis and foster metastasis. miR-221 and miR-222 act as a cluster, promoting epithelial-to-mesenchymal transition (EMT) and tumor invasiveness. Lastly, miR-143 has a strong tumor suppressive role and οbstruction of its cytoprotective properties in cell-cycle regulation and apoptotic balance mechanisms has been found to accelerate tumor progression. Cancerous and adjacent healthy tissues were obtained by 131 breast cancer patients that underwent tumor resection. Following tissue pulverization, total RNA was extracted using TRI Reagents. After spectrophotometric RNA purity assessment, 1 μg total RNA was polyadenylated at the 3’-end by E. coli poly(A) polymerase and reverse transcripted using a poly(T) primer and MMLV. SYBR Green-based qPCR assays were developed and validated for the quantification of miRNAs of interest using SNORD48 as reference gene for normalization purposes. Statistical analysis was performed with SPSS software. We have encountered a statistically significant (p<0,001) downregulated expression in all three miRNAs in tumor samples, compared to adjacent normal tissues. Reduced miR-221 (p=0,018) and miR-143 (p=0,017) levels were detected in high grade breast carcinomas. Both miR-221 and miR-143 were statistically reduced in patients with higher levels of the molecular marker Ki67 (miR-221: p=0.030; miR-143: p<0.001), as well as with CA15.3 levels (miR-221: p=0.034; miR-143: p=0.024). miR-222 expression showed also a negative correlation with FT4 rise (p=0,038). Focusing on disease outcome following treatment, a statistically significant (p=0,006) association between miR-221 overexpression and the risk for disease relapse, as well as adverse DFS of HER2/NEU-positive patients treated with Herceptin was observed. miR-221 and miR-143 appeal as promising biomarkers in the differential diagnosis of breast tumors that could contribute in monitoring breast cancer progression and therapeutic outcome.

----------------------------------------


45

Author Index Açan NL ...................................................................................................................................... 16 Adamopoulos PG ................................................................................................ 27, 28, 32, 37,38 Ajji A ............................................................................................................................................ 19 Alam S ......................................................................................................................................... 20 Alexopoulos L ............................................................................................................................. 30 Ardavanis A ............................................................................................................................ 41,44 Atlani C ........................................................................................................................................ 41 Avgeris M ................................................................................................................. 31, 39, 42, 44 Bouras N ..................................................................................................................................... 21 Bowen RAR ................................................................................................................................. 19 Boyages SC ................................................................................................................................ 24 Bradshaw N ................................................................................................................................. 36 Brandslund I ................................................................................................................................ 15 Büber E ....................................................................................................................................... 16 Cameron D .................................................................................................................................. 36 Çelikbıçak O ................................................................................................................................ 16 Cowieson A ................................................................................................................................. 36 Diamandis EP .................................................................................................................14, 33, 34 Diamantopoulos MA ................................................................................................................... 35 Dil EJ ........................................................................................................................................... 19 Dokou A ...................................................................................................................................... 41 Drizou M ...................................................................................................................................... 41 Ertürk Z ....................................................................................................................................... 16 Esguerra V .................................................................................................................................. 19 Fahnestock M ............................................................................................................................. 25 Falconer R ................................................................................................................................... 36 Fokialakis N.................................................................................................................................. 40 Garbis SD .................................................................................................................................... 29 Georgousaki A ............................................................................................................................ 40 Kempapidis T .............................................................................................................................. 36 Kim SC ........................................................................................................................................ 19 Kontes A ...................................................................................................................................... 42 Kontos CK .............................................................................................. 27, 28, 32, 35, 38, 40, 43 Koukouzeli FE ............................................................................................................................. 37 Levis P ........................................................................................................................................ 42 Magou E ...................................................................................................................................... 41 Mavrogiannis A ............................................................................................................................ 38 Nasi D .......................................................................................................................................... 41 Oellerich M .................................................................................................................................. 18 Özen H ........................................................................................................................................ 16 Panoutsopoulou K ....................................................................................................................... 39 Pantiora KV ................................................................................................................................. 40 Papachristopoulou G .................................................................................................................. 41 Papadimitriou MA ......................................................................................................................... 42 Papageorgiou SG ................................................................................................................. 35, 43 Pappa V ................................................................................................................................ 35, 43 Plebani M .................................................................................................................................... 22


46

Prassas I ..................................................................................................................................... 26 Saffar A ....................................................................................................................................... 19 Salih B ......................................................................................................................................... 16 Salvatore F .................................................................................................................................. 17 Sattayapiwat A ............................................................................................................................ 19 Scorilas A ................................................................................................... 27, 28, 31, 32, 35, 37-44 Sideris D ....................................................................................................................................... 38 Skourou PC ................................................................................................................................. 37 Stavrodimos K ............................................................................................................................. 42 Tokas T ....................................................................................................................................... 42 Tripodaki E .................................................................................................................................. 41 Tsiakanikas P .............................................................................................................................. 43 Varthali D .................................................................................................................................... 44 Yousef GM .................................................................................................................................. 23 Zare RN ....................................................................................................................................... 19 Zisi Z ............................................................................................................................................ 28


47

NOTES

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………


48

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

…………………………………………………………………………………………………………………………………………….

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………


49

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………


50

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

……………………………………………………………………………………………………………………………………………….

………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………

international society for enzymology annual meetingise.biol.uoa.gr/files/ise presentation...

Documents